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Sample records for vacuolar membraneassociated gra3

  1. Miopatia vacuolar do lupus eritematoso

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    José Antonio Levy

    1962-06-01

    Full Text Available Após considerações rápidas sôbre as miosites, particularmente sôbre a miopatia vacuolar do lúpus eritematoso, é relatado o caso de uma paciente no qual êsse diagnóstico pôde ser confirmado pelo exame histopatológico.

  2. Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells

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    Hyangju Kang

    2014-08-01

    Full Text Available Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs, which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

  3. Isolation and characterization of membrane-associated placental proteins.

    Science.gov (United States)

    Bohn, H; Winckler, W

    1991-01-01

    Membrane-associated proteins (MPs) of the human term placenta (afterbirth) were obtained by extracting the insoluble part of the tissue with solubilizing agents, after the soluble material had been removed by washing with saline. The insoluble residue was subsequently exhaustively extracted first with the nonionic detergent Triton X-100 and then with 6 M urea. In the Triton extract eleven new different membrane-associated antigens could be detected by immunochemical methods; they were designated as MP2A to MP2L. One of these proteins (MP2C) was found to be immunochemically identical with the already described soluble placental protein PP21 [3]. MP1 another antigen detected in the Triton extract later was identified as heart stable alkaline phosphatase. In the urea extract eight different membrane-associated antigens could be identified by immunochemical methods; they were designated as MP3 to MP10. MP3 later was found to be immunochemically identical with laminin. All these membrane-associated proteins have now been isolated to purity and characterized by their physico-chemical properties. Specific antisera to the new proteins were obtained by immunizing animals with the corresponding purified proteins. They were used to detect and quantitate the new proteins in extracts of placentas and other human tissues by immunochemical methods such as gel diffusion tests. The immunocytochemical localization of the new proteins as well as measurement of their concentrations in body fluids by sensitive radioimmunoassays or enzyme immunoassays are presently under investigation.

  4. Lipid nanotechnologies for structural studies of membrane-associated proteins.

    Science.gov (United States)

    Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

    2014-11-01

    We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

  5. Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

    Science.gov (United States)

    Zahn, J A; Bergmann, D J; Boyd, J M; Kunz, R C; DiSpirito, A A

    2001-12-01

    A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

  6. Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

    Science.gov (United States)

    Zahn, James A.; Bergmann, David J.; Boyd, Jeffery M.; Kunz, Ryan C.; DiSpirito, Alan A.

    2001-01-01

    A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 μmol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)+-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159–167, 1999; Vorholt et al., J. Bacteriol. 180:5351–5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b559/569 complex as the physiological electron acceptor. PMID:11698372

  7. Auxin regulates SNARE-dependent vacuolar morphology restricting cell size.

    Science.gov (United States)

    Löfke, Christian; Dünser, Kai; Scheuring, David; Kleine-Vehn, Jürgen

    2015-03-05

    The control of cellular growth is central to multicellular patterning. In plants, the encapsulating cell wall literally binds neighbouring cells to each other and limits cellular sliding/migration. In contrast to its developmental importance, growth regulation is poorly understood in plants. Here, we reveal that the phytohormone auxin impacts on the shape of the biggest plant organelle, the vacuole. TIR1/AFBs-dependent auxin signalling posttranslationally controls the protein abundance of vacuolar SNARE components. Genetic and pharmacological interference with the auxin effect on vacuolar SNAREs interrelates with auxin-resistant vacuolar morphogenesis and cell size regulation. Vacuolar SNARE VTI11 is strictly required for auxin-reliant vacuolar morphogenesis and loss of function renders cells largely insensitive to auxin-dependent growth inhibition. Our data suggests that the adaptation of SNARE-dependent vacuolar morphogenesis allows auxin to limit cellular expansion, contributing to root organ growth rates.

  8. Plant vacuole morphology and vacuolar trafficking

    Science.gov (United States)

    Zhang, Chunhua; Hicks, Glenn R.; Raikhel, Natasha V.

    2014-01-01

    Plant vacuoles are essential organelles for plant growth and development, and have multiple functions. Vacuoles are highly dynamic and pleiomorphic, and their size varies depending on the cell type and growth conditions. Vacuoles compartmentalize different cellular components such as proteins, sugars, ions and other secondary metabolites and play critical roles in plants response to different biotic/abiotic signaling pathways. In this review, we will summarize the patterns of changes in vacuole morphology in certain cell types, our understanding of the mechanisms of plant vacuole biogenesis, and the role of SNAREs and Rab GTPases in vacuolar trafficking. PMID:25309565

  9. The Amborella vacuolar processing enzyme family

    Directory of Open Access Journals (Sweden)

    Valérie ePoncet

    2015-08-01

    Full Text Available Most vacuolar proteins are synthesized on rough endoplasmic reticulum as proprotein precursors and then transported to the vacuoles, where they are converted into their respective mature forms by vacuolar processing enzymes (VPEs. In the case of the seed storage proteins, this process is of major importance, as it conditions the establishment of vigorous seedlings. Toward the goal of identifying proteome signatures that could be associated with the origin and early diversification of angiosperms, we previously characterized the 11S-legumin-type of seed storage proteins from Amborella trichopoda, a rainforest shrub endemic to New Caledonia that is also the probable sister to all other angiosperms (Amborella Genome Project, 2013. In the present study, proteomic and genomic approaches were used to characterize the VPE family in this species. Three genes were found to encode VPEs in the Amborella’s genome. Phylogenetic analyses showed that the Amborella sequences grouped within two major clades of angiosperm VPEs, indicating that the duplication that generated the ancestors of these clades occurred before the most recent common ancestor of living angiosperms. A further important duplication within the VPE family appears to have occurred in common ancestor of the core eudicots, while many more recent duplications have also occurred in specific taxa, including both Arabidopsis thaliana and Amborella. An analysis of natural genetic variation for each of the three Amborella VPE genes revealed the absence of selective forces acting on intronic and exonic single-nucleotide polymorphisms among several natural Amborella populations of in New Caledonia.

  10. Isolation of plasma membrane-associated membranes from rat liver.

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    Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

    2014-02-01

    Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

  11. Metabolic Disorders Presenting as Vacuolar Myopathy

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    Gayathri N

    1999-01-01

    Full Text Available Thirteen cases of vacuolar myopathy (6 males, 7 females, with age range of 4 months to 22 years and diagnosed over a period from 1986 to 1999, could be categorized into acid maltase deficiency (AMD (n=6, carnitine deficiency (CD (n=5, and mitochondria-lipid-glycogen myopathy (MLGM (n=2, cases of AMD presented as floppy infants with reparatory infection, while cases of carnitine deficiency presented with progressive motor weakness with normal initial milestones. Delayed motor milestones and proximal muscle weakness was the presenting complaints in MLGM. The diagnosis in all these cases was established based on the morphological findings on muscle biopsy, namely demonstration of PAS positive material within the vacuoles in AMD, ragged red fibers, vacuoles containing neutral fats and abnormal mitochondria in CD. MLGM was characterized by the presence of PAS positive material and neutral fat. The diagnosis was confirmed by identification of abnormal mitochondria under electron microscope. The storage product appears to affect not only the muscle metabolism but also the normal structure function relationship. The study highlights the importance of supplementing routine histopathology with muscle histochemistry and election microscopy to delineate the conditions, which look similar in routine histology.

  12. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

    Science.gov (United States)

    Zahn, J A; DiSpirito, A A

    1996-02-01

    An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.

  13. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G; Winther, Jakob R.

    1996-01-01

    The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide...... of proteinase A. We found that sorting of such a hybrid protein is dependent on the vacuolar protein-sorting receptor Vps10p. This was unexpected, as strains disrupted for VPS10 sort more than 85% of the proteinase A to the vacuole. Consistent with a role for Vps10p in sorting of proteinase A, we found that 1...

  14. Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1992-01-01

    The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. C...

  15. Hydrophilic C terminus of Salicornia europaea vacuolar Na /H ...

    Indian Academy of Sciences (India)

    2014-08-08

    Aug 8, 2014 ... from the cytosol to the external medium and the sequestration of Na. + into the vacuolar compartments. Na. +. /H. + ... Yeast strain and growth conditions. S. cerevisiae mutant GX1( ena1::HIS3::ena4, nhx1:: ..... Chinese Academy of Sciences, for kindly provid- ing the Saccharomyces cerevisiae mutant GX1.

  16. Cadmium-induced changes in vacuolar aspects of Arabidopsis thaliana.

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    Sharma, Shanti S; Yamamoto, Kotaro; Hamaji, Kohei; Ohnishi, Miwa; Anegawa, Aya; Sharma, Shashi; Thakur, Sveta; Kumar, Vijay; Uemura, Tomohiro; Nakano, Akihiko; Mimura, Tetsuro

    2017-05-01

    We have examined the changes due to Cd treatment in the vacuolar form in root tip cortical cells in Arabidopsis thaliana employing a transformant with GFP fused to a tonoplast protein. A Cd-induced enhancement in complexity with general expansion of vacuolar system within 24 h was evident. The changes in the vacuolar form were dependent on the applied Cd concentrations. Concomitantly, as revealed through dithizone staining, Cd accumulated in the seedling roots exhibiting abundance of Cd-dithizone complexes in root tip, root hairs and vasculature. To get insight into the involvement of SNARE protein-mediated vesicle fusion in Cd detoxification, the magnitude of Cd toxicity in a couple of knock out mutants of the vacuolar Qa-SNARE protein VAM3/SYP22 was compared with that in the wild type. The Cd toxicity appeared to be comparable in the mutants and the wild type. In order to analyze the Cd effects at cellular level, we treated the Arabidopsis suspension-cultured cells with Cd. Cd, however, did not induce a change in the vacuolar form in suspension-cultured cells although Cd measured with ICP-MS was obviously taken up into the cell. The V-ATPase activity in the microsomal fractions from vacuoles isolated from A. thaliana suspension cultured cells remained unaffected by Cd. Changes in the levels of certain metabolites of Cd-treated cells were also not so distinct except for those of glutathione. The significance of findings is discussed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Membrane-associated cargo recycling by tubule-based endosomal sorting

    NARCIS (Netherlands)

    van Weering, J.R.T.; Cullen, P.J.

    2014-01-01

    The endosome system is a collection of organelles that sort membrane-associated proteins and lipids for lysosomal degradation or recycling back to their target organelle. Recycling cargo is captured in a network of membrane tubules emanating from endosomes where tubular carriers pinch off. These

  18. Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase

    NARCIS (Netherlands)

    van Weely, S.; Aerts, J. M.; van Leeuwen, M. B.; Heikoop, J. C.; Donker-Koopman, W. E.; Barranger, J. A.; Tager, J. M.; Schram, A. W.

    1990-01-01

    The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts. Glucocerebrosidase is synthesised as a 62.5-kDa precursor with high-mannose-type oligosaccharide chains and an apparent

  19. Vacuolar myelinopathy in waterfowl from a North Carolina impoundment

    Science.gov (United States)

    Augspurger, T.; Fischer, John R.; Thomas, Nancy; Sileo, L.; Brannian, Roger E.; Miller, Kimberli J.; Rocke, Tonie E.

    2003-01-01

    Vacuolar myelinopathy was confirmed by light and electron microscopic examination of mallards (Anas platyrhynchos), ring-necked ducks (Aythya collaris), and buffleheads (Bucephala albeola) collected during an epizootic at Lake Surf in central North Carolina (USA) between November 1998 and February 1999. Clinical signs of affected birds were consistent with central nervous system impairment of motor function (incoordination, abnormal movement and posture, weakness, paralysis). This is the first report of this disease in wild waterfowl (Anseriformes).Aug

  20. Structure and Mechanism of GumK, a Membrane-associated Glucuronosyltransferase*S⃞

    OpenAIRE

    Barreras, Máximo; Salinas, Silvina R.; Patricia L. Abdian; Kampel, Matías A.; Ielpi, Luis

    2008-01-01

    Xanthomonas campestris GumK (β-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthes...

  1. An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast.

    Science.gov (United States)

    Hughes, Adam L; Gottschling, Daniel E

    2012-12-13

    Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.

  2. Attempts to reproduce vacuolar myelinopathy in domestic swine and chickens.

    Science.gov (United States)

    Lewis-Weis, Lynn A; Gerhold, Richard W; Fischer, John R

    2004-07-01

    Avian vacuolar myelinopathy (AVM) was first recognized as a cause of bald eagle (Haliaeetus leucocephalus) mortality in 1994 in Arkansas (USA) and has since caused over 90 bald eagle and numerous American coot (Fulica americana) mortalities in five southeastern states. The cause of AVM remains undetermined but is suspected to be a biotoxin. Naturally occurring AVM has been limited to wild waterbirds, raptors, and one species of shorebird, and has been reproduced experimentally in red-tailed hawks (Buteo jamaicensis). In this study, chickens and swine were evaluated for susceptibility to vacuolar myelinopathy with the intent of developing animal models for research and to identify specific tissues in affected coots that contain the causative agent. Additionally, submerged, aquatic vegetation, primarily hydrilla (Hydrilla verticillata), and associated material collected from a reservoir during an AVM outbreak was fed to chickens in an effort to reproduce the disease. In two separate experiments, six 4-wk-old leghorn chickens and ten 5-wk-old leghorn chickens were fed coot tissues. In a third experiment, five 3-mo-old domestic swine and one red-tailed hawk, serving as a positive control, were fed coot tissues. In these experiments, treatment animals received tissues (brain, fat, intestinal tract, kidney, liver, and/or muscle) from coots with AVM lesions collected at a lake during an AVM outbreak. Negative control chickens and one pig received tissues from coots without AVM lesions that had been collected at a lake where AVM has never been documented. In a fourth experiment, eight 3-wk-old leghorn chickens were fed aquatic vegetation material. Four chickens received material from the same lake from which coots with AVM lesions were collected for the previous experiments, and four control chickens were fed material from the lake where AVM has never been documented. Blood was collected and physical and neurologic exams were conducted on animals before and once per week

  3. Characteristics of fatty acid composition of lipids in higher plant vacuolar membranes.

    Science.gov (United States)

    Makarenko, S P; Konenkina, T A; Salyaev, R K

    2000-01-01

    The fatty acid composition of vacuolar membrane lipids from plant storage tissues and their genesis have been studied. A high content of unsaturated fatty acids (up to 77%) was observed in lipids of these membranes. Linoleic acid prevailed in vacuolar lipids of carrot and red beet (54.2 and 44.2%, respectively). Linolenic acid prevailed in vacuolar lipids of garden radish and turnip (39.7 and 33.9%, respectively). Regarding saturated fatty acids, vacuolar lipids of garden radish, carrot, and red beet contained predominantly palmitic acid (up to 20-24%). Unsaturated fatty acids, petroselinic (C18: 1omega12), cis-vaccenic (C18: 1omega7), hexatrien-7,-10,-13-oic (C16:3omega3) and others, were observed in vacuolar lipids of roots. These acids are usually synthesized in chloroplasts, and their presence in vacuolar lipids can be associated either with the transport of metabolites to the vacuole, or with endocytosis during vacuolar formation in the plant cell. The specific features of fatty acid composition of tonoplast lipids apparently are closely related to the tonoplast unique fluidity and mobility required for running osmotic processes in the cell and for forming transport protein assemblies.

  4. Function of Membrane-Associated Proteoglycans in the Regulation of Satellite Cell Growth.

    Science.gov (United States)

    Song, Yan

    2016-01-01

    Muscle growth can be divided into embryonic and postnatal periods. During the embryonic period, mesenchymal stem cells proliferate and differentiate to form muscle fibers. Postnatal muscle growth (hypertrophy) is characterized by the enlargement of existing muscle fiber size. Satellite cells (also known as adult myoblasts) are responsible for hypertrophy. The activity of satellite cells can be regulated by their extracellular matrix (ECM). The ECM is composed of collagens, proteoglycans, non-collagenous glycoproteins, cytokines and growth factors. Proteoglycans contain a central core protein with covalently attached glycosaminoglycans (GAGs: chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate) and N- or O-linked glycosylation chains. Membrane-associated proteoglycans attach to the cell membrane either through a glycosylphosphatidylinositol anchor or transmembrane domain. The GAGs can bind proteins including cytokines and growth factors. Both cytokines and growth factors play important roles in regulating satellite cell growth and development. Cytokines are generally associated with immune cells. However, cytokines can also affect muscle cell development. For instance, interleukin-6, tumor necrosis factor-α, and leukemia inhibitory factor have been reported to affect the proliferation and differentiation of satellite cells and myoblasts. Growth factors are potent stimulators or inhibitors of satellite cell proliferation and differentiation. The proper function of some cytokines and growth factors requires an interaction with the cell membrane-associated proteoglycans to enhance the affinity to bind to their primary receptors to initiate downstream signal transduction. This chapter is focused on the interaction of membrane-associated proteoglycans with cytokines and growth factors, and their role in satellite cell growth and development.

  5. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

    Science.gov (United States)

    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  6. Lysosomal and vacuolar sorting: not so different after all!

    Science.gov (United States)

    de Marcos Lousa, Carine; Denecke, Jurgen

    2016-06-15

    Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  7. Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Dan; Hammel, Ken

    2008-10-02

    Fungal lignin-degrading systems must include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, secretion of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Despite their importance, little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of them by shotgun liquid chromatography/tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein I.D. 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were up-regulated under ligninolytic conditions. Real time reverse transcription polymerase chain reaction assays showed that RNA transcripts encoding all of these proteins were also up-regulated in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium, and are therefore most likely associated with the outer envelope of the fungus.

  8. Avian vacuolar myelinopathy in the southeast: An ecoepidemiological assessment with emphasis on Lake Surf, North Carolina

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Between 2000 and 2005, the U.S. Fish and Wildlife Service (Service) and partners conducted an investigation of avian vacuolar myelinopathy (AVM), an unusual...

  9. Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

    Directory of Open Access Journals (Sweden)

    Helene Tournu

    Full Text Available The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs. Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

  10. Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

    Science.gov (United States)

    Tournu, Helene; Carroll, Jennifer; Latimer, Brian; Dragoi, Ana-Maria; Dykes, Samantha; Cardelli, James; Peters, Tracy L; Eberle, Karen E; Palmer, Glen E

    2017-01-01

    The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

  11. Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters.

    Science.gov (United States)

    Sloothaak, J; Odoni, D I; de Graaff, L H; Martins Dos Santos, V A P; Schaap, P J; Tamayo-Ramos, J A

    2015-01-01

    The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger

  12. siRNA-based Analysis of the Abrogation of the Protective Function of Membrane-associated Catalase of Tumor Cells.

    Science.gov (United States)

    Bauer, Georg

    2017-02-01

    Tumor cells, in contrast to non-malignant cells, show sustained expression of membrane-associated NADPH oxidase-1 and therefore generate extracellular superoxide anions and their dismutation product H2O2 In order to prevent intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling, tumor cells need to express membrane-associated catalase that interferes with HOCl and nitric oxide/peroxynitrite signaling. Catalase is attached to tumor cells through the activity of transglutaminase-2 and is prevented from superoxide anion-dependent inhibition through coexpression of membrane-associated superoxide dismutase. Therefore, specific inhibition of membrane-associated catalase should reactivate intercellular ROS/RNS-dependent apoptosis-inducing signaling. These processes are analyzed here through small interfering RNA-mediated knockdown of essential signaling compounds. This allows to establish a rather comprehensive picture of intercellular ROS/RNS signaling that may be instrumental for future therapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. Experimental vacuolar myelinopathy in red-tailed hawks.

    Science.gov (United States)

    Fischer, John R; Lewis-Weis, Lynn A; Tate, Cynthia M

    2003-04-01

    Avian vacuolar myelinopathy (AVM) was recognized in 1994 as a cause of wild bird mortality when 29 bald eagles (Haliaeetus leucocephalus) succumbed to the disease at DeGray Lake, Arkansas (USA). The cause of AVM and its source remain undetermined despite extensive diagnostic and research investigations. Two years later, when AVM killed 26 eagles in the same area in Arkansas, it became apparent that American coots (Fulica americana) had identical neurologic signs and lesions, and it was hypothesized that eagles acquired AVM via ingestion of affected coots. In order to test this hypothesis, we fed coot tissues (brain, liver, kidney, muscle, fat, and intestinal tract) to rehabilitated, non-releasable red-tailed hawks (Buteo jamaicensis). Five hawks received tissues from coots with AVM lesions, and one hawk received tissues from coots without brain lesions that had been collected at a site where AVM never has been documented. All hawks received 12-70 g/day (mean = 38 g) of coot tissues for 28 days. All six hawks remained clinically normal during the study. The birds were euthanatized on day 29 and microscopic lesions of AVM were found in all hawks that received tissues from affected coots, but not in the hawk that received tissues from unaffected coots. This marks the first time that AVM has been produced in birds under laboratory conditions and proves that birds of prey can acquire AVM via ingestion of tissues from affected coots.

  14. Clinical features of avian vacuolar myelinopathy in American coots

    Science.gov (United States)

    Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Tomlinson, L.; Thomas, N.J.; Stoskopf, M.K.

    2002-01-01

    Objectivea??To characterize clinical features of avian vacuolar myelinopathy (AVM) in American coots. Designa??Case-control study. Animalsa??26 AVM-affected American coots and 12 unaffected coots. Proceduresa??Complete physical, neurologic, hematologic, and plasma biochemical evaluations were performed. Affected coots received supportive care. All coots died or were euthanatized, and AVM status was confirmed via histopathologic findings. Resultsa??3 severely affected coots were euthanatized immediately after examination. Seventeen affected coots were found dead within 7 days of admission, but 5 affected coots survived > 21 days and had signs of clinical recovery. Abnormal physical examination findings appeared to be related to general debilitation. Ataxia (88%), decreased withdrawal reflexes (88%), proprioceptive deficits (81%), decreased vent responses (69%), beak or tongue weakness (42%), and head tremors (31%), as well as absent pupillary light responses (46%), anisocoria (15%), apparent blindness (4%), nystagmus (4%), and strabismus (4%) were detected. Few gross abnormalities were detected at necropsy, but histologically, all AVM-affected coots had severe vacuolation of white matter of the brain. None of the control coots had vacuolation. Conclusions and Clinical Relevancea??Although there was considerable variability in form and severity of clinical neurologic abnormalities, clinical signs common in AVM-affected birds were identified. Clinical recovery of some AVM-affected coots can occur when supportive care is administered. Until the etiology is identified, caution should be exercised when rehabilitating and releasing coots thought to be affected by AVM.

  15. TBK1 protects vacuolar integrity during intracellular bacterial infection.

    Directory of Open Access Journals (Sweden)

    Andrea L Radtke

    2007-03-01

    Full Text Available TANK-binding kinase-1 (TBK1 is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella, more extensive bacterial proliferation was observed in tbk1(-/- than tbk1(+/+ cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

  16. Imaging plant nuclei and membrane-associated cytoskeleton by field emission scanning electron microscopy.

    Science.gov (United States)

    Fišerová, Jindřiška; Goldberg, Martin W

    2014-01-01

    Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.

  17. Resolution of Dialyzer Membrane-Associated Thrombocytopenia with Use of Cellulose Triacetate Membrane: A Case Report

    Directory of Open Access Journals (Sweden)

    Feyisayo Olafiranye

    2011-01-01

    Full Text Available Blood and dialyzer membrane interaction can cause significant thrombocytopenia through the activation of complement system. The extent of this interaction determines the biocompatibility of the membrane. Although the newer synthetic membranes have been shown to have better biocompatibility profile than the cellulose-based membranes, little is known about the difference in biocompatibility between synthetic membrane and modified cellulose membrane. Herein, we report a case of a patient on hemodialysis who developed dialyzer-membrane-related thrombocytopenia with use of synthetic membrane (F200NR polysulfone. The diagnosis of dialyzer membrane-associated thrombocytopenia was suspected by the trend of platelet count before and after dialysis, and the absence of other possible causes of thrombocytopenia. We observed significant improvement in platelet count when the membrane was changed to modified cellulose membrane (cellulose triacetate. In patients at high risk for thrombocytopenia, the modified cellulose membrane could be a better alternative to the standard synthetic membranes during hemodialysis.

  18. Cryo-electron tomography: an ideal method to study membrane-associated proteins.

    Science.gov (United States)

    Dunstone, Michelle A; de Marco, Alex

    2017-08-05

    Cryo-electron tomography (cryo-ET) is a three-dimensional imaging technique that makes it possible to analyse the structure of complex and dynamic biological assemblies in their native conditions. The latest technological and image processing developments demonstrate that it is possible to obtain structural information at nanometre resolution. The sample preparation required for the cryo-ET technique does not require the isolation of a protein and other macromolecular complexes from its native environment. Therefore, cryo-ET is emerging as an important tool to study the structure of membrane-associated proteins including pores.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'. © 2017 The Author(s).

  19. Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions.

    Science.gov (United States)

    Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa

    2013-12-01

    Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is known about the nature and regulation of these membrane-associated reactions. Advanced proteomic and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions are reviewed. However, further research is still needed to fully understand the role of microsomes in fungal biodegradation and biotransformation reactions.

  20. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    Science.gov (United States)

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

  1. Discovery of New Membrane-Associated Proteins Overexpressed in Small-Cell Lung Cancer

    Science.gov (United States)

    Ocak, Sebahat; Friedman, David B.; Chen, Heidi; Ausborn, Jamie A.; Hassanein, Mohamed; Detry, Bruno; Weynand, Birgit; Aboubakar, Frank; Pilette, Charles; Sibille, Yves; Massion, Pierre P.

    2014-01-01

    Introduction Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may identify membrane-associated proteins (MAPs) specific to SCLC, advance our understanding of SCLC biology, and discover new biomarkers of SCLC. Methods MAP lysates were prepared from three SCLCs, three non–small-cell lung cancers, and three immortalized normal bronchial epithelial cell lines and coanalyzed by DIGE. Subsequent protein identification was performed by mass spectrometry. Proteins were submitted to Ingenuity Pathway Analysis. Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC). Results Principal component analysis on the global DIGE data set demonstrated that the four replicates derived from each of the nine cell lines clustered closely, as did samples within the same histological group. One hundred thirty-seven proteins were differentially expressed in SCLC compared with non–small-cell lung cancer and immortalized normal bronchial epithelial cells. These proteins were overrepresented in cellular/tissue morphology networks. Dihydropyrimidinase-related protein 2, guanine nucleotide–binding protein alpha-q, laminin receptor 1, pontin, and stathmin 1 were selected as candidate biomarkers among MAPs overexpressed in SCLC. Overexpression of all candidates but RSSA in SCLC was verified by WB and/or IHC on tissue microarrays. These proteins were significantly associated with SCLC histology and survival in univariables analyses. Conclusion DIGE analysis of a membrane-associated subproteome discovered overexpression of dihydropyrimidinase-related protein 2, guanine nucleotide–binding protein alpha-q, RUVB1, and stathmin 1 in SCLC. Results were verified by WB and/or IHC in primary tumors, suggesting that investigating their functional relevance in SCLC progression is warranted. Association with

  2. MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

    Directory of Open Access Journals (Sweden)

    Helena Bujalka

    Full Text Available The myelination of axons is a crucial step during vertebrate central nervous system (CNS development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf, as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

  3. Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress1

    Science.gov (United States)

    Baetz, Ulrike; Tohge, Takayuki; Martinoia, Enrico; De Angeli, Alexis

    2016-01-01

    The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na+ and Cl− are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl− channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl− and Na+. In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl− and Na+ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl− loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity. PMID:27503602

  4. Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

    Science.gov (United States)

    Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

  5. Interplay of vacuolar transporters for coupling primary and secondary active transport

    Directory of Open Access Journals (Sweden)

    Michèle Siek

    2016-10-01

    Full Text Available Secondary active transporters are driven by the proton motif force which is generated by primary active transporters such as the vacuolar proton pumps V-ATPase and V-PPase. The vacuole occupies up to 90 % of the mature cell and acidification of the vacuolar lumen is a challenging and energy-consuming task for the plant cell. Therefore, a direct coupling of primary and secondary active transporters is expected to enhance transport efficiency and to reduce energy consumption by transport processes across the tonoplast. This has been addressed by analyzing physical and functional interactions between the V-ATPase and a selection of vacuolar transporters including the primary active proton pump AVP1, the calcium ion/proton exchanger CAX1, the potassium ion/proton symporter KUP5, the sodium ion/proton exchanger NHX1, and the anion/proton exchanger CLC-c. Physical interaction was demonstrated in vivo for the V-ATPase and the secondary active transporters CAX1 and CLC-c, which are responsible for calcium- and anion-accumulation in the vacuole, respectively. Measurements of V-ATPase activity and vacuolar pH revealed a functional interaction of V-ATPase and CAX1, CLC-c that is likely caused by the observed physical interaction. The complex of the V-ATPase further interacts with the nitrate reductase 2, and as a result, nitrate assimilation is directly linked to the energization of vacuolar nitrate accumulation by secondary active anion/proton exchangers.

  6. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

    Science.gov (United States)

    Stransky, Laura A.; Forgac, Michael

    2015-01-01

    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  7. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

    2015-01-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

  8. Human skin basement membrane-associated heparan sulphate proteoglycan: distinctive differences in ultrastructural localization as a function of developmental age

    DEFF Research Database (Denmark)

    Horiguchi, Y; Fine, J D; Couchman, J R

    1991-01-01

    at different developmental ages using two monoclonal antibodies to a well-characterized basement membrane-associated heparan sulphate proteoglycan. A series of foetal skin specimens (range, 54-142 gestational days) were examined using an immunoperoxidase immunoelectron microscopic technique. In specimens...... representing very early developmental ages, very diffuse immunoreaction products were detected. However, by approximately 76 gestational days, some accentuation of heparan sulphate proteoglycan was noted along the lamina densa, and by 142 gestational days, the distribution of heparan sulphate proteoglycan...... was identical to that observed in neonatal and adult human skin. These findings demonstrate that active remodelling of the dermo-epidermal junction occurs during at least the first two trimesters, and affects not only basement membrane-associated structures but also specific antigens....

  9. The membrane-associated form of α(s1-casein interacts with cholesterol-rich detergent-resistant microdomains.

    Directory of Open Access Journals (Sweden)

    Annabelle Le Parc

    Full Text Available Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1-casein. These experiments reveal that the insolubility of α(s1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

  10. The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

    Science.gov (United States)

    Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

    2017-12-01

    The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

  11. The plasma membrane-associated NADH oxidase of spinach leaves responds to blue light

    Science.gov (United States)

    Morre, D. James; Penel, Claude; Greppin, Hubert; Morre, Dorothy M.

    2002-01-01

    The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks is characterized by oscillations in activity with a regular period length of ca. 24 min. Within a single population of plants exposed to light at the same time, NOX activities of all plants function synchronously. Exposure of plants transferred from darkness to blue light (495 nm, 2 min, 50 micromoles m-2 s-1) resulted in a complex response pattern but with a new maximum in the rate of NOX activity 36 (24+12) min after illumination and then with maxima in the rate of NOX activity every 24 min thereafter. Transient maxima in NOX activity were observed as well after 9.3 + /- 1.4 and 20.7 +/- 2.1 min. The blue light response differed from the response to red (650 nm, 10 min, 50 micromoles m-2 s-1) or white light where activity maxima were initiated 12 min after the light exposure followed by maxima every 24 min thereafter. Green or yellow light was ineffective. The light response was independent of the time in the 24-min NOX cycle when the light was given. The net effects of blue and red light were ultimately the same with a new maximum in the rate of NOX activity at 12+24=36 min (and every 24 min thereafter), but the mechanisms appear to be distinct.

  12. The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe.

    Science.gov (United States)

    Kawano-Kawada, Miyuki; Chardwiriyapreecha, Soracom; Manabe, Kunio; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

    2016-12-01

    Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3(∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3+ but was not completely rescued by the expression of avt3(∆1-270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

  13. Vacuolar invertase gene silencing in potato decreasing the frequency of sugar-end defects

    Science.gov (United States)

    Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one e...

  14. INHIBITION OF INWARD RECTIFYING TONOPLAST CHANNELS BY A VACUOLAR FACTOR - PHYSIOLOGICAL AND KINETIC IMPLICATIONS

    NARCIS (Netherlands)

    MAATHUIS, FJM; PRINS, HBA

    Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of young Vigna unguiculata seedlings show no or hardly any channel activity at tonoplast

  15. Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification

    NARCIS (Netherlands)

    Li, Y.; Provenzano, S.; Bliek, M.; Spelt, C.; Appelhagen, I.; Machado de Faria, L.; Verweij, W.; Schubert, A.; Sagasser, M.; Seidel, T.; Weisshaar, B.; Koes, R.; Quattrocchio, F.

    2016-01-01

    Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H+ P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As

  16. An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour

    NARCIS (Netherlands)

    Verweij, W.; Spelt, C.; di Sansebastiano, G.-P.; Vermeer, J.; Reale, L.; Ferranti, F.; Koes, R.; Quattrocchio, F.

    2008-01-01

    The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase1 (V-ATPase), which, in plants, act together with

  17. Vacuolar iron transporter BnMEB2 is involved in enhancing iron tolerance of Brassica napus

    Directory of Open Access Journals (Sweden)

    Wei Zhu

    2016-09-01

    Full Text Available Iron toxicity is a major nutrient disorder that severely affects crop development and yield. Vacuolar detoxification of metal stress is an important strategy for plants to survive and adapt to this adverse environment. Vacuolar iron transporter (VIT members are involved in this process and play essential roles in iron storage and transport. In this study, a rapeseed VIT gene BnMEB2 (BnaC07g30170D was identified. BnMEB2 is a homolog to Arabidopsis MEB2 (At5g24290 and acts as a detoxifier in vacuolar sequestration of divalent metal. Transient expression analysis revealed that BnMEB2 was localized to the vacuolar membrane. Q-PCR detection showed a high expression of BnMEB2 in mature (60-day-old leaves and could be obviously induced by exogenous iron stress in both roots and leaves. Over-expressed BnMEB2 in both Arabidopsis wild type and meb2 mutant seedlings resulted in greatly improved iron tolerability with no significant changes in the expression level of other vacuolar iron transporter genes. The mutant meb2 grew slowly and its root hair elongation was inhibited under high iron concentration condition while BnMEB2 over-expressed transgenic plants of the mutant restored the phenotypes with apparently higher iron storage in roots and dramatically increased iron content in the whole plant. Taken together, these results suggested that BnMEB2 was a VIT gene in rapeseed which was necessary for safe storage and vacuole detoxification function of excess iron to enhance the tolerance of iron toxicity. This research sheds light on a potentially new strategy for attenuating hazardous metal stress from environment and improving iron biofortification in Brassicaceae crops.

  18. Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Soracom Chardwiriyapreecha

    Full Text Available In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

  19. Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

    2015-01-01

    In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

  20. Structure and mechanism of GumK, a membrane-associated glucuronosyltransferase.

    Science.gov (United States)

    Barreras, Máximo; Salinas, Silvina R; Abdian, Patricia L; Kampel, Matías A; Ielpi, Luis

    2008-09-05

    Xanthomonas campestris GumK (beta-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9 angstroms resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28 angstroms resolution. We identified a number of catalytically important residues, including Asp157, which serves as the general base in the transfer reaction. Residues Met231, Met273, Glu272, Tyr292, Met306, Lys307, and Gln310 interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved alpha/beta/alpha structural motif of GumK.

  1. Structure and Mechanism of GumK, a Membrane-associated Glucuronosyltransferase*S⃞

    Science.gov (United States)

    Barreras, Máximo; Salinas, Silvina R.; Abdian, Patricia L.; Kampel, Matías A.; Ielpi, Luis

    2008-01-01

    Xanthomonas campestris GumK (β-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9Å resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28Å resolution. We identified a number of catalytically important residues, including Asp157, which serves as the general base in the transfer reaction. Residues Met231, Met273, Glu272, Tyr292, Met306, Lys307, and Gln310 interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved α/β/α structural motif of GumK. PMID:18596046

  2. Caspase-2 short isoform interacts with membrane-associated cytoskeleton proteins to inhibit apoptosis.

    Directory of Open Access Journals (Sweden)

    Chunhua Han

    Full Text Available Caspase-2 (casp-2 is the most conserved caspase across species, and is one of the initiator caspases activated by various stimuli. The casp-2 gene produces several alternative splicing isoforms. It is believed that the long isoform, casp-2L, promotes apoptosis, whereas the short isoform, casp-2S, inhibits apoptosis. The actual effect of casp-2S on apoptosis is still controversial, however, and the underlying mechanism for casp-2S-mediated apoptosis inhibition is unclear. Here, we analyzed the effects of casp-2S on DNA damage induced apoptosis through "gain-of-function" and "loss-of-function" strategies in ovarian cancer cell lines. We clearly demonstrated that the over-expression of casp-2S inhibited, and the knockdown of casp-2S promoted, the cisplatin-induced apoptosis of ovarian cancer cells. To explore the mechanism by which casp-2S mediates apoptosis inhibition, we analyzed the proteins which interact with casp-2S in cells by using immunoprecipitation (IP and mass spectrometry. We have identified two cytoskeleton proteins, Fodrin and α-Actinin 4, which interact with FLAG-tagged casp-2S in HeLa cells and confirmed this interaction through reciprocal IP. We further demonstrated that casp-2S (i is responsible for inhibiting DNA damage-induced cytoplasmic Fodrin cleavage independent of cellular p53 status, and (ii prevents cisplatin-induced membrane blebbing. Taken together, our data suggests that casp-2S affects cellular apoptosis through its interaction with membrane-associated cytoskeletal Fodrin protein.

  3. Structure and Mechanism of GumK, a Membrane-Associated Glucuronosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Barreras, M.; Salinas, S; Abdian, P; Kampel, M; Lelpi, L

    2008-01-01

    Xanthomonas campestris GumK (?-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9A resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28A resolution. We identified a number of catalytically important residues, including Asp157, which serves as the general base in the transfer reaction. Residues Met231, Met273, Glu272, Tyr292, Met306, Lys307, and Gln310 interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved ?/?/? structural motif of GumK.

  4. Saccharomyces cerevisiae mutants affected in vacuole assembly or vacuolar H+-ATPase are hypersensitive to lead (Pb) toxicity

    OpenAIRE

    Sousa, Cátia A.; Perez, Rita R.; Soares, Eduardo V.

    2014-01-01

    Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H+-ATPase (V-ATPas...

  5. Involvement of MoVMA11, a Putative Vacuolar ATPase c' Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Guoqing Chen

    Full Text Available Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c' subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae.

  6. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  7. Vsl1p cooperates with Fsv1p for vacuolar protein transport and homotypic fusion in Schizosaccharomyces pombe.

    Science.gov (United States)

    Hosomi, Akira; Higuchi, Yujiro; Yagi, Satoshi; Takegawa, Kaoru

    2015-01-01

    Members of the SNARE protein family participate in the docking-fusion step of several intracellular vesicular transport events. Saccharomyces cerevisiae Vam7p was identified as a SNARE protein that acts in vacuolar protein transport and membrane fusion. However, in Schizosaccharomyces pombe, there have been no reports regarding the counterpart of Vam7p. Here, we found that, although the SPCC594.06c gene has low similarity to Vam7p, the product of SPCC594.06c has a PX domain and SNARE motif like Vam7p, and thus we designated the gene Sch. pombe vsl1(+) (Vam7-like protein 1). The vsl1Δ cells showed no obvious defect in vacuolar protein transport. However, cells of the vsl1Δ mutant with a deletion of fsv1(+), which encodes another SNARE protein, displayed extreme defects in vacuolar protein transport and vacuolar morphology. Vsl1p was localized to the vacuolar membrane and prevacuolar compartment, and its PX domain was essential for proper localization. Expression of the fusion protein GFP-Vsl1p was able to suppress ZnCl2 sensitivity and the vacuolar protein sorting defect in the fsv1Δ cells. Moreover, GFP-Vsl1p was mislocalized in a pep12Δ mutant and in cells overexpressing fsv1(+). Importantly, overexpression of Sac. cerevisiae VAM7 could suppress the sensitivity to ZnCl2 of vsl1Δ cells and the vacuolar morphology defect of vsl1Δfsv1Δ cells in Sch. pombe. Taken together, these data suggest that Vsl1p and Fsv1p are required for vacuolar protein transport and membrane fusion, and they function cooperatively with Pep12p in the same membrane-trafficking step. © 2015 The Authors.

  8. Nanoparticulate vacuolar ATPase blocker exhibits potent host-targeted antiviral activity against feline coronavirus

    OpenAIRE

    Hu, Che-Ming Jack; Chang, Wei-Shan; Fang, Zih-Syun; Chen, You-Ting; Wang, Wen-Lin; Tsai, Hsiao-Han; Chueh, Ling-Ling; Takano, Tomomi; Hohdatsu, Tsutomu; Chen, Hui-Wen

    2017-01-01

    Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. The disease remains incurable, and there is no effective vaccine available. In light of the pathogenic mechanism of feline coronavirus that relies on endosomal acidification for cytoplasmic entry, a novel vacuolar ATPase blocker, diphyllin, and its nanoformulation are herein investigated for their antiviral activity against the type II feline infectious per...

  9. Vacuolar-Iron-Transporter1-Like proteins mediate iron homeostasis in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Julia Gollhofer

    Full Text Available Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1. Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140, AtVTL2 (At1g76800 or AtVTL5 (At3g25190 in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.

  10. Discovery, Structure and Tentative Functions of a C-terminal propeptide of Vacuolar Potato Lipases (Patatins)

    DEFF Research Database (Denmark)

    Welinder, Karen Gjesing; Jørgensen, Malene

    Potato tuber patatins amount to 25-40% of potato tuber protein. They are dimers of ca. 90 kDa with lipase/ esterase activity as shown by gel filtration followed by activity measurements, whereas the subunits are 40-42 kDa including glycans as demonstrated by non-reducing SDS-PAGE and MALDI-TOF-MS...... ligand of the vacuolar sorting receptor and processing enzyme responsible for patatin import and processing.  ...

  11. Vacuolar chloride transport in Mesembryanthemum crystallinum L. measured using the fluorescent dye lucigenin.

    Science.gov (United States)

    Wissing, F; Smith, J A

    2000-10-01

    To study vacuolar chloride (Cl(-)) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl(-) uptake into isolated tonoplast vesicles was measured using the Cl(-)-sensitive fluorescent dye lucigenin (N,N'-dimethyl-9,9'-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl(-), with a Stern-Volmer constant of 173 m(-1) in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl(-)-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K(m) of 17.2 mm and a V(max) of 4.8 mm min(-1). Vacuolar Cl(-) transport was not affected by sulfate, malate, or nitrate. In the presence of 250 microm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl(-) transport was actually significantly increased by 24%. To determine absolute fluxes of Cl(-) using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 x 10(7) m(-1). After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl(-) transport of 31 nmol m(-2) sec(-1) was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.

  12. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

    KAUST Repository

    Wang, Zhen-Yu

    2014-11-21

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

  13. Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

    Science.gov (United States)

    Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

    2013-09-01

    Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Cloning and characterization of a novel vacuolar Na+/H+ antiporter gene (Dgnhx1 from chrysanthemum.

    Directory of Open Access Journals (Sweden)

    Qing-Lin Liu

    Full Text Available Plant vacuolar Na(+/H(+ antiporter genes play significant roles in salt tolerance. However, the roles of the chrysanthemum vacuolar Na(+/H(+ antiporter genes in salt stress response remain obscure. In this study, we isolated and characterized a novel vacuolar Na(+/H(+ antiporter gene DgNHX1 from chrysanthemum. The DgNHX1 sequence contained 1920 bp with a complete open reading frame of 1533 bp encoding a putative protein of 510 amino acids with a predicted protein molecular weight of 56.3 kDa. DgNHX1 was predicted containing nine transmembrane domains. Its expression in the chrysanthemum was up-regulated by salt stress, but not by abscisic acid (ABA. To assess roles of DgNHX1 in plant salt stress responses, we performed gain-of-function experiment. The DgNHX1-overexpression tobacco plants showed significant salt tolerance than the wild type (WT. The transgenic lines exhibited more accumulation of Na(+ and K(+ under salt stress. These findings suggest that DgNHX1 plays a positive regulatory role in salt stress response.

  15. Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

    Science.gov (United States)

    Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

    2017-11-01

    Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

  16. Characterization of Gene Candidates for Vacuolar Sodium Transport from Hordeum Vulgare

    KAUST Repository

    Scheu, Arne Hagen August

    2017-05-01

    Soil salinity is a major abiotic stress for land plants, and multiple mechanisms of salt tolerance have evolved. Tissue tolerance is one of these mechanisms, which involves the sequestration of sodium into the vacuole to retain low cytosolic sodium concentrations. This enables the plant to maintain cellular functions, and ultimately maintain growth and yield. However, the molecular components involved in tissue tolerance remain elusive. Several candidate genes for vacuolar sodium sequestration have recently been identified by proteome analysis of vacuolar membranes purified from the salt-tolerant cereal Hordeum vulgare (barley). In this study, I aimed to characterize these candidates in more detail. I successfully cloned coding sequences for the majority of candidate genes with primers designed based on the barley reference genome sequence. During the course of this study a newer genome sequence with improved annotations was published, to which I also compared my observations. To study the candidate genes, I used the heterologous expression system Saccharomyces cerevisiae (yeast). I used several salt sensitive yeast strains (deficient in intrinsic sodium transporters) to test whether the candidate genes would affect their salt tolerance by mediating the sequestration of sodium into the yeast vacuole. I observed a reduction in growth upon expression for several of the gene candidate under salt-stress conditions. However, confocal microscopy suggests that most gene products are subject to degradation, and did not localize to the vacuolar membrane (tonoplast). Therefore, growth effects cannot be linked to protein function without further evidence. Various potential causes are discussed, including inaccuracies in the genome resource used as reference for primer design and issues inherent to the model system. Finally, I make suggestions on how to proceed to further characterize the candidate genes and hopefully identify novel sodium transporters from barley.

  17. Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

    Science.gov (United States)

    Mitchell, Gabriel

    2017-01-01

    Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

  18. Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mounia Kortebi

    2017-11-01

    Full Text Available Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs. Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC, preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

  19. Frameshift mutations of vacuolar protein sorting genes in gastric and colorectal cancers with microsatellite instability.

    Science.gov (United States)

    An, Chang Hyeok; Kim, Yoo Ri; Kim, Ho Shik; Kim, Sung Soo; Yoo, Nam Jin; Lee, Sug Hyung

    2012-01-01

    Vacuolar protein sorting plays crucial roles in the traffic of molecules between cellular organelles. Although involvement of vacuolar protein sorting proteins in cancer is known, genetic alterations of VPS genes have not been reported in cancers. We found that VPS4B, VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS37D, VPS41, and VPS54 have mononucleotide repeats in their coding sequences. To see whether these genes are mutated in cancers with microsatellite instability, we analyzed the mononucleotide repeats in 30 gastric cancers with high microsatellite instability, 13 gastric cancers with low microsatellite instability, and 45 gastric cancers with stable microsatellites and 40 colorectal cancers with high microsatellite instability, 14 colorectal cancers with low microsatellite instability, and 45 colorectal cancers with stable microsatellites by single-strand conformation polymorphism. We found mutations of VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS41, and VPS54 in 9, 3, 12, 3, 5, 9, 2, and 2 cancers, respectively, all in cancers with high microsatellite instability. The gastric cancers and colorectal cancers with high microsatellite instability harbored one or more mutations of the VPS genes in 53.3% and 50.0%, respectively. Loss of Vps13A expression was observed in 30% of the gastric cancers and 35% of the colorectal cancers, whereas loss of Vps35 was observed in 55% of the gastric cancers and 55% of the colorectal cancers. Our data indicate that frameshift mutations of VPS genes and losses of expression of Vps13A and Vps35 proteins are common in gastric cancers and colorectal cancers with high microsatellite instability and suggest that these alterations might contribute to development of cancers with high microsatellite instability by deregulating vacuolar protein sorting proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. ENV7 and YCK3, which encode vacuolar membrane protein kinases, genetically interact to impact cell fitness and vacuole morphology.

    Science.gov (United States)

    Manandhar, Surya P; Gharakhanian, Editte

    2014-05-01

    Saccharomyces cerevisiae vacuoles serve as a model for membrane fusion and fission. Yck3, a vacuolar membrane kinase, has been implicated in regulation of vacuole fusion. Recently, we established Env7 as another vacuolar membrane protein kinase with similar but nonredundant function to Yck3. Here, we report that native Env7 localizes to the vacuole independent of Yck3, where as its phosphorylation is YCK3 dependent. We also show that env7Δyck3Δ double mutant exhibits severely compromised fitness, altered cell size and bud vacuoles, and F-class vacuolar morphology. Our results establish negative genetic interactions between ENV7 and YCK3 and suggest cooperative roles for the two conserved genes in regulation of membrane dynamics. Such genetic buffering supports a critical role for membrane flux in global cell fitness. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    Science.gov (United States)

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance.

  2. A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

    Science.gov (United States)

    Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

    2015-10-23

    Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. TAI vacuolar invertase orthologs: the interspecific variability in tomato plants (Solanum section Lycopersicon).

    Science.gov (United States)

    Slugina, M A; Shchennikova, A V; Kochieva, E Z

    2017-06-20

    Understanding the genetic mechanisms underlying carbohydrate metabolism can promote the development of biotechnological advances in fruit plants. The flesh tomato fruit represents an ideal system for examining the role of sucrose cleavage enzymes in fruit development, and wild tomato species differing in storage sugars serve as an excellent research material for this purpose. Plant vacuolar invertase is a key enzyme of sucrose metabolism in the sink organs. In the present study, we identified complete gene sequences encoding the TAI vacuolar invertase in 11 wild and one cultivated tomato accessions of the Solanum section Lycopersicon. The average level of interspecific polymorphism in TAI genes was 8.58%; however, in the green-fruited tomatoes, the TAI genes contained 100 times more SNPs than those in the red-fruited accessions. The TAI proteins demonstrated 8% variability, whereas the red-fruited species had none. A TAI-based phylogenetic tree revealed two main clusters containing self-compatible and self-incompatible species, which concurs with the previous crossability-based division and demonstrates that the TAI genes reflect the evolutionary relationships between the red- and green-fruited tomatoes. Furthermore, we detected differential expression patterns of the TAI genes in the fruits of wild and cultivated tomatoes, which corresponded to sugar composition. The polymorphism analysis of the TAI acid invertases of Solanum section Lycopersicon species will contribute to the understanding of the genetic potential of TAI genes to impact tomato breeding through genetic engineering of the carbohydrate composition in the fruit.

  4. Saccharomyces cerevisiae vacuolar H+-ATPase regulation by disassembly and reassembly: one structure and multiple signals.

    Science.gov (United States)

    Parra, Karlett J; Chan, Chun-Yuan; Chen, Jun

    2014-06-01

    Vacuolar H(+)-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

    Science.gov (United States)

    Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

    1999-07-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

  6. A Tomato Vacuolar Invertase Inhibitor Mediates Sucrose Metabolism and Influences Fruit Ripening1

    Science.gov (United States)

    Zhu, Zhu; Wang, Weihao; Cai, Jianghua; Chen, Yong; Tian, Shiping

    2016-01-01

    Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value. PMID:27694342

  7. A Tomato Vacuolar Invertase Inhibitor Mediates Sucrose Metabolism and Influences Fruit Ripening.

    Science.gov (United States)

    Qin, Guozheng; Zhu, Zhu; Wang, Weihao; Cai, Jianghua; Chen, Yong; Li, Li; Tian, Shiping

    2016-11-01

    Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

    1991-01-01

    and intracellular sorting, and the stabilities in vivo and in vitro were studied. It was found that carbohydrate was not important for accurate vacuolar targeting of CPY, but that the rate of transport of the unglycosylated CPY through the secretory pathway to the vacuole was reduced. Tunicamycin, which inhibits...

  9. Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH

    Science.gov (United States)

    Cation exchangers CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3...

  10. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

  11. Crystallization and preliminary crystallographic characterization of GumK, a membrane-associated glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Barreras, Máximo [Fundación Instituto Leloir, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1405BWE Buenos Aires (Argentina); Bianchet, Mario A. [Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland 21205 (United States); Ielpi, Luis, E-mail: lielpi@leloir.org.ar [Fundación Instituto Leloir, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1405BWE Buenos Aires (Argentina)

    2006-09-01

    Crystallization of a membrane-associated glucuronosyltransferase. GumK is a membrane-associated inverting glucuronosyltransferase that is part of the biosynthetic route of xanthan, an industrially important exopolysaccharide produced by Xanthomonas campestris. The enzyme catalyzes the fourth glycosylation step in the pentasaccharide-P-P-polyisoprenyl assembly, an oligosaccharide diphosphate lipid intermediate in xanthan biosynthesis. GumK has marginal homology to other glycosyltransferases (GTs). It belongs to the CAZy family GT 70, for which no structure is currently available, and indirect biochemical evidence suggests that it also belongs to the GT-B structural superfamily. Crystals of recombinant GumK from X. campestris have been grown that diffract to 1.9 Å resolution. Knowledge of the crystal structure of GumK will help in understanding xanthan biosynthesis and its regulation and will also allow a subsequent rational approach to enzyme design and engineering. The multiwavelength anomalous diffraction approach will be used to solve the phase problem.

  12. Listeria monocytogenes infection in macrophages induces vacuolar-dependent host miRNA response.

    Science.gov (United States)

    Schnitger, Anna K D; Machova, Alzbeta; Mueller, Roman Ulrich; Androulidaki, Ariadne; Schermer, Bernhard; Pasparakis, Manolis; Krönke, Martin; Papadopoulou, Nikoletta

    2011-01-01

    Listeria monocytogenes is a gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels.

  13. Listeria monocytogenes infection in macrophages induces vacuolar-dependent host miRNA response.

    Directory of Open Access Journals (Sweden)

    Anna K D Schnitger

    Full Text Available Listeria monocytogenes is a gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post

  14. Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

    Science.gov (United States)

    Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

    2014-05-15

    Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

  15. Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus bath: evidence for a Diiron center.

    Science.gov (United States)

    Martinho, Marlène; Choi, Dong W; Dispirito, Alan A; Antholine, William E; Semrau, Jeremy D; Münck, Eckard

    2007-12-26

    Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the well-characterized soluble MMO contains a bis-mu-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the alpha subunit (pmoB) of the alphabetagamma trimer and a site at the interface of the betagamma subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1-2 iron atoms per alphabetagamma show the highest catalytic activity. We have employed Mössbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per alphabetagamma of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the "Zn site" that accommodates the diiron center in active pMMO.

  16. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex.

    Science.gov (United States)

    Hollopeter, Gunther; Lange, Jeffrey J; Zhang, Ying; Vu, Thien N; Gu, Mingyu; Ailion, Michael; Lambie, Eric J; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Jorgensen, Erik M

    2014-10-10

    The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

  17. The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

    Science.gov (United States)

    Bauer, Georg; Motz, Manfred

    2016-11-01

    Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  18. Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.

    Directory of Open Access Journals (Sweden)

    Gerrit Vortmeier

    Full Text Available The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.

  19. Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

    Science.gov (United States)

    Els-Heindl, Sylvia; Chollet, Constance; Scheidt, Holger A.; Beck-Sickinger, Annette G.; Meiler, Jens; Huster, Daniel

    2015-01-01

    The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide’s secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide’s positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8–17 form an α-helix, while residues 21–23 and 26–27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane. PMID:25803439

  20. Characterization of a teleost membrane-associated protein that is involved in the regulation of complement activation and bacterial infection.

    Science.gov (United States)

    Li, Mo-Fei; Sun, Li

    2018-02-01

    In mammals, membrane-associated complement regulatory proteins (MCRP) can protect host cells from the damaging of the activated complement. In teleost, few studies on the function of MCRP have been documented. In the present report, we identified a MCRP (named CsMCRP) from the teleost fish tongue sole Cynoglossus semilaevis and examined its immune function. CsMCRP shares moderate sequence identities with fish DAF-like molecules. CsMCRP was predicted to be a transmembrane protein with three short consensus repeats located in the extracellular region. CsMCRP expression occurred in nine different tissues, especially blood, and in peripheral blood leukocytes (PBL). Recombinant CsMCRP inhibited complement activation and interacted with bacterial pathogen, the latter in a highly selective manner. Antibody blocking the CsMCRP on PBL significantly inhibited bacterial infection of PBL. These results indicate that teleost CsMCRP is both a regulator of complement activation and a cellular receptor involved in bacterial invasion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

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    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

  2. Understanding the inhibitory effect of highly potent and selective archazolides binding to the vacuolar ATPase.

    Science.gov (United States)

    Dreisigacker, Sandra; Latek, Dorota; Bockelmann, Svenja; Huss, Markus; Wieczorek, Helmut; Filipek, Slawomir; Gohlke, Holger; Menche, Dirk; Carlomagno, Teresa

    2012-08-27

    Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the subnanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular docking, molecular dynamics simulations, and biochemical experiments, resulting in a sound model for the inhibitory mechanism of this class of putative anticancer agents. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded V(O)-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.

  3. Vacuolar SPX-MFS transporters are essential for phosphate adaptation in plants.

    Science.gov (United States)

    Liu, Jinlong; Fu, Shaomin; Yang, Lei; Luan, Mingda; Zhao, Fugeng; Luan, Sheng; Lan, Wenzhi

    2016-08-02

    To survive in most soils in which inorganic phosphate (Pi) levels are limited and constantly changing, plants universally use the vacuoles as cellular Pi "sink" and "source" to maintain Pi homeostasis. However, the transporters that mediate Pi sequestration into the vacuoles remain unknown. Recently, we and other 2 groups independently identified the members of SPS-MSF family as the candidates for tonoplast Pi transporters in Arabidopsis thaliana and Oryza sativa. We and Liu et al. demonstrated that one of SPS-MSF member, VPT1 (Vacuolar Phosphate Transporter 1), also named as PHT5;1 (Phosphate Transporter 5;1), plays a predominant role in Pi sequestration of vacuoles in Arabidopsis. Here we show that vpt1 mutants and VPT1-GFP overexpressing lines displayed sensitive to Pi stress under the hydroponic system containing the medium with low iron, supporting that VPT1 is essential for Arabidopsis to adapt phosphate stress.

  4. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    2010-02-01

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  5. Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates.

    Science.gov (United States)

    Bartholomew, Dolores M; Van Dyk, Drew E; Lau, Sze-Mei Cindy; O'Keefe, Daniel P; Rea, Philip A; Viitanen, Paul V

    2002-11-01

    Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H(+) gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H(+) gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H(+) gradient dependence of glucoside uptake. It is concluded that H(+) gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H(+) antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and

  6. Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus.

    Science.gov (United States)

    Han, Yong-Liang; Song, Hai-Xing; Liao, Qiong; Yu, Yin; Jian, Shao-Fen; Lepo, Joe Eugene; Liu, Qiang; Rong, Xiang-Min; Tian, Chang; Zeng, Jing; Guan, Chun-Yun; Ismail, Abdelbagi M; Zhang, Zhen-Hua

    2016-03-01

    Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8. © 2016 American Society of Plant Biologists. All Rights Reserved.

  7. Molecular cloning and characterization of a vacuolar H+₋pyrophosphatase from Dunaliella viridis.

    Science.gov (United States)

    Meng, Xiangzong; Xu, Zhengkai; Song, Rentao

    2011-06-01

    The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na(+). In plants, sequestration of Na(+) into the vacuole is driven by the electrochemical H(+) gradient generated by H(+) pumps, and this Na(+) sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H(+) pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V-H(+)-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V-H(+)-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V-H(+)-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na(+) hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V-H(+)-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V-H(+)-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V-H(+)-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.

  8. abc3+ encodes an iron-regulated vacuolar ABC-type transporter in Schizosaccharomyces pombe.

    Science.gov (United States)

    Pouliot, Benoît; Jbel, Mehdi; Mercier, Alexandre; Labbé, Simon

    2010-01-01

    Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in Saccharomyces cerevisiae, less information is available concerning the mobilization of vacuolar iron stores in Schizosaccharomyces pombe. In this study, we report the identification of a gene designated abc3(+) that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of abc3(+) is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of abc3(+) required a functional fep1(+) gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the abc3(+) promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1. abc3Delta cells exhibited increased levels of expression of the frp1(+)-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When abc3(+) was expressed using the nmt1(+) promoter system, its induction led to a reduced transcriptional activity of the frp1(+) gene. Because S. pombe does not possess vacuolar membrane-localized orthologs to S. cerevisiae Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency.

  9. abc3+ Encodes an Iron-Regulated Vacuolar ABC-Type Transporter in Schizosaccharomyces pombe▿

    Science.gov (United States)

    Pouliot, Benoît; Jbel, Mehdi; Mercier, Alexandre; Labbé, Simon

    2010-01-01

    Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in Saccharomyces cerevisiae, less information is available concerning the mobilization of vacuolar iron stores in Schizosaccharomyces pombe. In this study, we report the identification of a gene designated abc3+ that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of abc3+ is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of abc3+ required a functional fep1+ gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the abc3+ promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1. abc3Δ cells exhibited increased levels of expression of the frp1+-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When abc3+ was expressed using the nmt1+ promoter system, its induction led to a reduced transcriptional activity of the frp1+ gene. Because S. pombe does not possess vacuolar membrane-localized orthologs to S. cerevisiae Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency. PMID:19915076

  10. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

    1999-01-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  11. The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein.

    Science.gov (United States)

    Basu, Piku; Katterle, Bettina; Andersson, K Kristoffer; Dalton, Howard

    2003-01-15

    A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

  12. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gloria; Spring, Frances A.; Parons, Stephen F.; Mankelow, Tosti J.; Peters, Luanne L.; Koury, Mark J.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2003-02-18

    ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68 percent overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.

  13. Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain.

    Science.gov (United States)

    Hansen, Majbritt; Le Nours, Jérôme; Johansson, Eva; Antal, Torben; Ullrich, Alexandra; Löffler, Monika; Larsen, Sine

    2004-04-01

    The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two alpha-helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N-terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 A movement of the first residue in the first alpha-helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane-bound rat DHOD and membrane-associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.

  14. Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

    Science.gov (United States)

    Autieri, M V; Fresa, K L; Coffman, F D; Katz, M E; Cohen, S

    1990-12-01

    We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

  15. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    Directory of Open Access Journals (Sweden)

    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  16. Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

    Science.gov (United States)

    Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

    2014-11-03

    Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. The tetraspanin TSP3 of Neurospora crassa is a vacuolar membrane protein and shares characteristics with IDI proteins.

    Science.gov (United States)

    Heine, Daniela; Petereit, Linda; Schumann, Marcel R; Patzelt, Diana; Rachid, Leila; Brandt, Ulrike; Werner, Antonia; Pöggeler, Stefanie; Fleißner, André

    2016-01-01

    The fungal vacuole is an organelle, which adopts pleiotropic morphologies and functions. In aging and starving hyphae it is the compartment of degradation and recycling of cellular constituents. Here we identified TSP3, one of three tetraspanins present in the filamentous ascomycete fungus Neurospora crassa, as a vacuolar membrane protein. The protein is detected only in aging and starving cultures and under other conditions, which induce autophagy, such as vegetative incompatibility or the presence of the macrolide antibiotic rapamycin. Mutant analysis revealed that TSP3 is dispensable for growth and development of the fungus under laboratory conditions. Together these findings indicate that tsp3 shares characteristics with idi (induced during incompatibility) genes and might promote vacuolar functions related to autophagy. © 2016 by The Mycological Society of America.

  18. Mutational analysis of the vacuolar sorting signal of procarboxypeptidase Y in yeast shows a low requirement for sequence conservation

    DEFF Research Database (Denmark)

    van Voorst, F; Kielland-Brandt, Morten; Winther, Jakob R.

    1996-01-01

    The core of the vacuolar targeting signal of yeast carboxypeptidase Y (CPY) is recognized by the receptor Vps10p and consists of four contiguous amino acid residues, Gln24-Arg-Pro-Leu27, near the amino terminus of the propeptide (Valls, L.A., Winther, J. R., and Stevens, T. H. (1990) J. Cell Biol....... 111, 361-368; Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E., and Emr, S. D. (1994) Cell 77, 579-586). In order to determine the sequence specificity of the interaction with the sorting receptor, substitutions were introduced into this part of the propeptide by semirandom...... site-directed mutagenesis. The efficiency of vacuolar sorting by the mutants was determined by immunoprecipitation of CPY from pulse-labeled cells. It was found that amino acid residues Gln24 and Leu27 were the most important ones. While it appears that Gln24 is essential for proper function, Leu27 can...

  19. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds.

    Science.gov (United States)

    Appelhagen, Ingo; Nordholt, Niclas; Seidel, Thorsten; Spelt, Kees; Koes, Ronald; Quattrochio, Francesca; Sagasser, Martin; Weisshaar, Bernd

    2015-06-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+) -pyrophosphatases (H(+) -PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA-filled central vacuoles as observed in the wild-type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P-type H(+) -ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H(+) -PPase VHP1. Our findings indicate that the P3A -ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12-mediated transport of PA precursors to the vacuole. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  20. Heme Assimilation in Schizosaccharomyces pombe Requires Cell-surface-anchored Protein Shu1 and Vacuolar Transporter Abc3.

    Science.gov (United States)

    Mourer, Thierry; Normant, Vincent; Labbé, Simon

    2017-03-24

    The Schizosaccharomyces pombe shu1+ gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2.

    Science.gov (United States)

    Schulz, Alexander; Beyhl, Diana; Marten, Irene; Wormit, Alexandra; Neuhaus, Ekkehard; Poschet, Gernot; Büttner, Michael; Schneider, Sabine; Sauer, Norbert; Hedrich, Rainer

    2011-10-01

    The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  2. The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

    Science.gov (United States)

    De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

    2013-08-01

    Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

  3. A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis.

    Science.gov (United States)

    Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

    2016-01-01

    A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

  4. Transmissibility studies of vacuolar changes in the rostral colliculus of pigs

    Directory of Open Access Journals (Sweden)

    Spencer Yvonne I

    2009-09-01

    Full Text Available Abstract Background Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE. Feedstuff containing BSE-contaminated meat and bone meal (MBM may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. Results None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. Conclusion The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant

  5. Contribution of chitinase A's C-terminal vacuolar sorting determinant to the study of soluble protein compartmentation.

    Science.gov (United States)

    Stigliano, Egidio; Di Sansebastiano, Gian-Pietro; Neuhaus, Jean-Marc

    2014-06-18

    Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin's tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  6. Contribution of Chitinase A’s C-Terminal Vacuolar Sorting Determinant to the Study of Soluble Protein Compartmentation

    Directory of Open Access Journals (Sweden)

    Egidio Stigliano

    2014-06-01

    Full Text Available Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY (AlaPheValTyr and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  7. Differential changes in the activity of cytosolic and vacuolar trehalases along the growth cycle of Saccharomyces cerevisiae.

    Science.gov (United States)

    San Miguel, P F; Argüelles, J C

    1994-07-06

    Saccharomyces cerevisiae cells contain two intracellular and soluble trehalases with distinct subcellular location (cytosol and vacuoles, respectively). Both enzymes showed an opposite pattern of activity along the growth cycle. Activity of the cytosolic trehalase was high in cells growing exponentially on fermentable sugars (glucose, mannose or galactose) and sharply decayed as the cultures enter stationary phase coinciding with the beginning of trehalose biosynthesis. By contrast, vacuolar trehalase was only detectable in glucose-grown resting cells or in cultures growing on respiratory substrates (glycerol or ethanol). This enzyme was partially derepressed in the mutant hex2, which is deficient in glucose repression. Addition of fresh YPD medium to stationary-phase cultures induced the sudden reactivation of cytosolic trehalase with the concomitant slower inactivation of vacuolar trehalase. However, addition of glucose or various nitrogen sources alone had only a minor effect on both activities. The presence of cycloheximide had no effect on cytosolic trehalase, whereas completely blocked the appearance of vacuolar trehalase suggesting the requirement of protein synthesis 'de novo'.

  8. Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-07-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.

  9. Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport1[W

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-01-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3′,4′-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4+ and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H+ gradient preestablished across the tonoplast by either vacuolar H+-ATPase or vacuolar H+-pyrophosphatase. The initial rates of H+ gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H+ antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  10. FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates its Delivery to the Vacuolar Degradation Pathway.

    Science.gov (United States)

    Belda-Palazon, Borja; Rodriguez, Lesia; Fernandez, Maria A; Castillo, Mari-Cruz; Anderson, Erin A; Gao, Caiji; González-Guzmán, Miguel; Peirats-Llobet, Marta; Zhao, Qiong; De Winne, Nancy; Gevaert, Kris; De Jaeger, Geert; Jiang, Liwen; Leon, Jose; Mullen, Robert T; Rodriguez, Pedro L

    2016-08-05

    Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana. This suggested that ubiquitylated ABA receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knock-down fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA. Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling. © 2016 American Society of Plant Biologists. All rights reserved.

  11. Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

    Science.gov (United States)

    Bauer, Georg; Zarkovic, Neven

    2015-04-01

    Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases.

    Science.gov (United States)

    Greening, Chris; Berney, Michael; Hards, Kiel; Cook, Gregory M; Conrad, Ralf

    2014-03-18

    In the Earth's lower atmosphere, H2 is maintained at trace concentrations (0.53 ppmv/0.40 nM) and rapidly turned over (lifetime ≤ 2.1 y(-1)). It is thought that soil microbes, likely actinomycetes, serve as the main global sink for tropospheric H2. However, no study has ever unambiguously proven that a hydrogenase can oxidize this trace gas. In this work, we demonstrate, by using genetic dissection and sensitive GC measurements, that the soil actinomycete Mycobacterium smegmatis mc(2)155 constitutively oxidizes subtropospheric concentrations of H2. We show that two membrane-associated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H2 at a range of concentrations [Vmax(app) = 12 nmol⋅g⋅dw(-1)⋅min(-1); Km(app) = 180 nM; threshold = 130 pM in the Δhyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [Vmax(app) = 2.5 nmol⋅g⋅dw(-1)⋅min(-1); Km(app) = 50 nM; threshold = 50 pM in the Δhyd13 (Hyd2 only) strain]. These observations strongly support previous studies that have linked group 5 [NiFe] hydrogenases (e.g., Hyd2) to the oxidation of tropospheric H2 in soil ecosystems. We further reveal that group 2a [NiFe] hydrogenases (e.g., Hyd1) can contribute to this process. Hydrogenase expression and activity increases in carbon-limited cells, suggesting that scavenging of trace H2 helps to sustain dormancy. Distinct physiological roles for Hyd1 and Hyd2 during the adaptation to this condition are proposed. Soil organisms harboring high-affinity hydrogenases may be especially competitive, given that they harness a highly dependable fuel source in otherwise unstable environments.

  13. Promoter regions of potato vacuolar invertase gene in response to sugars and hormones.

    Science.gov (United States)

    Ou, Yongbin; Song, Botao; Liu, Xun; Xie, Conghua; Li, Meng; Lin, Yuan; Zhang, Huiling; Liu, Jun

    2013-08-01

    Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from -118 to -551, -551 to -1021, and -1021 to -1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  14. Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues.

    Science.gov (United States)

    Chen, Tai-Hung; Huang, Yu-Chiao; Yang, Chii-Shen; Yang, Chien-Chih; Wang, Ai-Yu; Sung, Hsien-Yi

    2009-01-01

    Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobetafruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K(m) values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k(cat) values than the wild-type enzyme, but an increase in the k(cat) value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.

  15. Clinicopathologic correlations of HIV-1-associated vacuolar myelopathy: an autopsy-based case-control study.

    Science.gov (United States)

    Dal Pan, G J; Glass, J D; McArthur, J C

    1994-11-01

    To determine the clinical correlates of HIV-1-associated vacuolar myelopathy (VM), we designed a case-control study based on 215 AIDS autopsies in which we examined the spinal cord. We defined a case as an individual dying with AIDS and with VM present at autopsy; we defined a control as an individual dying with AIDS without VM. VM was found in 100 of 215 (46.5%) autopsies, with no apparent temporal trends. A higher number of AIDS-defining illnesses was strongly associated with the likelihood of VM (trend chi-square = 26.52, p cases than in controls (odds ratio = 3.68, 95% CI = 1.73 to 7.47, p cases than in controls (odds ratio = 5.00, 95% CI = 1.35 to 18.5, p cases with VM had detailed neurologic evaluations, but only 15 (26.8%) had signs and symptoms of myelopathy. The presence of symptomatic myelopathy was related to the pathologic severity: none of 17 cases with grade 1, five of 26 with grade 2, and 10 of 13 with grade 3 had clinical features of myelopathy (trend chi-square = 21.16, p < 0.005). VM is a common neuropathologic finding that is frequently unrecognized during life. The association with the number of systemic illnesses, M avium-intracellulare infection, and P carinii pneumonia suggests that the development of VM is related to the severity of immunosuppression.

  16. Formation of vacuolar tannin deposits in the chlorophyllous organs of Tracheophyta: from shuttles to accretions.

    Science.gov (United States)

    Brillouet, Jean-Marc; Romieu, Charles; Lartaud, Marc; Jublanc, Elodie; Torregrosa, Laurent; Cazevieille, Chantal

    2014-11-01

    Most Tracheophyta synthesize-condensed tannins (also called proanthocyanidins), polymers of catechins, which appear in the vacuole as uniformly stained deposits-termed tannin accretions-lining the inner face of the tonoplast. A large body of evidence argues that tannins are formed in recently described thylakoid-derived organelles, the tannosomes, which are packed in membrane-bound shuttles (Brillouet et al. 2013); it has been suggested that shuttles agglomerate into tannin accretions. The aim of the study was to describe the ontogenesis of tannin accretions in members of the Tracheophyta. For this purpose, fresh specimens of young tissues from diverse Tracheophyta were cut, gently lacerated in paraformaldehyde, and examined using light, epifluorescence, confocal, and transmission electron microscopy. Fresh samples were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Our observations showed that vacuolar accretions (1 → 40 μm), that constitute the typical form of tannin storage in tannin-producing Tracheophyta, are formed by agglomeration (not fusion) of shuttles containing various proportions of chlorophylls and tannins.

  17. Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato.

    Science.gov (United States)

    Bhaskar, Pudota B; Wu, Lei; Busse, James S; Whitty, Brett R; Hamernik, Andy J; Jansky, Shelley H; Buell, C Robin; Bethke, Paul C; Jiang, Jiming

    2010-10-01

    Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

  18. Suppression of the vacuolar invertase gene delays senescent sweetening in chipping potatoes.

    Science.gov (United States)

    Wiberley-Bradford, Amy E; Bethke, Paul C

    2018-01-01

    Potato chip processors require potato tubers that meet quality specifications for fried chip color, and color depends largely upon tuber sugar contents. At later times in storage, potatoes accumulate sucrose, glucose, and fructose. This developmental process, senescent sweetening, manifests as a blush of color near the center of the fried chip, becomes more severe with time, and limits the storage period. Vacuolar invertase (VInv) converts sucrose to glucose and fructose and is hypothesized to play a role in senescent sweetening. To test this hypothesis, senescent sweetening was quantified in multiple lines of potato with reduced VInv expression. Chip darkening from senescent sweetening was delayed by about 4 weeks for tubers with reduced VInv expression. A strong positive correlation between frequency of dark chips and tuber hexose content was observed. Tubers with reduced VInv expression had lower hexose to sucrose ratios than controls. VInv activity contributes to reducing sugar accumulation during senescent sweetening. Sucrose breakdown during frying may contribute to chip darkening. Suppressing VInv expression increases the storage period of the chipping potato crop, which is an important consideration, as potatoes with reduced VInv expression are entering commercial production in the USA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  19. SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

    Science.gov (United States)

    Baster, Paweł; Robert, Stéphanie; Kleine-Vehn, Jürgen; Vanneste, Steffen; Kania, Urszula; Grunewald, Wim; De Rybel, Bert; Beeckman, Tom; Friml, Jiří

    2013-01-01

    The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-boxTIR1/AFB (SCFTIR1/AFB)-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth. PMID:23211744

  20. Vacuolar H+-ATPase: An Essential Multitasking Enzyme in Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    L. Shannon Holliday

    2014-01-01

    Full Text Available Vacuolar H+-ATPases (V-ATPases are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (prorenin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.

  1. Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy

    Directory of Open Access Journals (Sweden)

    Fais Stefano

    2010-05-01

    Full Text Available Abstract The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

  2. Candida albicans SH3-domain proteins involved in hyphal growth, cytokinesis, and vacuolar morphology.

    Science.gov (United States)

    Reijnst, Patrick; Jorde, Sigyn; Wendland, Jürgen

    2010-08-01

    This report describes the analyses of three Candida albicans genes that encode Src Homology 3 (SH3)-domain proteins. Homologs in Saccharomyces cerevisiae are encoded by the SLA1, NBP2, and CYK3 genes. Deletion of CYK3 in C. albicans was not feasible, suggesting it is essential. Promoter shutdown experiments of CaCYK3 revealed cytokinesis defects, which are in line with the localization of GFP-tagged Cyk3 at septal sites. Deletion of SLA1 resulted in strains with decreased ability to form hyphal filaments. The number of cortical actin patches was strongly reduced in Deltasla1 strains during all growth stages. Sla1-GFP localizes in patches that are found concentrated at the hyphal tip. Deletion of the first two SH3-domains of Sla1 still resulted in cortical localization of the truncated protein. However, the actin cytoskeleton in this strain was aberrant like in the Deltasla1 deletion mutant indicating a function of these SH3 domains to recruit actin nucleation to sites of endocytosis. Deletion of NBP2 resulted in a defect in vacuolar fusion in hyphae. Germ cells of Deltanbp2 strains lacked a large vacuole but initiated several germ tubes. The mutant phenotypes of Deltanbp2 and Deltasla1 could be corrected by reintegration of the wild-type genes.

  3. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  4. Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Rubinstein, John L

    2016-07-01

    Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases.

  5. Efficient ammonium uptake and mobilization of vacuolar arginine by Saccharomyces cerevisiae wine strains during wine fermentation.

    Science.gov (United States)

    Crépin, Lucie; Sanchez, Isabelle; Nidelet, Thibault; Dequin, Sylvie; Camarasa, Carole

    2014-08-19

    Under N-limiting conditions, Saccharomyces cerevisiae strains display a substantial variability in their biomass yield from consumed nitrogen -in particular wine yeasts exhibit high growth abilities- that is correlated with their capacity to complete alcoholic fermentation, a trait of interest for fermented beverages industries. The aim of the present work was to assess the contribution of nitrogen availability to the strain-specific differences in the ability to efficiently use N-resource for growth and to identify the underlying mechanisms. We compared the profiles of assimilation of several nitrogen sources (mostly ammonium, glutamine, and arginine) for high and low biomass-producing strains in various conditions of nitrogen availability. We also analyzed the intracellular fate of nitrogen compounds. Strains clustered into two groups at initial nitrogen concentrations between 85 and 385 mg N.L(-1): high biomass producers that included wine strains, were able to complete fermentation of 240 g.L(-1) glucose and quickly consume nitrogen, in contrast to low biomass producers. The two classes of strains exhibited distinctive characteristics that contributed to their differential capacity to produce biomass. The contribution of each characteristic varied according to nitrogen availability. In high biomass producers, the high rate of ammonium uptake resulted in an important consumption of this preferred nitrogen source that promoted the growth of these yeasts when nitrogen was provided in excess. Both classes of yeast accumulated poor nitrogen sources, mostly arginine, in vacuoles during the first stages of growth. However, at end of the growth phase when nitrogen had become limiting, high biomass producers more efficiently used this vacuolar nitrogen fraction for protein synthesis and further biomass formation than low biomass producers. Overall, we demonstrate that the efficient management of the nitrogen resource, including efficient ammonium uptake and efficient

  6. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

    2010-01-01

    Full Text Available The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  7. The vacuolar protein sorting genes in insects: A comparative genome view.

    Science.gov (United States)

    Li, Zhaofei; Blissard, Gary

    2015-07-01

    In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Identification of a new target of miR-16, Vacuolar Protein Sorting 4a.

    Directory of Open Access Journals (Sweden)

    Neeta Adhikari

    Full Text Available The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs to discover pathways in heart failure (HF.The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD.BEDTools v2.14.3 was used to discriminate SNPs within predicted 3'UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001. MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a expression in HEK 293T cells (p<0.01. The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3'UTR of VPS4a (p<0.05. VPS4a was increased in the circulation of end-stage HF patients (p<0.001, and led to a decrease in the number of HEK 293T cells in vitro (p<0.001.We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.

  9. Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression

    Science.gov (United States)

    Ibrahim, Safaa A.; Katara, Gajendra K.; Kulshrestha, Arpita; Jaiswal, Mukesh K.; Amin, Magdy A.; Beaman, Kenneth D.

    2015-01-01

    In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy. PMID:26460736

  10. Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H+-ATPase Inhibitors

    Directory of Open Access Journals (Sweden)

    Renukadevi Patil

    2017-09-01

    Full Text Available The vacuolar (H+-ATPases (V-ATPases are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V as an initial hit from a similarity search using four known V-ATPase inhibitors (I–IV. Based on the initial hit (V, we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a–e. All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7 and ovarian cancer (A2780, Cis-A2780, and PA-1 cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM. Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

  11. Dissociation of the vacuolar and macroautophagic cytopathology from the cytotoxicity induced by the lipophilic local anesthetic bupivacaine.

    Science.gov (United States)

    Morissette, Guillaume; Bawolak, Marie-Thérèse; Marceau, François

    2011-07-01

    Local anesthetics, like many other cationic drugs, induce a vacuolar and macroautophagic cytopathology that has been observed in vivo and in various cell types; some also induce cytotoxicity of mitochondrial origin (apoptosis and necrosis) and it is not known whether the 2 types of toxicity overlap or interact. We compared bupivacaine with a more hydrophilic agent, lidocaine, for morphological, functional, and toxicological responses in a previously exploited nonneuronal system, primary smooth muscle cells. Bupivacaine induced little vacuolization (≥2.5 mmol/L, 4 h), but elicited autophagic accumulation (≥0.5 mmol/L, 4 h) and was massively cytotoxic at 2.5-5 mmol/L (4-24 h), the latter effect being unabated by the V-ATPase inhibitor bafilomycin A1. Lidocaine exerted little cytotoxicity at and below 5 mmol/L for 24 h, but intensely induced the V-ATPase-dependent vacuolar and autophagic cytopathology. Bupivacaine was more potent than lidocaine in disrupting mitochondrial potential, as judged by Mitotracker staining (significant proportions of cells affected in the 1-5 and 5-10 mmol/L concentration ranges, respectively). The addition of mitochondrial-inactivating toxins antimycin A and oligomycin to lidocaine (2.5 mmol/L) reproduced the profile of bupivacaine action (low intensity of vacuolization and retained autophagic accumulation). The high potency of bupivacaine as a mitochondrial toxicant eclipses the benign vacuolar and autophagic response seen with more hydrophilic local anesthetics.

  12. Vacuolar Na+/H+ NHX-Type Antiporters Are Required for Cellular K+ Homeostasis, Microtubule Organization and Directional Root Growth

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    Tyler McCubbin

    2014-08-01

    Full Text Available Na+/H+ antiporters (NHXs are integral membrane transporters that catalyze the electroneutral exchange of K+ or Na+ for H+ and are implicated in cell expansion, development, pH and ion homeostasis and salt tolerance. Arabidopsis contains four vacuolar NHX isoforms (NHX1–NHX4, but only the functional roles for NHX1 and NHX2 have been assessed thus far. Colocalization studies indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4, lacking all vacuolar NHXs, was generated. Seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K+, but not Na+, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton might be perturbed. Whole mount immunolabeling of cortical microtubules indicated that high K+ caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (Taxol and destabilizing (propyzamide drugs, we found that the effect of K+ on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of Taxol, whereas elevated K+ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K+ homeostasis leads to an increase in the dynamics of cortical microtubule reorganization in nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone.

  13. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Motawie, Mohammed Saddik; Olsen, Carl Erik

    2016-01-01

    for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated...... experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O...

  14. The acidocalcisome vacuolar transporter chaperone 4 catalyzes the synthesis of polyphosphate in insect-stages of Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Ulrich, Paul N; Lander, Noelia; Kurup, Samarchith P; Reiss, Laura; Brewer, Jessica; Soares Medeiros, Lia C; Miranda, Kildare; Docampo, Roberto

    2014-01-01

    Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  15. Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.

    Science.gov (United States)

    Pick, E; Bromberg, Y; Shpungin, S; Gadba, R

    1987-12-05

    Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113

  16. Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

    OpenAIRE

    Seaman, Matthew N.J.; Marcusson, Eric G.; Cereghino, Joan Lin; Emr, Scott D

    1997-01-01

    Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble va...

  17. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

    Science.gov (United States)

    Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

  18. Using biomass of starch-rich transgenic Arabidopsis vacuolar as feedstock for fermentative hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Yung-Chung; Cheng, Chieh-Lun; Chen, Chun-Yen [National Cheng Kung Univ., Tainan, Taiwan (China). Dept. of Chemical Engineering; Huang, Li-Fen; Chang, Jo-Shu [Yuan Ze Univ., Tao-yuan, Taiwan (China). Graduate School of Biotechnology and Bioengineering

    2010-07-01

    Cellulose is the major constitute of plant biomass and highly available in agricultural wastes and industrial effluents, thereby being a cost-effective feedstock for bioenergy production. However, most hydrogen producing bacteria (HPB) could not directly convert cellulosic materials (such as rice husk and rice straw) into hydrogen whereas most HPB could utilize sugar and starch for hydrogen production. In this work, we used an indigenous bacterial isolate Clostridium butyricum CGS2 as HPB, which could directly convert soluble starch into H2 with a maximum H2 production rate and a H2 yield of 205.07 ml H2/h/l and 6.46 mmol H2/g starch, respectively. However, C. butyricum CGS2 could not ferment pure cellulosic materials such as carboxymethyl cellulose and xylan. Moreover, we found that C. butyricum CGS2 could utilize rich husk to produce H2 at a rate of 13.19 ml H2/h/l due to the starch content in rice husk (H2 yield = 1.49 mmol H2/g rice husk). In contrast, since lacking starch content, rice straw cannot be converted to H2 by C. butyricum CGS2. The foregoing results suggest that increasing the starch content in the natural agricultural wastes may make them better feedstock for fermentative H2 production. Hence, a genetically modified plant (Arabidopsis vacuolar) was constructed to enhance its starch concentration. The starch concentration of mutant plant S1 increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using mutant plant S1 as carbon source, C. butyricum CGS2 was able to give a high cumulative H2 production and H2 production rate of 285.4 ml H2/l and 43.6 ml/h/l, respectively. The cumulative H2 production and H2 production rate both increased when the concentration of the transgenic plant was increased. Therefore, this study successful demonstrated the feasibility of expressing starch on genetically-modified plants to create a more effective feedstock for dark H2 fermentation. (orig.)

  19. Interplay of sugar, light and gibberellins in expression of Rosa hybrida vacuolar invertase 1 regulation.

    Science.gov (United States)

    Rabot, Amélie; Portemer, Virginie; Péron, Thomas; Mortreau, Eric; Leduc, Nathalie; Hamama, Latifa; Coutos-Thévenot, Pierre; Atanassova, Rossitza; Sakr, Soulaiman; Le Gourrierec, José

    2014-10-01

    Our previous findings showed that the expression of the Rosa hybrida vacuolar invertase 1 gene (RhVI1) was tightly correlated with the ability of buds to grow out and was under sugar, gibberellin and light control. Here, we aimed to provide an insight into the mechanistic basis of this regulation. In situ hybridization showed that RhVI1 expression was localized in epidermal cells of young leaves of bursting buds. We then isolated a 895 bp fragment of the promoter of RhVI1. In silico analysis identified putative cis-elements involved in the response to sugars, light and gibberellins on its proximal part (595 bp). To carry out functional analysis of the RhVI1 promoter in a homologous system, we developed a direct method for stable transformation of rose cells. 5' deletions of the proximal promoter fused to the uidA reporter gene were inserted into the rose cell genome to study the cell's response to exogenous and endogenous stimuli. Deletion analysis revealed that the 468 bp promoter fragment is sufficient to trigger reporter gene activity in response to light, sugars and gibberellins. This region confers sucrose- and fructose-, but not glucose-, responsive activation in the dark. Inversely, the -595 to -468 bp region that carries the sugar-repressive element (SRE) is required to down-regulate the RhVI1 promoter in response to sucrose and fructose in the dark. We also demonstrate that sugar/light and gibberellin/light act synergistically to up-regulate β-glucuronidase (GUS) activity sharply under the control of the 595 bp pRhVI1 region. These results reveal that the 127 bp promoter fragment located between -595 and -468 bp is critical for light and sugar and light and gibberellins to act synergistically. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

    Science.gov (United States)

    Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

    2016-01-01

    Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

  1. Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs.

    Directory of Open Access Journals (Sweden)

    Yujin Tang

    Full Text Available Vacuolar processing enzymes (VPEs have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD, which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177, Cys (219] and substrate binding pocket [Arg (112, Arg (389, Ser (395], except that Ser (395 in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF, close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

  2. Crystallization and Preliminary Crystallographic Characterization of GumK, A Membrane-Associated Gluocuronosyltransferase from Xanthomonas campestris Required for Xanthan Polysaccharide Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Barreras,M.; Bianchet, M.; Ielpi, L.; Tong, L.

    2006-01-01

    GumK is a membrane-associated inverting glucuronosyltransferase that is part of the biosynthetic route of xanthan, an industrially important exopolysaccharide produced by Xanthomonas campestris. The enzyme catalyzes the fourth glycosylation step in the pentasaccharide-P-P-polyisoprenyl assembly, an oligosaccharide diphosphate lipid intermediate in xanthan biosynthesis. GumK has marginal homology to other glycosyltransferases (GTs). It belongs to the CAZy family GT 70, for which no structure is currently available, and indirect biochemical evidence suggests that it also belongs to the GT-B structural superfamily. Crystals of recombinant GumK from X. campestris have been grown that diffract to 1.9 {angstrom} resolution. Knowledge of the crystal structure of GumK will help in understanding xanthan biosynthesis and its regulation and will also allow a subsequent rational approach to enzyme design and engineering. The multiwavelength anomalous diffraction approach will be used to solve the phase problem.

  3. Crystallization and preliminary crystallographic characterization of GumK, a membrane-associated glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis.

    Science.gov (United States)

    Barreras, Máximo; Bianchet, Mario A; Ielpi, Luis

    2006-09-01

    GumK is a membrane-associated inverting glucuronosyltransferase that is part of the biosynthetic route of xanthan, an industrially important exopolysaccharide produced by Xanthomonas campestris. The enzyme catalyzes the fourth glycosylation step in the pentasaccharide-P-P-polyisoprenyl assembly, an oligosaccharide diphosphate lipid intermediate in xanthan biosynthesis. GumK has marginal homology to other glycosyltransferases (GTs). It belongs to the CAZy family GT 70, for which no structure is currently available, and indirect biochemical evidence suggests that it also belongs to the GT-B structural superfamily. Crystals of recombinant GumK from X. campestris have been grown that diffract to 1.9 A resolution. Knowledge of the crystal structure of GumK will help in understanding xanthan biosynthesis and its regulation and will also allow a subsequent rational approach to enzyme design and engineering. The multiwavelength anomalous diffraction approach will be used to solve the phase problem.

  4. Ameliorative effect of membrane-associated estrogen receptor G protein coupled receptor 30 activation on object recognition memory in mouse models of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Takashi Kubota

    2016-07-01

    Full Text Available Membrane-associated estrogen receptor “G protein-coupled receptor 30” (GPR30 has been implicated in spatial recognition memory and protection against neuronal death. The present study investigated the role of GPR30 in object recognition memory in an Alzheimer's disease (AD mouse model (5XFAD by using novel object recognition (NOR test. Impairment of long-term (24 h recognition memory was observed in both male and female 5XFAD mice. Selective GPR30 agonist, G-1, ameliorated this impairment in female 5XFAD mice, but not in male mice. Our study demonstrated the ameliorative role of GPR30 in NOR memory impaired by AD pathology in female mice.

  5. Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553.

    Science.gov (United States)

    Albouy, D; Sturgis, J N; Feiler, U; Nitschke, W; Robert, B

    1997-02-18

    Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.

  6. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

    Science.gov (United States)

    Capell, Anja; Liebscher, Sabine; Fellerer, Katrin; Brouwers, Nathalie; Willem, Michael; Lammich, Sven; Gijselinck, Ilse; Bittner, Tobias; Carlson, Aaron M; Sasse, Florenz; Kunze, Brigitte; Steinmetz, Heinrich; Jansen, Rolf; Dormann, Dorothee; Sleegers, Kristel; Cruts, Marc; Herms, Jochen; Van Broeckhoven, Christine; Haass, Christian

    2011-02-02

    Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

  7. In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.

    Science.gov (United States)

    Hovasse, Agnès; Alayi, Tchilabalo Dilezitoko; Van Dorsselaer, Alain; Marchal, Richard; Jégou, Sandrine; Schaeffer-Reiss, Christine

    2016-06-01

    Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Starvation-Dependent Regulation of Golgi Quality Control Links the TOR Signaling and Vacuolar Protein Sorting Pathways

    Directory of Open Access Journals (Sweden)

    Niv Dobzinski

    2015-09-01

    Full Text Available Upon amino acid (AA starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.

  9. Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jørgensen, M U; Emr, S D; Winther, Jakob R.

    1999-01-01

    Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand...... binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p....... The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p...

  10. The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

    Science.gov (United States)

    Steele-Mortimer, Olivia

    2012-01-01

    Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

  11. Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa).

    Science.gov (United States)

    Cho, Jung-Il; Burla, Bo; Lee, Dae-Woo; Ryoo, Nayeon; Hong, Soon-Kwan; Kim, Hyun-Bi; Eom, Joon-Seob; Choi, Sang-Bong; Cho, Man-Ho; Bhoo, Seong Hee; Hahn, Tae-Ryong; Neuhaus, H Ekkehard; Martinoia, Enrico; Jeon, Jong-Seong

    2010-05-01

    In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice.

  12. Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids▿ † ¶

    Science.gov (United States)

    Hazelwood, Lucie A.; Walsh, Michael C.; Pronk, Jack T.; Daran, Jean-Marc

    2010-01-01

    The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators. PMID:19915041

  13. High Boron-induced Ubiquitination Regulates Vacuolar Sorting of the BOR1 Borate Transporter in Arabidopsis thaliana*

    Science.gov (United States)

    Kasai, Koji; Takano, Junpei; Miwa, Kyoko; Toyoda, Atsushi; Fujiwara, Toru

    2011-01-01

    Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles. PMID:21148314

  14. Molecular characterization and functional analysis of a vacuolar Na(+)/H(+) antiporter gene (HcNHX1) from Halostachys caspica.

    Science.gov (United States)

    Guan, Bo; Hu, Youzhen; Zeng, Youling; Wang, Yan; Zhang, Fuchun

    2011-03-01

    According to sequences of several vacuolar Na(+)/H(+) antiporter genes from Xinjiang halophytic plants, a new vacuolar Na(+)/H(+) antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR using primers corresponding to conserved regions of the coding sequences. The obtained HcNHX1 cDNA was 1,983 bp and contained a 1,656 bp open reading frame encoding a deduced protein of 551 amino acid residues. The deduced amino acid sequence showed high identity with other NHX1 we have cloned previously from halophyte in Xinjiang desert area. The phylogenetic analysis showed that HcNHX1 formed a clade with NHX homologs of Chenopodiaceae. Expression profiles under salt treatment and ABA induction were investigated, and the results revealed that expression of HcNHX1 was induced by NaCl and ABA. To compare the degree of salt tolerance, we over-expressed HcNHX1 in Arabidopsis. Two transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na(+) in the leaves compared with wild-type plants. Together, these results suggest that the products of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na(+)/H(+) antiporter.

  15. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

    Science.gov (United States)

    McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

    2013-01-01

    Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS. © 2012 Blackwell Publishing Ltd.

  16. A Maize Vacuolar Invertase, IVR2, Is Induced by Water Stress. Organ/Tissue Specificity and Diurnal Modulation of Expression1

    Science.gov (United States)

    Kim, Jae-Yean; Mahé, Aline; Brangeon, Judy; Prioul, Jean-Louis

    2000-01-01

    The expression of invertases was analyzed in vegetative organs of well-watered and water-stressed maize (Zea mays) plants. Early changes in sucrose metabolism and in acid soluble invertase expression were observed in vegetative sink and source organs under mild water stress. The organ-specific induction of acid invertase activity was correlated with an increase in the Ivr2 gene transcripts and in the vacuolar invertase proteins. In addition diurnal changes in activity and Ivr2 transcripts for vacuolar invertase were noted in shoots. Hexoses (glucose and fructose) accumulated in all organs examined from water-stressed plants. In situ localization studies showed that glucose accumulation, vacuolar invertase activity, invertase protein, and the Ivr2 transcripts colocalized specifically in bundle sheath and vascular tissue cells of mature stressed leaf; in primary roots the stress-induced increase of Ivr2 transcripts was detected only in root tips. Based on these results different regulatory roles are proposed in sink and source organs for the stress induced Ivr2 vacuolar invertase. PMID:10982423

  17. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

    Science.gov (United States)

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

  18. Recombinant vacuolar iron transporter family homologue PfVIT from human malaria-causing Plasmodium falciparum is a Fe2+/H+exchanger.

    Science.gov (United States)

    Labarbuta, Paola; Duckett, Katie; Botting, Catherine H; Chahrour, Osama; Malone, John; Dalton, John P; Law, Christopher J

    2017-02-15

    Vacuolar iron transporters (VITs) are a poorly understood family of integral membrane proteins that can function in iron homeostasis via sequestration of labile Fe2+ into vacuolar compartments. Here we report on the heterologous overexpression and purification of PfVIT, a vacuolar iron transporter homologue from the human malaria-causing parasite Plasmodium falciparum. Use of synthetic, codon-optimised DNA enabled overexpression of functional PfVIT in the inner membrane of Escherichia coli which, in turn, conferred iron tolerance to the bacterial cells. Cells that expressed PfVIT had decreased levels of total cellular iron compared with cells that did not express the protein. Qualitative transport assays performed on inverted vesicles enriched with PfVIT revealed that the transporter catalysed Fe2+/H+ exchange driven by the proton electrochemical gradient. Furthermore, the PfVIT transport function in this system did not require the presence of any Plasmodium-specific factor such as post-translational phosphorylation. PfVIT purified as a monomer and, as measured by intrinsic protein fluorescence quenching, bound Fe2+ in detergent solution with low micromolar affinity. This study of PfVIT provides material for future detailed biochemical, biophysical and structural studies to advance understanding of the vacuolar iron transporter family of membrane proteins from important human pathogens.

  19. Vacuolar H+-ATPase subunit Vma1p functions as the molecular ligand in the vacuole-targeting fungicidal activity of polymyxin B.

    Science.gov (United States)

    Iida, Maki; Yamada, Keiichi; Nango, Yoshiya; Yamaguchi, Yoshihiro; Ogita, Akira; Fujita, Ken-Ichi; Tanaka, Toshio

    2017-04-01

    Polymyxin B (PMB) is a cationic cyclic peptide that can selectively inhibit the growth of Gram-negative bacteria by disrupting the outer membrane permeability barrier through binding to lipopolysaccharide (LPS). Here, a fluorescent PMB derivative (PMB-Ds) was applied to visually confirm the vacuole as a direct lethal target of PMB against fungal cells, which lack LPS. PMB-Ds could be visualized in the normal rounded vacuolar membrane of Saccharomyces cerevisiae cells, suggesting the presence of a molecular ligand assisting the vacuole-targeting mobilization of the peptide in the organism. Vma1p, a cytoplasmic subunit constituent of the yeast vacuolar-type ATPase, was identified as one of the PMB-binding proteins by means of mass spectrometry. Mutant cells carrying a deletion of Vma1p but not those with deletions in two separate PMB-binding proteins were shown to be resistant to the vacuolar membrane disruptive action of PMB. Furthermore, the mutant cells were resistant to PMB lethality even when treated with PMB in combination with allicin, an allyl sulfur compound, which can selectively enhance the vacuole-targeting fungicidal activity of the peptide. In contrast, the parent cells were not made resistant to the vacuolar membrane disruptive action of PMB even if cells were pre-treated with bafilomycin A1, a specific inhibitor of the yeast vacuolar-type H+-ATPase. However, the parent cells were rendered more resistant to PMB consequent to Vma1p-GFP localization in the cytoplasm. These findings suggested a role for Vma1p in the vacuole-targeting fungicidal activity of PMB comparable to that of LPS in the outer membrane of Gram-negative bacteria.

  20. A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

    Science.gov (United States)

    Momonoi, Kazumi; Yoshida, Kumi; Mano, Shoji; Takahashi, Hideyuki; Nakamori, Chihiro; Shoji, Kazuaki; Nitta, Akira; Nishimura, Mikio

    2009-08-01

    Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

  1. Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

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    Gogarten Johann

    2002-04-01

    Full Text Available Abstract Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

  2. Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

    Science.gov (United States)

    Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

    2010-02-01

    We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

  3. RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

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    Huiying Zhang

    Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

  4. Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

    Science.gov (United States)

    Tang, G Q; Lüscher, M; Sturm, A

    1999-02-01

    To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.

  5. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

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    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  6. Genetic suppression analysis in novel vacuolar processing enzymes reveals their roles in controlling sugar accumulation in tomato fruits.

    Science.gov (United States)

    Ariizumi, Tohru; Higuchi, Kenji; Arakaki, Shoko; Sano, Tsunenori; Asamizu, Erika; Ezura, Hiroshi

    2011-05-01

    In plant cells, many vacuolar proteins are synthesized as precursors in the endoplasmic reticulum and are subsequently transported to the vacuole. These precursors are subject to post-translational modifications to allow the active mature forms to be produced. Vacuolar processing enzyme (VPE) has been identified as a family of cysteine proteases involved in protein maturation in the vacuole. In this study, novel VPE genes were isolated from tomato (Solanum lycopersicum), and they were designated SlVPE1-SlVPE5. Phylogenic analysis suggested that SlVPE1 and SlVPE2 were categorized as the seed coat type, SlVPE4 was categorized as the seed type, and both SlVPE3 and SlVPE5 were categorized as the vegetative type. Expression analysis demonstrated that these genes were expressed during fruit development, and that their expression profiles agreed with this classification. High VPE enzyme activity was observed during tomato fruit development; the enzyme activity was correlated with the SlVPE mRNA levels, indicating that the SlVPE encoded active VPE proteins. The total sugar content was higher in RNA interference (RNAi) lines compared with the control plants, suggesting negative roles for SlVPE in sugar accumulation. The quantitative expression analysis of each SlVPE gene in the RNAi lines suggested that the suppression of SlVPE5 probably had the strongest effect on the sugar accumulation observed. The suppression of SlVPE did not influence the total amino acid content, suggesting that the molecular targets of SlVPE were mainly involved in sugar accumulation.

  7. Structural studies of alternative oxidase (AOX) from moniliophthora perniciosa, the causal agent of witches' broom disease in cacao: a membrane-associated protein

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, J.F.; Prado, P.F.V.; Tiezzi, H.O.; Dias, S.M.G.; Ambrosio, A.L.B. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Thomazella, D.P.T.; Teixeira, P.J.P.L.; Pereira, G.A.G. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2012-07-01

    Full text: Alternative oxidase (AOX) is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain. This protein is present in higher plants, pathogenic fungi and some parasites. The structural characterization of AOX becomes interesting due to its potential as a fungicide target. AOX is predicted to be a monotopic interfacial membrane protein interacting with a single leaflet of the lipid bilayer, rather than transmembrane. Amino acid sequence analysis reveals the presence of two conserved glutamate-histidine motifs, identifying it as a member of the diiron carboxylate protein family. The AOX model is defined by two pairs of helices forming a four helix bundle and an additional hydrophobic connecting sequence between the two helical pairs is proposed to act as the membrane anchoring region. In this work we aim at production, purification and crystallization of the AOX protein from M. perniciosa for further structural studies of this membrane-associated protein, by X-ray protein crystallography (author)

  8. A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors

    Science.gov (United States)

    Krebs, Christopher J.; Jarvis, Erich D.; Chan, Johnny; Lydon, John P.; Ogawa, Sonoko; Pfaff, Donald W.

    2000-01-01

    The ventromedial hypothalamus (VMH) plays a central role in the regulation of the female reproductive behavior lordosis, a behavior dependent upon the sequential activation of receptors for the ovarian steroid hormones estradiol (E) and progesterone (P). These receptors function as transcription factors to alter the expression of target genes. To discover behaviorally relevant genes targeted by E and P in the VMH, we used the differential display PCR to identify messenger RNAs that are differentially expressed in the hypothalamus of ovariectomized (ovx) rats treated with E alone compared with ovariectomized rats treated with E and P. We show here that one interesting mRNA within the hypothalamus that is repressed by P after E priming encodes the protein 25-Dx, the rat homolog of the human membrane-associated P-binding protein Hpr6.6. Neurons in the brain containing the highest levels of 25-Dx are located in several nuclei of the basal forebrain, including the VMH. 25-Dx expression is also higher in the hypothalamus of female P receptor “knockout” mice than in their wild-type littermates. These findings suggest a mechanism in which the activation of nuclear P receptor represses expression of a membrane P receptor, 25-Dx, during lordosis facilitation. PMID:11070092

  9. The membrane-associated transient receptor potential vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.

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    Zohar Bromberg

    Full Text Available The heat shock response (HSR is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1, however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV. We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX, upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.

  10. Identification of a Novel Function of Adipocyte Plasma Membrane-Associated Protein (APMAP) in Gestational Diabetes Mellitus by Proteomic Analysis of Omental Adipose Tissue.

    Science.gov (United States)

    Ma, Yuhang; Gao, Jing; Yin, Jiajing; Gu, Liping; Liu, Xing; Chen, Su; Huang, Qianfang; Lu, Huifang; Yang, Yuemin; Zhou, Hu; Wang, Yufan; Peng, Yongde

    2016-02-05

    Gestational diabetes mellitus (GDM) is considered as an early stage of type 2 diabetes mellitus. In this study, we compared demographic and clinical data between six GDM subjects and six normal glucose tolerance (NGT; healthy controls) subjects and found that homeostasis model of assessment for insulin resistance index (HOMA-IR) increased in GDM. Many previous studies demonstrated that omental adipose tissue dysfunction could induce insulin resistance. Thus, to investigate the cause of insulin resistance in GDM, we used label-free proteomics to identify differentially expressed proteins in omental adipose tissues from GDM and NGT subjects (data are available via ProteomeXchange with identifier PXD003095). A total of 3528 proteins were identified, including 66 significantly changed proteins. Adipocyte plasma membrane-associated protein (APMAP, a.k.a. C20orf3), one of the differentially expressed proteins, was down-regulated in GDM omental adipose tissues. Furthermore, mature 3T3-L1 adipocytes were used to simulate omental adipocytes. The inhibition of APMAP expression by RNAi impaired insulin signaling and activated NFκB signaling in these adipocytes. Our study revealed that the down-regulation of APMAP in omental adipose tissue may play an important role in insulin resistance in the pathophysiology of GDM.

  11. Effect of preparations Methyure and Ivine on Са(2+)-ATPases activity in plasma and vacuolar membrane of corn seedling roots under salt stress conditions.

    Science.gov (United States)

    Rudnytska, M V; Palladina, T A

    2017-01-01

    Ca2+-ATPases regulate the functioning of Ca2+-dependent signaling pathway SOS which provides removal of Na+ from the cytoplasm of cells via Na+/H+-antiporters in saline conditions. The influence of synthetic preparations Methyure and Ivine on the Ca2+-ATPase activity was investigated. It was shown that exposition of corn seedlings in the presence of 0.1 M NaCl rather enhanced hydrolytic than transport activity of Ca2+-ATPases in plasma and vacuolar membrane of root cells. It was found that seed treatment with such preparations, especially Methyure, caused intensification of the both activities of Ca2+-ATPases, mainly in vacuolar membrane. The results indicate than salt protective activity of preparations, especially Methyure, is associated with increased Ca2+-ATPase activity, which regulates the functioning of Na+/H+-antiporters.

  12. Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1 and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana.

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    Sagarika Mishra

    Full Text Available Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata, an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1 contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93 was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.

  13. Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Mishra, Sagarika; Alavilli, Hemasundar; Lee, Byeong-ha; Panda, Sanjib Kumar; Sahoo, Lingaraj

    2014-01-01

    Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.

  14. Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects.

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    Zhu, Xiaobiao; Richael, Craig; Chamberlain, Patrick; Busse, James S; Bussan, Alvin J; Jiang, Jiming; Bethke, Paul C

    2014-01-01

    Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

  15. Vacuolar invertase gene silencing in potato (Solanum tuberosum L. improves processing quality by decreasing the frequency of sugar-end defects.

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    Xiaobiao Zhu

    Full Text Available Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

  16. A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance.

    Science.gov (United States)

    Makrantoni, Vasso; Dennison, Paul; Stark, Michael J R; Coote, Peter J

    2007-12-01

    In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

  17. Early salt stress effects on the differential expression of vacuolar H(+)-ATPase genes in roots and leaves of Mesembryanthemum crystallinum.

    Science.gov (United States)

    Löw, R; Rockel, B; Kirsch, M; Ratajczak, R; Hörtensteiner, S; Martinoia, E; Lüttge, U; Rausch, T

    1996-01-01

    In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H(+)-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H(+)-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.

  18. The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

    Science.gov (United States)

    Choi, Dong-W; Kunz, Ryan C; Boyd, Eric S; Semrau, Jeremy D; Antholine, William E; Han, J-I; Zahn, James A; Boyd, Jeffrey M; de la Mora, Arlene M; DiSpirito, Alan A

    2003-10-01

    Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

  19. αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

    Directory of Open Access Journals (Sweden)

    Le Parc Annabelle

    2010-08-01

    Full Text Available Abstract Background Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes. Conclusions These experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.

  20. Medically Relevant Acinetobacter Species Require a Type II Secretion System and Specific Membrane-Associated Chaperones for the Export of Multiple Substrates and Full Virulence.

    Science.gov (United States)

    Harding, Christian M; Kinsella, Rachel L; Palmer, Lauren D; Skaar, Eric P; Feldman, Mario F

    2016-01-01

    Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion.

  1. Medial prefrontal cortical estradiol rapidly alters memory system bias in female rats: ultrastructural analysis reveals membrane-associated estrogen receptors as potential mediators.

    Science.gov (United States)

    Almey, Anne; Cannell, Elizabeth; Bertram, Kyla; Filardo, Edward; Milner, Teresa A; Brake, Wayne G

    2014-11-01

    High plasma levels of estradiol (E2) are associated with use of a place memory system over a response memory system. We examined whether infusing estradiol into the medial prefrontal cortex (mPFC) or anterior cingulate cortex (AC) could affect memory system bias in female rats. We also examined the ultrastructural distribution of estrogen receptor (ER)-α, ERβ, and G protein-coupled estrogen receptor 1 (GPER1) in the mPFC of female rats as a mechanism for the behavioral effects of E2 in the mPFC. Each rat was infused bilaterally with either E2 (0.13 μg) or vehicle into the mPFC or AC. The majority of E2 mPFC rats used place memory. In contrast, the majority of mPFC vehicle rats and AC E2 or vehicle rats used response memory. These data show that mPFC E2 rapidly biases females to use place memory. Electron microscopic analysis demonstrated that ERα, ERβ, and GPER1 are localized in the mPFC, almost exclusively at extranuclear sites. This is the first time that GPER1 has been localized to the mPFC of rats and the first time that ERα and ERβ have been described at extranuclear sites in the rat mPFC. The majority of receptors were observed on axons and axon terminals, suggesting that estrogens alter presynaptic transmission in the mPFC. This provides a mechanism via which ERs could rapidly alter transmission in the mPFC to alter PFC-dependent behaviors, such as memory system bias. The discrete nature of immunolabeling for these membrane-associated ERs may explain the discrepancy in previous light microscopy studies.

  2. A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

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    Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko; Endo, Toshiya [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan); Nishikawa, Shuh-ichi, E-mail: shuh@biochem.chem.nagoya-u.ac.jp [Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 (Japan)

    2010-03-12

    Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

  3. Suppression of the Vacuolar Invertase Gene Prevents Cold-Induced Sweetening in Potato12[W][OA

    Science.gov (United States)

    Bhaskar, Pudota B.; Wu, Lei; Busse, James S.; Whitty, Brett R.; Hamernik, Andy J.; Jansky, Shelley H.; Buell, C. Robin; Bethke, Paul C.; Jiang, Jiming

    2010-01-01

    Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding. PMID:20736383

  4. The polyphenol oxidase gene family in poplar: phylogeny, differential expression and identification of a novel, vacuolar isoform.

    Science.gov (United States)

    Tran, Lan T; Constabel, C Peter

    2011-10-01

    Polyphenol oxidases (PPOs) are oxidative enzymes that convert monophenols and o-diphenols to o-quinones using molecular oxygen. The quinone products are highly reactive following tissue damage and can interact with cellular constituents and cause oxidative browning and cross-linking. The induction of PPO in some plants as a result of wounding, herbivore attack, or pathogen infection has implicated them in defense. However, PPO-like enzymes that act as specific hydroxylases, for example in lignan and pigment biosynthesis, have also been discovered. Here, we present the first genome-enabled analysis of a PPO gene family. The Populus trichocarpa genome was found to contain a minimum of nine complete PPO genes, and seven of these were characterized further. The PPO gene family includes both recently duplicated and divergent sequences that are 36-98% identical at the amino acid level. Gene expression profiling in poplar tissues and organs revealed that the PPO genes are all differentially expressed during normal development, but that only a small subset of PPO genes are significantly upregulated by wounding, methyl jasmonate or pathogen infection. Our studies also identified PtrPPO13, a novel PPO gene that is predicted to encode an N-terminal signal peptide. Transient expression of green fluorescent protein fusions demonstrated its localization to the vacuolar lumen. Together, our findings show that the poplar PPO family is diverse and is likely linked to diverse physiological functions.

  5. Silicon ameliorates chromium toxicity through phytochelatin-mediated vacuolar sequestration in the roots of Oryza sativa (L.).

    Science.gov (United States)

    Huda, A K M Nazmul; Haque, Mohammad Anwarul; Zaman, Reshma; Swaraz, A M; Kabir, Ahmad Humayan

    2017-03-04

    High chromium (Cr) in rice causes reduced yield and health hazards. This work investigates how Si alleviates Cr toxicity in rice. Addition of Si under Cr stress restored the growth parameters, total protein content, and membrane stability along with reduced Cr content in shoots, confirming that Si plays critical roles in Cr detoxification in rice. However, Si supplementation under Cr stress caused no significant changes in root Cr content but decreased shoot Cr concentrations compared with Cr-stressed plants, indicating that alleviation of Cr toxicity might be associated with Cr sequestration in roots. Further, concentration of Fe and expression of Fe transporter (OsIRT1) showed no significant changes due to Si supplementation under Cr stress, implying that Fe regulation is not involved with Si-mediated mitigation of Cr toxicity in rice. Further, phytochelatin accumulation and OsPCS1 (phytochelatin synthase) transcripts strongly induced due to the dual treatment of Si and Cr compared with Cr-stressed plants, suggesting that phytochelatin might bind to Cr, which leads to vacuolar sequestration in roots. Furthermore, increased glutathione reductase activity in roots implies that active involvement of ROS scavenging partially ameliorates Cr toxicity in rice plants. The study illustrates first evidences on the effect of Si alleviating Cr toxicity in rice plants.

  6. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

    Science.gov (United States)

    Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H; Møller, Birger Lindberg; Rook, Fred

    2016-11-14

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

  7. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2014-09-01

    Full Text Available The vacuolar (H+-ATPase (V-ATPase of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.

  8. Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

    Science.gov (United States)

    Myers, D E; Jun, X; Waddick, K G; Forsyth, C; Chelstrom, L M; Gunther, R L; Tumer, N E; Bolen, J; Uckun, F M

    1995-10-10

    CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

  9. Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath.

    Science.gov (United States)

    Choi, Dong W; Antholine, William E; Do, Young S; Semrau, Jeremy D; Kisting, Clint J; Kunz, Ryan C; Campbell, Damon; Rao, Vinay; Hartsel, Scott C; DiSpirito, Alan A

    2005-10-01

    Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3b(T) or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min(-1) (mg protein)(-1) range, activities approaching 400 nmol propylene oxidized min(-1) (mg protein)(-1) were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity. The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0.5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0.4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0.4 and 0.9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH4 and/or O2). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O2 and decreased the residual free radical following the addition of O2 plus CH4. The increase in pMMO activity and EPR spectral changes

  10. Effect of bafilomycin and NAADP on membrane-associated ATPases and respiration of isolated mitochondria of the murine Nemeth-Kellner lymphoma.

    Science.gov (United States)

    Hreniukh, V; Bychkova, S; Kulachkovsky, O; Babsky, A

    2016-12-01

    The goal of the study was to estimate the effect of a selective V-type H(+) -ATPase inhibitor bafilomycin A1 and nicotinic acid adenine dinucleotide phosphate (NAADP) on energetic processes in NK/Ly cell by directly measuring the respiration of isolated mitochondria and ATPase activities. NAADP (7 μM) increased the activity of Na(+) /K(+) -ATPase in the postmitochondrial fraction of NK/Ly cells, but lower concentration of NAADP decreased it (0.1 and 1 μM). The increase the activity of plasma membrane Ca(2)(+) ATPase (PMCA) under NAADP application (1 and 7 μM) was observed. However, NAADP (1 μM) decreased activities of sarcoendoplasmic reticulum Ca(2)(+) -ATPase (SERCA) and basal Mg(2)(+) -ATPase. Bafilomycin A1 (1 μM) increased the activity of Na(+) /K(+) -ATPase and potentiated the effect of NAADP (1 μM) on this pump. At the same time, bafilomycin A1 (1 μM) completely prevented all effects of NAADP (1 μM) on activities of PMCA, SERCA, and basal Mg(2)(+) -ATPase, confirming that these effects are dependent on acidic stores. Bafilomycin A1 or NAADP decreased respiratory and oxidative phosphorylation rates in NK/Ly mitochondria when α-ketoglutarate was used as substrate in contrast to succinate. Thus, α-ketoglutarate oxidation is more sensitive to bafilomycin A1 and NAADP influences compared with succinate oxidation. However, bafilomycin A1 + NAADP and any of these compounds separately lead to full uncoupling of mitochondria after ADP addition irrespectively to substrate used. Bafilomycin A1 affects isolated tumor mitochondria more effectively in combination with NAADP. Bafilomycin and NAADP alter some membrane-associated ATPases and inhibit respiration in mitochondria of the Nemeth-Kellner lymphoma. Bafilomycin A1 potentiates the effect of NAADP by inhibiting the mitochondrial energetic process in lymphoma cells and activity of Na(+) /K(+) -ATPase. The obtained data show promising possibility to use bafilomycin A1 and NAADP as chemotherapeutic

  11. Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro.

    Science.gov (United States)

    Gu, Wenwen; Dong, Nian; Wang, Peng; Shi, Changgen; Yang, Jun; Wang, Jian

    2017-04-01

    The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER+) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER+ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER+ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66-/PR-/HER2-) and ER-α36+/GPER1+ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER+ breast cancer.

  12. Hydrogen sulfide alleviates cadmium toxicity through regulations of cadmium transport across the plasma and vacuolar membranes in Populus euphratica cells.

    Science.gov (United States)

    Sun, Jian; Wang, Ruigang; Zhang, Xuan; Yu, Yicheng; Zhao, Rui; Li, Zongyun; Chen, Shaoliang

    2013-04-01

    Hydrogen sulfide (H2S) is emerging as a novel signalling molecule involved in plant growth and responses against abiotic stresses. However, little information is known about its role in cadmium (Cd) detoxification. In the present study, the effects of H2S on Cd toxicity were investigated in Populus euphratica cells using fluorescence imaging technique and a non-invasive vibrating ion-selective microelectrode. Pretreatment with a H2S donor, sodium hydrosulfide (NaHS), significantly mitigated the Cd-induced programmed cell death in P. euphratica cells. The alleviation effect of NaHS was more pronounced at 50-100 μM as compared to low (25 μM) and high doses (200 μM). Under Cd stress, total activities of antioxidant enzymes, such as ascorbate peroxidase, catalase and glutathione reductase, were significantly enhanced in NaHS-treated cells, leading to a decline of H2O2 accumulation and lipid peroxidation. Moreover, NaHS reduced Cd accumulation in the cytoplasm but increased the fraction of Cd in the vacuole. Cd flux profiles revealed that H2S inhibited the Cd influx through the plasma membrane (PM) calcium channels that activated by H2O2. NaHS enhanced Cd influx into the vacuole, and the Cd influx was dependent on the pH gradients across the tonoplast. Taken together, these results suggest that H2S alleviates Cd toxicity via the improvement of antioxidant system and cellular Cd homeostasis. The up-regulation of antioxidant enzymes by H2S reduced the accumulation of H2O2, and thus decreased Cd influx through the H2O2-activated PM calcium channels. The H2S-simulated vacuolar Cd sequestration was presumably due to the activation of tonoplast Cd(2+)/H(+) antiporters. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  13. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis.

    Science.gov (United States)

    Klemens, Patrick A W; Patzke, Kathrin; Deitmer, Joachim; Spinner, Lara; Le Hir, Rozenn; Bellini, Catherine; Bedu, Magali; Chardon, Fabien; Krapp, Anne; Neuhaus, H Ekkehard

    2013-11-01

    Here, we report that SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET16) from Arabidopsis (Arabidopsis thaliana) is a vacuole-located carrier, transporting glucose (Glc), fructose (Fru), and sucrose (Suc) after heterologous expression in Xenopus laevis oocytes. The SWEET16 gene, similar to the homologs gene SWEET17, is mainly expressed in vascular parenchyma cells. Application of Glc, Fru, or Suc, as well as cold, osmotic stress, or low nitrogen, provoke the down-regulation of SWEET16 messenger RNA accumulation. SWEET16 overexpressors (35SPro:SWEET16) showed a number of peculiarities related to differences in sugar accumulation, such as less Glc, Fru, and Suc at the end of the night. Under cold stress, 35SPro:SWEET16 plants are unable to accumulate Fru, while under nitrogen starvation, both Glc and Fru, but not Suc, were less abundant. These changes of individual sugars indicate that the consequences of an increased SWEET16 activity are dependent upon the type of external stimulus. Remarkably, 35SPro:SWEET16 lines showed improved germination and increased freezing tolerance. The latter observation, in combination with the modified sugar levels, points to a superior function of Glc and Suc for frost tolerance. 35SPro:SWEET16 plants exhibited increased growth efficiency when cultivated on soil and showed improved nitrogen use efficiency when nitrate was sufficiently available, while under conditions of limiting nitrogen, wild-type biomasses were higher than those of 35SPro:SWEET16 plants. Our results identify SWEET16 as a vacuolar sugar facilitator, demonstrate the substantial impact of SWEET16 overexpression on various critical plant traits, and imply that SWEET16 activity must be tightly regulated to allow optimal Arabidopsis development under nonfavorable conditions.

  14. Experimental feeding of Hydrilla verticillata colonized by stigonematales cyanobacteria induces vacuolar myelinopathy in painted turtles (Chrysemys picta.

    Directory of Open Access Journals (Sweden)

    Albert D Mercurio

    Full Text Available Vacuolar myelinopathy (VM is a neurologic disease primarily found in birds that occurs when wildlife ingest submerged aquatic vegetation colonized by an uncharacterized toxin-producing cyanobacterium (hereafter "UCB" for "uncharacterized cyanobacterium". Turtles are among the closest extant relatives of birds and many species directly and/or indirectly consume aquatic vegetation. However, it is unknown whether turtles can develop VM. We conducted a feeding trial to determine whether painted turtles (Chrysemys picta would develop VM after feeding on Hydrilla (Hydrilla verticillata, colonized by the UCB (Hydrilla is the most common "host" of UCB. We hypothesized turtles fed Hydrilla colonized by the UCB would exhibit neurologic impairment and vacuolation of nervous tissues, whereas turtles fed Hydrilla free of the UCB would not. The ability of Hydrilla colonized by the UCB to cause VM (hereafter, "toxicity" was verified by feeding it to domestic chickens (Gallus gallus domesticus or necropsy of field collected American coots (Fulica americana captured at the site of Hydrilla collections. We randomly assigned ten wild-caught turtles into toxic or non-toxic Hydrilla feeding groups and delivered the diets for up to 97 days. Between days 82 and 89, all turtles fed toxic Hydrilla displayed physical and/or neurologic impairment. Histologic examination of the brain and spinal cord revealed vacuolations in all treatment turtles. None of the control turtles exhibited neurologic impairment or had detectable brain or spinal cord vacuolations. This is the first evidence that freshwater turtles can become neurologically impaired and develop vacuolations after consuming toxic Hydrilla colonized with the UCB. The southeastern United States, where outbreaks of VM occur regularly and where vegetation colonized by the UCB is common, is also a global hotspot of freshwater turtle diversity. Our results suggest that further investigations into the effect of the

  15. Post-transcriptional regulation of fruit ripening and disease resistance in tomato by the vacuolar protease SlVPE3.

    Science.gov (United States)

    Wang, Weihao; Cai, Jianghua; Wang, Peiwen; Tian, Shiping; Qin, Guozheng

    2017-03-07

    Proteases represent one of the most abundant classes of enzymes in eukaryotes and are known to play key roles in many biological processes in plants. However, little is known about their functions in fruit ripening and disease resistance, which are unique to flowering plants and required for seed maturation and dispersal. Elucidating the genetic mechanisms of fruit ripening and disease resistance is an important goal given the biological and dietary significance of fruit. Through expression profile analyses of genes encoding tomato (Solanum lycopersicum) cysteine proteases, we identify a number of genes whose expression increases during fruit ripening. RNA interference (RNAi)-mediated repression of SlVPE3, a vacuolar protease gene, results in alterations in fruit pigmentation, lycopene biosynthesis, and ethylene production, suggesting that SlVPE3 is necessary for normal fruit ripening. Surprisingly, the SlVPE3 RNAi fruit are more susceptible to the necrotrophic pathogen Botrytis cinerea. Quantitative proteomic analysis identified 314 proteins that differentially accumulate upon SlVPE3 silencing, including proteins associated with fruit ripening and disease resistance. To identify the direct SlVPE3 targets and mechanisms contributing to fungal pathogen resistance, we perform a screening of SlVPE3-interacting proteins using co-immunoprecipitation coupled with mass spectrometry. We show that SlVPE3 is required for the cleavage of the serine protease inhibitor KTI4, which contributes to resistance against the fungal pathogen B. cinerea. Our findings contribute to elucidating gene regulatory networks and mechanisms that control fruit ripening and disease resistance responses.

  16. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

    Science.gov (United States)

    Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Overexpression of the Vacuolar Sugar Carrier AtSWEET16 Modifies Germination, Growth, and Stress Tolerance in Arabidopsis1[W

    Science.gov (United States)

    Klemens, Patrick A.W.; Patzke, Kathrin; Deitmer, Joachim; Spinner, Lara; Le Hir, Rozenn; Bellini, Catherine; Bedu, Magali; Chardon, Fabien; Krapp, Anne; Neuhaus, H. Ekkehard

    2013-01-01

    Here, we report that SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET16) from Arabidopsis (Arabidopsis thaliana) is a vacuole-located carrier, transporting glucose (Glc), fructose (Fru), and sucrose (Suc) after heterologous expression in Xenopus laevis oocytes. The SWEET16 gene, similar to the homologs gene SWEET17, is mainly expressed in vascular parenchyma cells. Application of Glc, Fru, or Suc, as well as cold, osmotic stress, or low nitrogen, provoke the down-regulation of SWEET16 messenger RNA accumulation. SWEET16 overexpressors (35SPro:SWEET16) showed a number of peculiarities related to differences in sugar accumulation, such as less Glc, Fru, and Suc at the end of the night. Under cold stress, 35SPro:SWEET16 plants are unable to accumulate Fru, while under nitrogen starvation, both Glc and Fru, but not Suc, were less abundant. These changes of individual sugars indicate that the consequences of an increased SWEET16 activity are dependent upon the type of external stimulus. Remarkably, 35SPro:SWEET16 lines showed improved germination and increased freezing tolerance. The latter observation, in combination with the modified sugar levels, points to a superior function of Glc and Suc for frost tolerance. 35SPro:SWEET16 plants exhibited increased growth efficiency when cultivated on soil and showed improved nitrogen use efficiency when nitrate was sufficiently available, while under conditions of limiting nitrogen, wild-type biomasses were higher than those of 35SPro:SWEET16 plants. Our results identify SWEET16 as a vacuolar sugar facilitator, demonstrate the substantial impact of SWEET16 overexpression on various critical plant traits, and imply that SWEET16 activity must be tightly regulated to allow optimal Arabidopsis development under nonfavorable conditions. PMID:24028846

  18. The m6A methyltransferase Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology in haploid yeast cells.

    Science.gov (United States)

    Yadav, Pradeep Kumar; Rajasekharan, Ram

    2017-08-18

    N6-Methyladenosine (m6A) is among the most common modifications in eukaryotic mRNA. The role of yeast m6A methyltransferase, Ime4, in meiosis and sporulation in diploid strains is very well studied, but its role in haploid strains has remained unknown. Here, with the help of an immunoblotting strategy and Ime4-GFP protein localization studies, we establish the physiological role of Ime4 in haploid cells. Our data showed that Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology through the long-chain fatty acyl-CoA synthetase Faa1, independently of the RNA methylation complex (MIS complex). The MIS complex consists of the Ime4, Mum2, and Slz1 proteins. Our affinity enrichment strategy (methylated RNA immunoprecipitation assays) using m6A polyclonal antibodies coupled with mRNA isolation, quantitative real-time PCR, and standard PCR analyses confirmed the presence of m6A-modified FAA1 transcripts in haploid yeast cells. The term "epitranscriptional regulation" encompasses the RNA modification-mediated regulation of genes. Moreover, we demonstrate that the Aft2 transcription factor up-regulates FAA1 expression. Because the m6A methylation machinery is fundamentally conserved throughout eukaryotes, our findings will help advance the rapidly emerging field of RNA epitranscriptomics. The metabolic link identified here between m6A methylation and triacylglycerol metabolism via the Ime4 protein provides new insights into lipid metabolism and the pathophysiology of lipid-related metabolic disorders, such as obesity. Because the yeast vacuole is an analogue of the mammalian lysosome, our findings pave the way to better understand the role of m6A methylation in lysosome-related functions and diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The Vacuolar Import and Degradation Pathway Merges with the Endocytic Pathway to Deliver Fructose-1,6-bisphosphatase to the Vacuole for Degradation*

    Science.gov (United States)

    Brown, C. Randell; Wolfe, Allison B.; Cui, Dongying; Chiang, Hui-Ling

    2008-01-01

    The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the ε-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Δvph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway. PMID:18660504

  20. The vacuolar import and degradation pathway merges with the endocytic pathway to deliver fructose-1,6-bisphosphatase to the vacuole for degradation.

    Science.gov (United States)

    Brown, C Randell; Wolfe, Allison B; Cui, Dongying; Chiang, Hui-Ling

    2008-09-19

    The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the epsilon-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.

  1. Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

    Science.gov (United States)

    Brummell, David A; Chen, Ronan K Y; Harris, John C; Zhang, Huaibi; Hamiaux, Cyril; Kralicek, Andrew V; McKenzie, Marian J

    2011-06-01

    Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.

  2. The assembly of metals chelation by thiols and vacuolar compartmentalization conferred increased tolerance to and accumulation of cadmium and arsenic in transgenic Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jiangbo [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China); Inner Mongolia Key Laboratory of Biomass-Energy Conversion, The Institute of Bioengineering and Technology, Inner Mongolia University of Science and Technology, Baotou 040100 (China); Xu, Wenzhong [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China); Ma, Mi, E-mail: mami@ibcas.ac.cn [Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093 (China)

    2012-01-15

    Highlights: Black-Right-Pointing-Pointer Simultaneous transformation of AsPCS1 and ScYCF1 into Arabidopsis thaliana which is sensitive to heavy metals, leads to transgenic plants tolerant to Arsenic and cadmium. Black-Right-Pointing-Pointer Dual-gene transgenic Arabidopsis showed higher accumulation of Arsenic and cadmium than single and non-transgenic plants. Black-Right-Pointing-Pointer Our results proved that improved thiol peptides synthesis and vacuolar compartmentation in plant dramatically boosted the survival rates of plants when exposed to heavy metals. Black-Right-Pointing-Pointer A new strategy for efficient phytoremediation of heavy metals by stacking genes transformation in plants was developed in this article. - Abstract: Transgenic Arabidopsis thaliana were developed to increase tolerance for and accumulation of heavy metals and metalloids by simultaneous overexpression of AsPCS1 and YCF1 (derived from garlic and baker's yeast) based on the fact that chelation of metals and vacuolar compartmentalization are the main strategies for heavy metals/metalloids detoxification and tolerance in plants. Dual-gene transgenic lines had the longest roots and the highest accumulation of Cd and As than single-gene transgenic lines and wildtype. When grown on cadmium or arsenic (arsenite/arsenate), Dual-gene transgenic lines accumulated over 2-10 folds cadmium/arsenite and 2-3 folds arsenate than wild type or plants expressing AsPCS1 or YCF1 alone. Such stacking modified genes involved in chelation of toxic metals and vacuolar compartmentalization represents a highly promising new tool for use in phytoremediation efforts.

  3. Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

    Science.gov (United States)

    Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

    2014-06-01

    Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. Salt regulation of transcript levels for the c subunit of a leaf vacuolar H(+)-ATPase in the halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Tsiantis, M S; Bartholomew, D M; Smith, J A

    1996-05-01

    The halophyte Mesembryanthemum crystallinum is an inducible crassulacean acid metabolism (CAM) plant native to seasonally arid coastal environments that has been widely used to study plant responses to environmental stress. On exposure of plants to salt, the activities of both the tonoplast (vacuolar) H(+)-ATPase (V-ATPase) and Na+/H+ antiporter increase in leaf cells, thereby energizing vacuolar salt accumulation. To investigate the molecular basis of this response, a cDNA (Vmac1) encoding the H(+)-conducting c subunit (16.6 kDa) of an M. crystallinum V-ATPase has been cloned. Northern analysis of RNA from leaves of plants treated with NaCl or with isoosmotic mannitol solutions demonstrated (i) that NaCl increased steady-state transcript levels for the V-ATPase c subunit, and (ii) that this effect was caused by the ionic rather than the osmotic component of salt stress. Southern analysis of genomic DNA suggested the probable existence of more than one gene for this subunit of the V-ATPase in M. crystallinum. Expression studies using the 3'-untranslated region of the Vmac1 cDNa as a probe showed that the corresponding salt-inducible transcript was preferentially expressed in leaves. Induction by salt was also observed in juvenile plants in addition to adult ones. These findings, as well as the inability of mannitol to upregulate mRNA levels for this gene, clearly differentiate between the induction of transcript for the V-ATPase c subunit and for genes involved in the CAM pathway in M. crystallinum. Further, the plant growth regulator abscisic acid (ABA) was able to mimic the effect of salt on transcript levels for the V-ATPase c subunit, suggesting the possible involvement of ABA in a distinct signal-transduction pathway linked to vacuolar salt accumulation in this highly salt-tolerant species.

  5. Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells.

    Science.gov (United States)

    Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J

    2016-07-22

    Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448-19457). This study capitalized on the mechanisms suppressing vacuolar H(+)-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Plant regeneration and Agrobacterium-mediated transformation of Vacuolar H+-ATPase c Subunit Gene in hybrid poplar populus davidiana Dode × P. bollena Lauche

    Directory of Open Access Journals (Sweden)

    Han Xue

    2017-01-01

    Full Text Available An efficient regeneration and transformation system was developed for hybrid poplar populus davidiana Dode × P. bollena Lauche. Several factors, such as a competent regeneration protocol, antibiotics, bacterial concentration, infection time have been shown to affect transformation efficiency. It is the first report describing an efficient protocol for Vacuolar H+-ATPase c Subunit Gene from salt tolerant plant Puccinellia chinampoensis, which transformed into populus. Stable transgene integration was confirmed by the expression of GFP fusion proteins in leaves, stem and root for populus.

  7. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole.

    OpenAIRE

    Stack, J H; Herman, P. K.; Schu, P V; Emr, S D

    1993-01-01

    The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Over-production of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vps15 kinase domain mutants, suggesting that Vps15p and Vps34p functionally interact. Subcellular fractionation and sucrose density gradients indicate that Vps15p is responsible for the association of Vps34p with an intracellular membrane f...

  8. The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation

    Directory of Open Access Journals (Sweden)

    Babatunde A. Ayodele

    2017-12-01

    Full Text Available Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2 were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase V0 subunit d2 (ATP6V0D2, was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium. In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

  9. Evolution of vacuolar proton pyrophosphatase domains and volutin granules: clues into the early evolutionary origin of the acidocalcisome

    Directory of Open Access Journals (Sweden)

    Valerio Alejandro

    2011-10-01

    Full Text Available Abstract Background Volutin granules appear to be universally distributed and are morphologically and chemically identical to acidocalcisomes, which are electron-dense granular organelles rich in calcium and phosphate, whose functions include storage of phosphorus and various metal ions, metabolism of polyphosphate, maintenance of intracellular pH, osmoregulation and calcium homeostasis. Prokaryotes are thought to differ from eukaryotes in that they lack membrane-bounded organelles. However, it has been demonstrated that as in acidocalcisomes, the calcium and polyphosphate-rich intracellular "volutin granules (polyphosphate bodies" in two bacterial species, Agrobacterium tumefaciens, and Rhodospirillum rubrum, are membrane bound and that the vacuolar proton-translocating pyrophosphatases (V-H+PPases are present in their surrounding membranes. Volutin granules and acidocalcisomes have been found in organisms as diverse as bacteria and humans. Results Here, we show volutin granules also occur in Archaea and are, therefore, present in the three superkingdoms of life (Archaea, Bacteria and Eukarya. Molecular analyses of V-H+PPase pumps, which acidify the acidocalcisome lumen and are diagnostic proteins of the organelle, also reveal the presence of this enzyme in all three superkingdoms suggesting it is ancient and universal. Since V-H+PPase sequences contained limited phylogenetic signal to fully resolve the ancestral nodes of the tree, we investigated the divergence of protein domains in the V-H+PPase molecules. Using Protein family (Pfam database, we found a domain in the protein, PF03030. The domain is shared by 31 species in Eukarya, 231 in Bacteria, and 17 in Archaea. The universal distribution of the V-H+PPase PF03030 domain, which is associated with the V-H+PPase function, suggests the domain and the enzyme were already present in the Last Universal Common Ancestor (LUCA. Conclusion The importance of the V-H+PPase function and the

  10. Cloning and expression analysis of GhDET3, a vacuolar H+ -ATPase subunit C gene, from cotton.

    Science.gov (United States)

    Xiao, Zhongyi; Tan, Kunling; Hu, Mingyu; Liao, Peng; Chen, Kuijun; Luo, Ming

    2008-05-01

    Vacuolar H(+)-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypeptide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

  11. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P

    2001-01-01

    Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase alpha and beta subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na......+,K+-ATPase alpha (1) subunit, and iii) Na+,K+-ATPase beta (1) subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmoregulatory ability as judged by their ability to regulate plasma [Cl-] in a 24-hr...... seawater challenge test (35 ppt). Gill Na+,K+-ATPase alpha (1) and beta (1) subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased...

  12. Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Taisuke; Furukawa, Soichi; Kitamoto, Katsuhiko; Takatsuki, Akira; Hirata, Ryogo; Ogihara, Hirokazu; Yamasaki, Makari

    2005-11-25

    As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.

  13. Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

    Science.gov (United States)

    Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

    1995-10-01

    Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

  14. Involvement of vacuolar processing enzyme SlVPE5 in post-transcriptional process of invertase in sucrose accumulation in tomato.

    Science.gov (United States)

    Wang, Ning; Duhita, Narendra; Ariizumi, Toru; Ezura, Hiroshi

    2016-11-01

    Enhancing the flavor of fruits plays a fundamental role in improving fruit quality, and volatile compositions as well as acid and sugar accumulation are significant factors that have an impact on the acceptability of sensory responses by human beings. Vacuoles in plants not only function as cell compartments that store amino acids, sugars and other metabolites but also act as lytic organelles where vacuolar proteins are post-translationally processed into mature forms or degraded by the action of vacuolar processing enzyme (VPE). We have previously characterized VPE genes (SlVPE1-5) during fruit development in tomato and discovered that the VPE enzyme activity negatively interfered with sugar accumulation in mature fruits. Comparative proteomic analysis demonstrated that acid invertase was one of the molecular targets of SlVPE5, which is involved in the hydrolysis of sucrose. This study also showed that decreased VPE enzyme activity due to suppression of SlVPE5 by RNAi strategy (RNAi-SlVPE5) accompanied with decreased enzyme activity of acid invertase. Further, we identified the enzyme activity of acid invertase was not well correlated with mRNA levels in the RNAi-SlVPE5 line. These results suggest that SlVPE5 regulates post-transcriptional processing through de novo synthesis of the acid invertase protein to suppress enzyme activity, thereby eventually ensuring sucrose hydrolysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion.

    Science.gov (United States)

    Takeda, Kozue; Cabrera, Margarita; Rohde, Jan; Bausch, Dirk; Jensen, Ole N; Ungermann, Christian

    2008-04-30

    At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6 and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion.

  16. The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

    DEFF Research Database (Denmark)

    Takeda, Kozue; Cabrera, Margarita; Rohde, Jan

    2008-01-01

    At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate...... Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6...

  17. Gene expression of H+-pumps in plasma and vacuolar membranes of corn root cells under the effect of sodium ions and bioactive preparations.

    Science.gov (United States)

    Kovalenko, N O; Palladina, T A

    2016-01-01

    Four isoforms of H+-ATPase of plasma membrane: MHA1, MHA2, MHA3, MHA4 are expressed in the corn seedling roots with prevalence of genes MHA3 і MHA4. The exposure of seedlings in the presence of 0.1 M NaCl activated the expression of MHA4 gene isoform, that demonstrates its important role in the processes of adaptation to salinization conditions. In vacuolar membrane, where potential is created by two Н+-pumps, sodium ions activated gene expression of only Н+-АТРase of V-type, taking no effect on the expression of Н+-pyrophosphatase. The seeds pretreatment by synthetic preparations Methyure and Ivine did not affect gene expression of Н+-pumps. Thus we can suppose that the ability of the above preparations to activate functioning of Н+-pumps in the presence of sodium ions is realized at the post-tranlation level.

  18. cDNA sequence and expression of subunit E of the vacuolar H(+)-ATPase in the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum.

    Science.gov (United States)

    Dietz, K J; Arbinger, B

    1996-06-11

    A cDNA coding for subunit E of the vacuolar H(+)-ATPase was cloned from Mesembryanthemum crystallinum, a plant which switches from C3-photosynthesis to Crassulacean acid metabolism under saline growth conditions. Sequence homology between the three subunit E-polypeptides of different higher plant species varied between 77.6 and 73.3%; peptide length was between 226 and 230 amino acid residues, 43 of which are invariant in all seven subunit E-polypeptides known so far from animals, fungi and plants. The deduced relative molecular mass of subunit E in Mesembryanthemum crystallinum is 26162 Da. Subunit E is present both in C3- and CAM-plants. mRNA levels increased severalfold in leaves of CAM-induced plants. This was accompanied by a less pronounced increase in subunit E protein. Obviously, expression is stimulated under conditions of increased requirement for tonoplast H(+)-pumping activity.

  19. Mathematical Modeling of the Central Carbohydrate Metabolism in Arabidopsis Reveals a Substantial Regulatory Influence of Vacuolar Invertase on Whole Plant Carbon Metabolism1[W

    Science.gov (United States)

    Nägele, Thomas; Henkel, Sebastian; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G.

    2010-01-01

    A mathematical model representing metabolite interconversions in the central carbohydrate metabolism of Arabidopsis (Arabidopsis thaliana) was developed to simulate the diurnal dynamics of primary carbon metabolism in a photosynthetically active plant leaf. The model groups enzymatic steps of central carbohydrate metabolism into blocks of interconverting reactions that link easily measurable quantities like CO2 exchange and quasi-steady-state levels of soluble sugars and starch. When metabolite levels that fluctuate over diurnal cycles are used as a basic condition for simulation, turnover rates for the interconverting reactions can be calculated that approximate measured metabolite dynamics and yield kinetic parameters of interconverting reactions. We used experimental data for Arabidopsis wild-type plants, accession Columbia, and a mutant defective in vacuolar invertase, AtβFruct4, as input data. Reducing invertase activity to mutant levels in the wild-type model led to a correct prediction of increased sucrose levels. However, additional changes were needed to correctly simulate levels of hexoses and sugar phosphates, indicating that invertase knockout causes subsequent changes in other enzymatic parameters. Reduction of invertase activity caused a decline in photosynthesis and export of reduced carbon to associated metabolic pathways and sink organs (e.g. roots), which is in agreement with the reported contribution of vacuolar invertase to sink strength. According to model parameters, there is a role for invertase in leaves, where futile cycling of sucrose appears to have a buffering effect on the pools of sucrose, hexoses, and sugar phosphates. Our data demonstrate that modeling complex metabolic pathways is a useful tool to study the significance of single enzyme activities in complex, nonintuitive networks. PMID:20207708

  20. Mathematical modeling of the central carbohydrate metabolism in Arabidopsis reveals a substantial regulatory influence of vacuolar invertase on whole plant carbon metabolism.

    Science.gov (United States)

    Nägele, Thomas; Henkel, Sebastian; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G

    2010-05-01

    A mathematical model representing metabolite interconversions in the central carbohydrate metabolism of Arabidopsis (Arabidopsis thaliana) was developed to simulate the diurnal dynamics of primary carbon metabolism in a photosynthetically active plant leaf. The model groups enzymatic steps of central carbohydrate metabolism into blocks of interconverting reactions that link easily measurable quantities like CO(2) exchange and quasi-steady-state levels of soluble sugars and starch. When metabolite levels that fluctuate over diurnal cycles are used as a basic condition for simulation, turnover rates for the interconverting reactions can be calculated that approximate measured metabolite dynamics and yield kinetic parameters of interconverting reactions. We used experimental data for Arabidopsis wild-type plants, accession Columbia, and a mutant defective in vacuolar invertase, AtbetaFruct4, as input data. Reducing invertase activity to mutant levels in the wild-type model led to a correct prediction of increased sucrose levels. However, additional changes were needed to correctly simulate levels of hexoses and sugar phosphates, indicating that invertase knockout causes subsequent changes in other enzymatic parameters. Reduction of invertase activity caused a decline in photosynthesis and export of reduced carbon to associated metabolic pathways and sink organs (e.g. roots), which is in agreement with the reported contribution of vacuolar invertase to sink strength. According to model parameters, there is a role for invertase in leaves, where futile cycling of sucrose appears to have a buffering effect on the pools of sucrose, hexoses, and sugar phosphates. Our data demonstrate that modeling complex metabolic pathways is a useful tool to study the significance of single enzyme activities in complex, nonintuitive networks.

  1. Evolutionary divergence of plant borate exporters and critical amino acid residues for the polar localization and boron-dependent vacuolar sorting of AtBOR1

    KAUST Repository

    Wakuta, Shinji

    2015-01-24

    Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  2. Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

    Science.gov (United States)

    Wakuta, Shinji; Mineta, Katsuhiko; Amano, Taro; Toyoda, Atsushi; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

    2015-05-01

    Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. Mielite transversa como manifestação clínica inicial de linfoma não Hodgkin disseminado e mielopatia vacuolar associada ao HIV: relato de caso Transverse myelitis as initial symptom of disseminated non-Hodgkin lymphoma and HIV-associated vacuolar myelopathy: case report

    Directory of Open Access Journals (Sweden)

    Leandro P. de Moura

    1996-06-01

    Full Text Available Linfomas não Hodgkin de alto grau são comumente relatados em pacientes com a síndrome da imunodeficiência adquirida (AIDS. Comprometendo com grande freqüência o sistema nervoso central, particularmente as leptomeninges e os hemisférios cerebrais. O acometimento epidural é pouco freqüente, variando de 3,5% a 8,3% de acordo com os registros da literatura. Os autores relatam o caso de um paciente de 27 anos de idade com AIDS, cuja manifestação clínica inicial da doença linfomatosa disseminada foi a mielite transversa associada à mielopatia vacuolar. Destaca-se a importância do diagnóstico diferencial precoce das mielopatias na AIDS, em virtude da alta malignidade da neoplasia e da evolução extremamente rápida nesses pacientes.Non-Hodgkin lymphoma is frequently seen in AIDS patients usually affecting the central nervous system (CNS, especially the leptomeninges and the cerebral hemispheres. The epidural involvement is rarely described, ranging from 3.5% to 8.3% among the CNS sites. The authors present a case of disseminated non Hodgkin lymphoma associated to vacuolar myelopathy in a 27 years-old male patient with AIDS emphasizing the importance of this differential diagnosis in the myelopathies of AIDS.

  4. Retraction: 'An ~400 kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4' by Susana Rivas, Tatiana Mucyn, Harrold A. van den Burg, Jacques Vervoort and Jonathan D. G. Jones.

    Science.gov (United States)

    2017-06-01

    The above article, published online on 26 March 2002 in Wiley Online Library (wileyonlinelibrary.com), and in Volume 29, pp. 783-796 has been retracted by agreement between the authors, the journal Editor-in-Chief, Christoph Benning, and John Wiley & Sons Ltd. The authors have requested that it be retracted because it contains a substantial number of inappropriate image manipulations. These manipulations consist of the reuse or duplication of images, both within the above article and with another manuscript published previously in The Plant Cell (Rivas et al., 2002b). Specific problems with the manuscripts include: The duplication of immunoblot bands in The Plant Journal Figure 4C and The Plant Cell Figure 3 row 3, The Plant Cell Figure 4A row 1, The Plant Cell Figure 4C. The Plant Journal Figure 4B Cf-9 c-myc band is duplicated in The Plant Journal Figure 4C Cf-9:TAP band under the 475 kDa marker. The first four lanes of Figure 4A row 3 in The Plant Journal are a duplication of lanes 23, 24, 25, and 26 in Figure 4A row 2 in The Plant Cell. The Plant Journal Figure 7B lanes 1&2 and lanes 4&5 are duplicates of each other and are duplicated from Figure 7B lanes 1 and 2 in The Plant Cell. The Plant Journal Figure 7C is a duplication from Figure 7C in The Plant Cell. Rivas, S., Mucyn, T., van den Burg, H.A., Vervoort, J. and Jones, J.D.G. (2002a) An ∼400 kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4. Plant J. 29, 783-796. https://doi.org/10.1046/j.1365-313X.2002.01254.x. Rivas, S., Romeis, T. and Jones, J.D.G. (2002b) The Cf-9 disease resistance protein is present in an ∼420-Kilodalton heteromultimeric membrane-associated complex at one molecule per complex. Plant Cell, 14, 689-702. https://doi.org/10.1105/tpc.010357. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  5. Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

    Science.gov (United States)

    Sepúlveda-González, M Eugenia; Parra-Ortega, Berenice; Betancourt-Cervantes, Yuliana; Hernández-Rodríguez, César; Xicohtencatl-Cortes, Juan; Villa-Tanaca, Lourdes

    2016-01-01

    The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen. Copyright © 2014 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  6. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    Science.gov (United States)

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

    Science.gov (United States)

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-02-03

    Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

  8. New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion.

    Science.gov (United States)

    Wurmser, A E; Sato, T K; Emr, S D

    2000-10-30

    The class C subset of vacuolar protein sorting (Vps) proteins (Vps11, Vps18, Vps16 and Vps33) assembles into a vacuole/prevacuole-associated complex. Here we demonstrate that the class C-Vps complex contains two additional proteins, Vps39 and Vps41. The COOH-terminal 148 amino acids of Vps39 direct its association with the class C-Vps complex by binding to Vps11. A previous study has shown that a large protein complex containing Vps39 and Vps41 functions as a downstream effector of the active, GTP-bound form of Ypt7, a rab GTPase required for the fusion of vesicular intermediates with the vacuole (Price, A., D. Seals, W. Wickner, and C. Ungermann. 2000. J. Cell Biol. 148:1231-1238). Here we present data that indicate that this complex also functions to stimulate nucleotide exchange on Ypt7. We show that Vps39 directly binds the GDP-bound and nucleotide-free forms of Ypt7 and that purified Vps39 stimulates nucleotide exchange on Ypt7. We propose that the class C-Vps complex both promotes Vps39-dependent nucleotide exchange on Ypt7 and, based on the work of Price et al., acts as a Ypt7 effector that tethers transport vesicles to the vacuole. Thus, the class C-Vps complex directs multiple reactions during the docking and fusion of vesicles with the vacuole, each of which contributes to the overall specificity and efficiency of this transport process.

  9. In Vivo 31P-Nuclear Magnetic Resonance Studies of Glyphosate Uptake, Vacuolar Sequestration, and Tonoplast Pump Activity in Glyphosate-Resistant Horseweed1[W

    Science.gov (United States)

    Ge, Xia; d’Avignon, D. André; Ackerman, Joseph J.H.; Sammons, R. Douglas

    2014-01-01

    Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. 31P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. 31P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells. PMID:25185124

  10. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants

    Science.gov (United States)

    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-01-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na+/H+) antiporter that transports Na+ into the vacuole and exports H+ into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na+/H+ antiporter that exports Na+ to the extracellular space and imports H+ into the plant cell. Plants rely on these enzymes either to keep Na+ out of the cell or to sequester Na+ into vacuoles to avoid the toxic level of Na+ in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. PMID:26985021

  11. Disruption of the vacuolar-type H(+)-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

    Science.gov (United States)

    Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul

    2017-04-03

    The vacuolar-type H(+)-translocating ATPase (v-H(+)-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H(+)-ATPase and MTORC1, we destablilized v-H(+)-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H(+)-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H(+)-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H(+)-ATPase-mediated regulation of MTORC1.

  12. A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes.

    Directory of Open Access Journals (Sweden)

    Shigeaki Matsuda

    Full Text Available Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1. VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H(+-ATPase (V-ATPase, the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC, which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.

  13. Evidence That High Activity of Vacuolar Invertase Is Required for Cotton Fiber and Arabidopsis Root Elongation through Osmotic Dependent and Independent Pathways, Respectively1[C][W][OA

    Science.gov (United States)

    Wang, Lu; Li, Xiao-Rong; Lian, Heng; Ni, Di-An; He, Yu-ke; Chen, Xiao-Ya; Ruan, Yong-Ling

    2010-01-01

    Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively. PMID:20699399

  14. Silencing the vacuolar invertase gene GhVIN1 blocks cotton fiber initiation from the ovule epidermis, probably by suppressing a cohort of regulatory genes via sugar signaling.

    Science.gov (United States)

    Wang, Lu; Cook, Akiko; Patrick, John W; Chen, Xiao-Ya; Ruan, Yong-Ling

    2014-05-01

    Cotton fibers, the most important source of cellulose for the global textile industry, are single-celled trichomes derived from the ovule epidermis at or just prior to anthesis. Despite progress in understanding cotton fiber elongation and cell-wall biosynthesis, knowledge regarding the molecular basis of fiber cell initiation, the first step of fiber development determining the fiber yield potential, remains elusive. Here, we provide evidence that expression of a vacuolar invertase (VIN) is an early event that is essential for cotton fiber initiation. RNAi-mediated suppression of GhVIN1, a major VIN gene that is highly expressed in wild-type fiber initials, resulted in significant reduction of VIN activity and consequently a fiberless seed phenotype in a dosage dependent manner. The absence of a negative effect on seed development in these fiberless seeds indicates that the phenotype is unlikely to be due to lack of carbon nutrient. Gene expression analyses coupled with in vitro ovule culture experiments revealed that GhVIN1-derived hexose signaling may play an indispensable role in cotton fiber initiation, probably by regulating the transcription of several MYB transcription factors and auxin signaling components that were previously identified as required for fiber initiation. Together, the data represent a significant advance in understanding the mechanisms of cotton fiber initiation, and provide the first indication that VIN-mediated hexose signaling may act as an early event modulating the expression of regulatory genes and hence cell differentiation from the ovule epidermis. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  15. Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

    Science.gov (United States)

    Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen

    2010-03-01

    The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

  16. The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex.

    Science.gov (United States)

    Stroupe, Christopher

    2012-04-01

    A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.

  17. Alternative expression of vacuolar iron transporter and ferritin genes leads to blue/purple coloration of flowers in tulip cv. 'Murasakizuisho'.

    Science.gov (United States)

    Shoji, Kazuaki; Momonoi, Kazumi; Tsuji, Tosiaki

    2010-02-01

    Flowers of tulip cv. 'Murasakizuisho' have a purple perianth except for the bottom region, which is blue in color even though it has the same anthocyanin, delphinidin 3-O-rutinoside, as the entire perianth. The development of the blue coloration in the perianth bottom is due to complexation by anthocyanin, flavonol and iron (Fe), as well as a vacuolar iron transporter, TgVit1. Although transient expression of TgVit1 in the purple cells led to a color change to light blue, the coloration of the transformed cells did not coincide with the dark blue color of the cells of the perianth bottom. We thought that another factor is required for the blue coloration of the cells of perianth bottom. To examine the effect of ferritin (FER), an Fe storage protein, on blue color development, we cloned an FER gene (TgFER1) and performed expression analyses. TgFER1 transcripts were found in the cells located in the upper region of the petals along with purple color development by anthocyanin and were not found in the blue cells of the perianth bottom. This gene expression is in contrast to that of TgVit1, expressed only in the cells of the perianth bottom. Co-expression of TgVIT1 and TgFER-RNAi, constructed for suppressing endogenous TgFER1 by RNA interference (RNAi), changed the purple petal cells to a dark blue color similar to that of the natural perianth bottom. These results strongly suggest that TgVit1 expression and TgFER1 suppression are critical for the development of blue color in the perianth bottom.

  18. Specific expression of the vacuolar iron transporter, TgVit, causes iron accumulation in blue-colored inner bottom segments of various tulip petals.

    Science.gov (United States)

    Momonoi, Kazumi; Tsuji, Toshiaki; Kazuma, Kohei; Yoshida, Kumi

    2012-01-01

    Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.

  19. The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

    Science.gov (United States)

    Castro, Mariana M; Kim, Bongki; Hill, Eric; Fialho, Maria C Q; Puga, Luciano C H P; Freitas, Mariella B; Breton, Sylvie; Machado-Neves, Mariana

    2017-01-01

    Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

  20. Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

    Science.gov (United States)

    Uchiyama, M; Komiyama, M; Yoshizawa, H; Shimizu, N; Konno, N; Matsuda, K

    2012-05-01

    The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the

  1. Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral organs.

    Science.gov (United States)

    Tyagi, Wricha; Rajagopal, Divya; Singla-Pareek, Sneh Lata; Reddy, Malireddy K; Sopory, Sudhir K

    2005-08-01

    We have cloned and characterized the cDNA, genomic clone and upstream promoter region of a vacuolar ATPase (V-ATPase) c subunit (PgVHA-c1) from Pennisetum glaucum. The deduced amino acid sequence shows 98-71% sequence identity with V-ATPase from rice and Arabidopsis, and is a highly hydrophobic protein with four transmembrane regions. PgVHA-c1-GFP fusion protein is expressed in BY2 cells on the endo-membranes surrounding vacuoles; however, PgVHA-c1 could not functionally complement V-ATPase-c deletion mutants of yeast. The sequence analysis of the genomic clone revealed the presence of two introns in the coding region, and the splice junctions followed the typical canonical GU-AG consensus sequence. The transcript analysis showed that the expression of PgVHA-c1 was stimulated more in response to salinity stress and very marginally in response to drought and low temperature stress. Exogenous application of abscisic acid, salicylic acid and calcium stimulated the transcript level in the absence of stress. We have cloned the 5'-flanking regions of PgVHA-c1 and mapped its transcript start site at 78 bp upstream of ATG. Transgenic tobacco with promoter::GUS constructs showed that the region -288/+78 was sufficient for GUS expression. The expression of the reporter gene even with the full-length promoter was limited to shoot hairs and to male and female reproductive organs. The dehydration-responsive element (DRE) and ABA-responsive element (ABRE) in the promoter did not show consensus flanking regions; however, gel mobility shift assays showed that Pennisetum has specific transacting factors that showed binding to the core DRE, ABRE and TCA elements.

  2. Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION.

    Science.gov (United States)

    Stam, Nicholas J; Wilkens, Stephan

    2017-02-03

    Eukaryotic vacuolar H + -ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V 1 -ATPase and membrane-integral V o proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V o The structure indicated that, when V 1 is released from V o , the N-terminal cytoplasmic domain of subunit a (a NT ) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a NT -d interface. Here we reconstituted V o into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a NT and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a NT -d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V o revealed that V o is halted in rotational state 3. Combined with our prior work that showed autoinhibited V 1 -ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V 1 and V o functions to prevent unintended reassembly of holo V-ATPase when activity is not needed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Vacuolar ATPase ‘a2’ isoform exhibits distinct cell surface accumulation and modulates matrix metalloproteinase activity in ovarian cancer

    Science.gov (United States)

    Kulshrestha, Arpita; Katara, Gajendra K.; Ibrahim, Safaa; Pamarthy, Sahithi; Jaiswal, Mukesh K.; Sachs, Alice Gilman; Beaman, Kenneth D.

    2015-01-01

    Tumor associated vacuolar H+-ATPases (V-ATPases) are multi-subunit proton pumps that acidify tumor microenvironment, thereby promoting tumor invasion. Subunit ‘a’ of its V0 domain is the major pH sensing unit that additionally controls sub-cellular targeting of V-ATPase and exists in four different isoforms. Our study reports an elevated expression of the V-ATPase-V0a2 isoform in ovarian cancer(OVCA) tissues and cell lines(A2780, SKOV-3 and TOV-112D). Among all V0’a’ isoforms, V0a2 exhibited abundant expression on OVCA cell surface while normal ovarian epithelia did not. Sub-cellular distribution of V-ATPase-V0a2 confirmed its localization on plasma-membrane, where it was also co-associated with cortactin, an F-actin stabilizing protein at leading edges of cancer cells. Additionally, V0a2 was also localized in early and late endosomal compartments that are sites for modulations of several signaling pathways in cancer. Targeted inhibition of V-ATPase-V0a2 suppressed matrix metalloproteinase activity(MMP-9 & MMP-2) in OVCA cells. In conclusion, V-ATPase-V0a2 isoform is abundantly expressed on ovarian tumor cell surface in association with invasion assembly related proteins and plays critical role in tumor invasion by modulating the activity of matrix-degrading proteases. This study highlights for the first time, the importance of V-ATPase-V0a2 isoform as a distinct biomarker and possible therapeutic target for treatment of ovarian carcinoma. PMID:25686833

  4. Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought.

    Science.gov (United States)

    Li, Xiaojuan; Guo, Chengjin; Gu, Juntao; Duan, Weiwei; Zhao, Miao; Ma, Chunying; Du, Xiaoming; Lu, Wenjing; Xiao, Kai

    2014-02-01

    Establishing crop cultivars with strong tolerance to P and N deprivation, high salinity, and drought is an effective way to improve crop yield and promote sustainable agriculture worldwide. A vacuolar H+-pyrophosphatase (V-H+-PPase) gene in wheat (TaVP) was functionally characterized in this study. TaVP cDNA is 2586-bp long and encodes a 775-amino-acid polypeptide that contains 10 conserved membrane-spanning domains. Transcription of TaVP was upregulated by inorganic phosphate (Pi) and N deprivation, high salinity, and drought. Transgene analysis revealed that TaVP overexpression improved plant growth under normal conditions and specifically under Pi and N deprivation stresses, high salinity, and drought. The improvement of growth of the transgenic plants was found to be closely related to elevated V-H+-PPase activities in their tonoplasts and enlarged root systems, which possibly resulted from elevated expression of auxin transport-associated genes. TaVP-overexpressing plants showed high dry mass, photosynthetic efficiencies, antioxidant enzyme activities, and P, N, and soluble carbohydrate concentrations under various growth conditions, particularly under the stress conditions. The transcription of phosphate and nitrate transporter genes was not altered in TaVP-overexpressing plants compared with the wild type, suggesting that high P and N concentrations regulated by TaVP were caused by increased root absorption area instead of alteration of Pi and NO3- acquisition kinetics. TaVP is important in the tolerance of multiple stresses and can serve as a useful genetic resource to improve plant P- and N-use efficiencies and to increase tolerance to high salinity and drought.

  5. Evidence that high activity of vacuolar invertase is required for cotton fiber and Arabidopsis root elongation through osmotic dependent and independent pathways, respectively.

    Science.gov (United States)

    Wang, Lu; Li, Xiao-Rong; Lian, Heng; Ni, Di-An; He, Yu-ke; Chen, Xiao-Ya; Ruan, Yong-Ling

    2010-10-01

    Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively.

  6. Impact of AtNHX1, a vacuolar Na+/H+ antiporter, upon gene expression during short- and long-term salt stress in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Blumwald Eduardo

    2007-04-01

    Full Text Available Abstract Background AtNHX1, the most abundant vacuolar Na+/H+ antiporter in Arabidopsis thaliana, mediates the transport of Na+ and K+ into the vacuole, influencing plant development and contributing to salt tolerance. In this report, microarray expression profiles of wild type plants, a T-DNA insertion knockout mutant of AtNHX1 (nhx1, and a 'rescued' line (NHX1::nhx1 were exposed to both short (12 h and 48 h and long (one and two weeks durations of a non-lethal salt stress to identify key gene transcripts associated with the salt response that are influenced by AtNHX1. Results 147 transcripts showed both salt responsiveness and a significant influence of AtNHX1. Fifty-seven of these genes showed an influence of the antiporter across all salt treatments, while the remaining genes were influenced as a result of a particular duration of salt stress. Most (69% of the genes were up-regulated in the absence of AtNHX1, with the exception of transcripts encoding proteins involved with metabolic and energy processes that were mostly down-regulated. Conclusion While part of the AtNHX1-influenced transcripts were unclassified, other transcripts with known or putative roles showed the importance of AtNHX1 to key cellular processes that were not necessarily limited to the salt stress response; namely calcium signaling, sulfur metabolism, cell structure and cell growth, as well as vesicular trafficking and protein processing. Only a small number of other salt-responsive membrane transporter transcripts appeared significantly influenced by AtNHX1.

  7. Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly.

    Science.gov (United States)

    Chan, Chun-Yuan; Parra, Karlett J

    2014-07-11

    V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V1 domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field

    KAUST Repository

    Schilling, Rhiannon K.

    2013-11-22

    Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H+-PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high-throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse-grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mm NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild-type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse- or field-grown plants. This study validates our greenhouse-based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  9. ALTERNATE FOOD-CHAIN TRANSFER OF THE TOXIN LINKED TO AVIAN VACUOLAR MYELINOPATHY AND IMPLICATIONS FOR THE ENDANGERED FLORIDA SNAIL KITE (ROSTRHAMUS SOCIABILIS).

    Science.gov (United States)

    Dodd, Shelley R; Haynie, Rebecca S; Williams, Susan M; Wilde, Susan B

    2016-04-28

    Avian vacuolar myelinopathy (AVM) is a neurologic disease causing recurrent mortality of Bald Eagles ( Haliaeetus leucocephalus ) and American Coots ( Fulica americana ) at reservoirs and small impoundments in the southern US. Since 1994, AVM is considered the cause of death for over 170 Bald Eagles and thousands of American Coots and other species of wild birds. Previous studies link the disease to an uncharacterized toxin produced by a recently described cyanobacterium, Aetokthonos hydrillicola gen. et sp. nov. that grows epiphytically on submerged aquatic vegetation (SAV). The toxin accumulates, likely in the gastrointestinal tract of waterbirds that consume SAV, and birds of prey are exposed when feeding on the moribund waterbirds. Aetokthonos hydrillicola has been identified in all reservoirs where AVM deaths have occurred and was identified growing abundantly on an exotic SAV hydrilla ( Hydrilla verticillata ) in Lake Tohopekaliga (Toho) in central Florida. Toho supports a breeding population of a federally endangered raptor, the Florida Snail Kite ( Rostrhamus sociabilis ) and a dense infestation of an exotic herbivorous aquatic snail, the island applesnail ( Pomacea maculata ), a primary source of food for resident Snail Kites. We investigated the potential for transmission in a new food chain and, in laboratory feeding trials, confirmed that the AVM toxin was present in the hydrilla/A. hydrillicola matrix collected from Toho. Additionally, laboratory birds that were fed apple snails feeding on hydrilla/A. hydrillicola material from a confirmed AVM site displayed clinical signs (3/5), and all five developed brain lesions unique to AVM. This documentation of AVM toxin in central Florida and the demonstration of AVM toxin transfer through invertebrates indicate a significant risk to the already diminished population of endangered Snail Kites.

  10. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  11. StInvInh2 as an inhibitor of StvacINV1 regulates the cold-induced sweetening of potato tubers by specifically capping vacuolar invertase activity.

    Science.gov (United States)

    Liu, Xun; Lin, Yuan; Liu, Jun; Song, Botao; Ou, Yongbin; Zhang, Huiling; Li, Meng; Xie, Conghua

    2013-06-01

    Reducing sugar (RS) accumulation in cold-stored potato tubers, known as cold-induced sweetening (CIS), is a crucial factor causing unacceptable colour changes and acrylamide formation of fried products. The activity of vacuolar invertase (StvacINV1) is proved important for the CIS process, and invertase inhibitors are speculated to play roles in the post-translational regulation of StvacINV1 activity. In our previous research, two putative inhibitors (StInvInh2A and StInvInh2B) of StvacINV1 were implied to be involved in potato CIS. Here, we further reported that StInvInh2A and StInvInh2B had similar function that specifically inhibited StvacINV1 activity in potatoes. The genetic transformation of these inhibitor genes in potatoes by overexpression in CIS-sensitive and RNAi-silenced in CIS-resistant genotypes showed that StvacINV1 activity was strongly regulated by alteration of the transcripts of the inhibitors without impacting on the expression of StvacINV1. A negative power relationship was found between the transcripts of the inhibitors and StvacINV1 activity, suggesting 1) a transcriptional determination of the inhibitory capacity of StInvInh2A and StInvInh2B and 2) a significant inhibitory role of these inhibitors in post-translational modulation of StvacINV1. The results also demonstrated that depression of StvacINV1 activity through overexpression of StInvInh2A and StInvInh2B weakened accumulation of RS and acrylamide in cold-stored tubers and consequently improved the chip quality. The present research strongly suggest that both StInvInh2A and StInvInh2B function as inhibitors of StvacINV1 and play similar roles in regulating potato CIS by capping StvacINV1 activity. These inhibitors could be novel genetic resources applicable for improving quality of potato processing products. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  12. Expression of a2 Vacuolar ATPase in Spermatozoa is Associated with Semen Quality and Chemokine-Cytokine Profiles in Infertile Men

    Science.gov (United States)

    Ota, Kuniaki; Jaiswal, Mukesh Kumar; Ramu, Sivakumar; Jeyendran, Rajasinjham; Kwak-Kim, Joanne; Gilman-Sachs, Alice; Beaman, Kenneth D.

    2013-01-01

    Background A number of laboratory tests have been developed to determine properties of spermatozoa quality but few have been adopted into routine clinical use in place of the WHO semen analysis. We investigated whether Atp6v0a2 (a2 isoform of vacuolar ATPase) is associated with abnormal semen quality and changes in chemokine-cytokine profiles in infertile men. Patients and Methods Semen samples were collected from 35 healthy donors and 35 infertile men at the Andrology laboratory from August 2011 to June 2012. The levels of Atp6v0a2 mRNA and protein, and its localization in spermatozoa were determined. a2NTD (the N-terminal portion of Atp6v0a2) and secreted chemokine-cytokine profiles in seminal fluid were measured. Results Atp6v0a2 protein (P<0.05) and mRNA (P<0.05) in spermatozoa from infertile men were significantly lower than those from fertile men. Fluorescent microscopy revealed that Atp6v0a2 is mainly expressed in the acrosomal region. Infertile men’s seminal fluid had significantly lower G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) levels when compared to the seminal fluid from fertile men. Seminal fluid a2NTD levels were significantly correlated with G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) which are key molecules during the onset of pregnancy. Conclusion These results suggested that a critical level of Atp6v0a2 is required for the fertile spermatozoa and its decreased level in spermatozoa could be used to predict male infertility. This study provides a possibility that Atp6v0a2 could be potentially used as a diagnostic marker for the evaluation of male infertility. PMID:23936208

  13. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François, E-mail: francois.marceau@crchul.ulaval.ca

    2013-07-15

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion

  14. Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase.

    Science.gov (United States)

    Yeung, Ching-Hei; Breton, Sylvie; Setiawan, Iwan; Xu, Yaoxian; Lang, Florian; Cooper, Trevor G

    2004-06-01

    Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO. Copyright 2004 Wiley-Liss, Inc.

  15. Enhanced salt tolerance conferred by the complete 2.3 kb cDNA of the rice vacuolar Na+/H+antiporter gene compared to 1.9 kb coding region with 5' UTR in transgenic lines of Rice

    Directory of Open Access Journals (Sweden)

    U.S. Mahzabin eAmin

    2016-01-01

    Full Text Available Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1 from the rice landrace (Pokkali and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5ʹ UTR with CDS and the other of 2.3 kb, including 5ʹ UTR, CDS and the 3ʹ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5ʹ and 3ʹ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P 1.9 kb> and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P<0.01 compared respectively to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

  16. Sugar metabolism, chip color, invertase activity, and gene expression during long-term cold storage of potato (Solanum tuberosum) tubers from wild-type and vacuolar invertase silencing lines of Katahdin.

    Science.gov (United States)

    Wiberley-Bradford, Amy E; Busse, James S; Jiang, Jiming; Bethke, Paul C

    2014-11-16

    Storing potato tubers at low temperatures minimizes sprouting and disease but can cause an accumulation of reducing sugars in a process called cold-induced sweetening. Tubers with increased amounts of reducing sugars produce dark-colored, bitter-tasting fried products with elevated amounts of acrylamide, a possible carcinogen. Vacuolar invertase (VInv), which converts sucrose produced by starch breakdown to glucose and fructose, is the key determinant of reducing sugar accumulation during cold-induced sweetening. In this study, wild-type tubers and tubers in which VInv expression was reduced by RNA interference were used to investigate time- and temperature-dependent changes in sugar contents, chip color, and expression of VInv and other genes involved in starch metabolism in tubers during long-term cold storage. VInv activities and tuber reducing sugar contents were much lower, and tuber sucrose contents were much higher, in transgenic than in wild-type tubers stored at 3-9°C for up to eight months. Large differences in VInv mRNA accumulation were not observed at later times in storage, especially at temperatures below 9°C, so differences in invertase activity were likely established early in the storage period and maintained by stability of the invertase protein. Sugar contents, chip color, and expression of several of the studied genes, including AGPase and GBSS, were affected by storage temperature in both wild-type and transgenic tubers. Though transcript accumulation for other sugar-metabolism genes was affected by storage temperature and duration, it was essentially unaffected by invertase silencing and altered sugar contents. Differences in stem- and bud-end sugar contents in wild-type and transgenic tubers suggested different compartmentalization of sucrose at the two ends of stored tubers. VInv silencing significantly reduced cold-induced sweetening in stored potato tubers, likely by means of differential VInv expression early in storage. Transgenic

  17. Membrane-associated signaling in human B-lymphoma lines

    Energy Technology Data Exchange (ETDEWEB)

    Tauzin, Sebastien; Ding, Heidrun; Burdevet, Dimitri [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Borisch, Bettina [Department of Social and Preventive Medicine, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland); Hoessli, Daniel C., E-mail: danielhoessli@gmail.com [Department of Pathology and Immunology, Centre medical universitaire, 1, rue Michel-Servet, 1211 Geneva 11 (Switzerland)

    2011-01-15

    In B-non-Hodgkin lymphomas, Lyn and Cbp/PAG constitute the core of an oncogenic signalosome that captures the Phosphatidylinositol-3-kinase, the Spleen tyrosine kinase and the Signal transducer and activator of transcription-3 to generate pro-survival and proliferative signals. Lymphoma lines corresponding to follicular, mantle-cell and Burkitt-derived lymphomas display type-specific signalosome organizations that differentially activate PI3K, Syk and STAT3. In the follicular lymphoma line, PI3K, Syk and STAT3 were optimally activated upon association with the Lyn-Cbp/PAG signalosome, while in the Burkitt lymphoma-derived line, the association with Cbp/PAG and activation of PI3K were interfered with by the latent membrane proteins encoded by the Epstein-Barr virus. In the Jeko-1 mantle-cell line, a weak association of Syk with the Lyn-Cbp/PAG signalosome resulted in poor activation of Syk, but in those cells, as in the follicular and Burkitt-derived lines, efficient apoptosis induction by the Syk inhibitor R406 indicated that Syk is nonetheless an important prosurvival element and therefore a valuable therapeutic target. In all configurations described herein is the Lyn-Cbp/PAG signalosome independent of external signals and provides efficient means of activation for its associated lipid and protein kinases. In follicular and Burkitt-derived lines, Syk appears to be activated following binding to Cbp/PAG and no longer requires B-cell receptor-associated activation motifs for activation. Assessment of the different modalities of Lyn-Cbp/PAG signalosome organization could help in selecting the appropriate combination of kinase inhibitors to eliminate a particular type of lymphoma cells.

  18. Trichophyton rubrum secreted and membrane-associated carboxypeptidases

    National Research Council Canada - National Science Library

    Zaugg, Christophe; Jousson, Olivier; Léchenne, Barbara; Staib, Peter; Monod, Michel

    2008-01-01

    .... Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA...

  19. Vacuolar-type H+-ATPase and Na+, K+-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

    DEFF Research Database (Denmark)

    Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo

    2001-01-01

    Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days......-subunit mRNA levels, although not to the same degree as hypercapnic treatment alone. Hyperoxia generally increased Na+, K+-ATPase alpha- and beta-subunit mRNA levels, whereas hypercapnia reduced mRNA levels in presmolts (beta) and smolts (alpha and beta). Despite these changes in mRNA levels, whole tissue...... response in order to minimise intracellular HCO3- formation in epithelial cells. Udgivelsesdato: 2001-Dec...

  20. Metabolism of the vacuolar pathogen Legionella and implications for virulence

    Directory of Open Access Journals (Sweden)

    Christian eManske

    2014-09-01

    Full Text Available Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called Legionnaires’ disease. To establish its intracellular niche termed the Legionella-containing vacuole (LCV, L. pneumophila employs a type IV secretion system and translocates ~300 different effector proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria.Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen.

  1. Hydrophilic C terminus of Salicornia europaea vacuolar Na/H ...

    Indian Academy of Sciences (India)

    ... Li2 Yurong Wang2 3. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People's Republic of China; Yaohua High School, Tianjin, 300040, People's Republic of China ...

  2. Cloning of a vacuolar H -pryophosphatase gene from emphemeral ...

    African Journals Online (AJOL)

    aghomotsegin

    2013-12-04

    Dec 4, 2013 ... Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance.

  3. Manganese tolerance in yeasts involves polyphosphate, magnesium, and vacuolar alterations.

    Science.gov (United States)

    Ryazanova, Lubov; Zvonarev, Anton; Rusakova, Tatiana; Dmitriev, Vladimir; Kulakovskaya, Tatiana

    2016-07-01

    Basidiomycetous and ascomycetous yeast species were tested for manganese tolerance. Basidiomycetous Cryptococcus humicola, Cryptococcus terricola, Cryptococcus curvatus and ascomycetous Candida maltosa, Kluyveromyces marxianus, Kuraishia capsulata, Lindnera fabianii and Sacharomyces cerevisiae were able to grow at manganese excess (2.5 mmol/L), while the growth of basidiomycetous Rhodotorula bogoriensis was completely suppressed. The lag phase duration increased and the exponential growth rate decreased at manganese excess. The increase of cell size and enlargement of vacuoles were characteristics for the cells grown at manganese excess. The alterations in inorganic polyphosphate content and cellular localization were studied. L. fabianii, K. capsulata, C. maltosa, and Cr. humicola accumulated the higher amounts of inorganic polyphosphates, while Cr. terricola and Cr. curvatus demonstrated no such accumulation. The polyphosphate content in the cell wall tested by DAPI staining increased in all species under the study; however, this effect was more pronounced in Cr. terricola and Cr. curvatus. The accumulation of Mg(2+) in the cell wall under Mn(2+) excess was observed in Cr. humicola, Cr. curvatus and Cr. terricola. The accumulation of polyphosphate and magnesium in the cell wall was supposed to be a factor of manganese tolerance in yeasts.

  4. Metabolism of the vacuolar pathogen Legionella and implications for virulence.

    Science.gov (United States)

    Manske, Christian; Hilbi, Hubert

    2014-01-01

    Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called "Legionnaires' disease." To establish its intracellular niche termed the "Legionella-containing vacuole" (LCV), L. pneumophila employs a type IV secretion system and translocates ~300 different "effector" proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria. Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen.

  5. RNAi-based silencing of genes encoding the vacuolar- ATPase ...

    African Journals Online (AJOL)

    2016-11-09

    Nov 9, 2016 ... Searching the PROSITE database revealed no common motifs for both subunits sequences. The phylogenetic tree analysis shows that the V-ATPase subunit a of both. P. gossypiella and Tribolium castaneum are located on the same branch as Bactrocera dorsalis with 94% bootstrap support (Figure 1a).

  6. Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM).

    Science.gov (United States)

    Zhuo, Yue; Choi, Ji Sun; Marin, Thibault; Yu, Hojeong; Harley, Brendan A; Cunningham, Brian T

    2016-11-01

    Adhesion is a critical cellular process that contributes to migration, apoptosis, differentiation, and division. It is followed by the redistribution of cellular materials at the cell membrane or at the cell-surface interface for cells interacting with surfaces, such as basement membranes. Dynamic and quantitative tracking of changes in cell adhesion mass redistribution is challenging because cells are rapidly moving, inhomogeneous, and nonequilibrium objects, whose physical and mechanical properties are difficult to measure or predict. Here, we report a novel biosensor based microscopy approach termed Photonic Crystal Enhanced Microscopy (PCEM) that enables the movement of cellular materials at the plasma membrane of individual live cells to be dynamically monitored and quantitatively imaged. PCEM utilizes a photonic crystal biosensor surface, which can be coated with arbitrary extracellular matrix materials to facilitate cellular interactions, within a modified brightfield microscope with a low intensity non-coherent light source. Benefiting from the high sensitivity, narrow resonance peak, and tight spatial confinement of the evanescent field atop the photonic crystal biosensor, PCEM enables label-free live cell imaging with high sensitivity and high lateral and axial spatial-resolution, thereby allowing dynamic adhesion phenotyping of single cells without the use of fluorescent tags or stains. We apply PCEM to investigate adhesion and the early stage migration of different types of stem cells and cancer cells. By applying image processing algorithms to analyze the complex spatiotemporal information generated by PCEM, we offer insight into how the plasma membrane of anchorage dependent cells is dynamically organized during cell adhesion. The imaging and analysis results presented here provide a new tool for biologists to gain a deeper understanding of the fundamental mechanisms involved with cell adhesion and concurrent or subsequent migration events.

  7. Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica

    Directory of Open Access Journals (Sweden)

    Kawamoto Eiichi

    2011-03-01

    Full Text Available Abstract Background Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system. Results The RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA. Conclusions The results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

  8. Increased Soluble EGF after Ischemis is Accompanied by a Decrease in Membrane-Associated Precursors

    Science.gov (United States)

    1993-01-01

    1761. 1949. release of biologically active EGF from its membrane- i!i, Jorgensen. U.. E. Nexo , and S. S. Poulsen. Thv- niumnbru•nt, bound precursors...S. S. Poulsen, and E. Nexo . eral nephrectomy (21). The levels of mRNA for HGF as The urinaryv excrewi,,n of epidermal grn,,h tac or hnei . raTa, well

  9. Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM)

    Science.gov (United States)

    Zhuo, Yue; Choi, Ji Sun; Marin, Thibault; Yu, Hojeong; Harley, Brendan A.; Cunningham, Brian T.

    2017-01-01

    Adhesion is a critical cellular process that contributes to migration, apoptosis, differentiation, and division. It is followed by the redistribution of cellular materials at the cell membrane or at the cell-surface interface for cells interacting with surfaces, such as basement membranes. Dynamic and quantitative tracking of changes in cell adhesion mass redistribution is challenging because cells are rapidly moving, inhomogeneous, and nonequilibrium objects, whose physical and mechanical properties are difficult to measure or predict. Here, we report a novel biosensor based microscopy approach termed Photonic Crystal Enhanced Microscopy (PCEM) that enables the movement of cellular materials at the plasma membrane of individual live cells to be dynamically monitored and quantitatively imaged. PCEM utilizes a photonic crystal biosensor surface, which can be coated with arbitrary extracellular matrix materials to facilitate cellular interactions, within a modified brightfield microscope with a low intensity non-coherent light source. Benefiting from the high sensitivity, narrow resonance peak, and tight spatial confinement of the evanescent field atop the photonic crystal biosensor, PCEM enables label-free live cell imaging with high sensitivity and high lateral and axial spatial-resolution, thereby allowing dynamic adhesion phenotyping of single cells without the use of fluorescent tags or stains. We apply PCEM to investigate adhesion and the early stage migration of different types of stem cells and cancer cells. By applying image processing algorithms to analyze the complex spatiotemporal information generated by PCEM, we offer insight into how the plasma membrane of anchorage dependent cells is dynamically organized during cell adhesion. The imaging and analysis results presented here provide a new tool for biologists to gain a deeper understanding of the fundamental mechanisms involved with cell adhesion and concurrent or subsequent migration events. PMID:28649149

  10. Isolation and characterization of MUC15, a novel cell membrane-associated mucin

    DEFF Research Database (Denmark)

    Pallesen, Lone Tjener; Berglund, Lars; Rasmussen, Lone Kjær

    2002-01-01

    The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length c......-like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino-acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane-bound mucins, such as a serine, threonine, and proline-rich extracellular region with several...

  11. Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters

    NARCIS (Netherlands)

    Sloothaak, J.; Odoni, D.I.; Graaff, de L.H.; Martins dos Santos, V.A.P.; Schaap, P.J.; Tamayo Ramos, J.A.

    2015-01-01

    BACKGROUND: The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic

  12. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    Science.gov (United States)

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. The mechanism of membrane-associated steps in tail-anchored protein insertion

    Energy Technology Data Exchange (ETDEWEB)

    Mariappan, Malaiyalam; Mateja, Agnieszka; Dobosz, Malgorzata; Bove, Elia; Hegde, Ramanujan S.; Keenan, Robert J. (NIH); (UC)

    2012-06-19

    Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.

  14. Lanthanide Chelates as Bilayer Alignment Tools in NMR Studies of Membrane-Associated Peptides

    Science.gov (United States)

    Prosser, R. S.; Bryant, H.; Bryant, R. G.; Vold, Regitze R.

    1999-12-01

    Theequimolar complex, consisting of the lipid-like, amphiphilic chelating agent 1,11-bis[distearylamino]-diethylenetriamine pentaacetic acid (DTPA-18) and Tm3+, is shown by deuterium (2H) NMR to be useful in aligning bicelle-like model membranes, consisting of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). As shown previously (1996, R. S. Prosser et al., J. Am. Chem. Soc. 118, 269-270), in the absence of chelate, the lanthanide ions bind loosely with the lipid phosphate groups and confer the membrane with a sufficient positive magnetic anisotropy to result in parallel alignment (i.e., average bilayer normal along the field). Apparently, DTPA-18 sequesters the lanthanide ions and inserts into the phospholipid bilayer in such a manner that bilayer morphology is preserved over a wide temperature range (35-70°C). The inherent paramagnetic shifts and line broadening effects are illustrated by 2H NMR spectra of the membrane binding peptide, Leu-enkephalin (Lenk-d2, Tyr-(Gly-d2)-Gly-Phe-Leu-OH), in the presence of varying concentrations of Tm3+, and upon addition of DTPA-18. Two conclusions could be drawn from this study: (1) The addition of Tm3+ to the bicelle system is consistent with a conformational change in the surface associated peptide, and this effect is shown to be reversed by addition of the chelate, and (2) The paramagnetic shifts are shown to be significantly reduced by addition of chelate.

  15. Membrane-associated cytotoxicity induced by realgar in promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Ye, Han-Qing; Gan, Lu; Yang, Xiang-Liang; Xu, Hui-Bi

    2006-02-20

    Realgar has been shown to have a therapeutic effect against acute promyelocytic leukemia (APL) by inducing apoptosis. However, there is little data about the effects of it on plasma membrane. In the present study, the cytotoxicity of realgar to HL-60 cells including its inhibiting cell growth, inducing apoptosis and bringing about membrane toxicity was investigated. It was suggested that realgar could significantly suppress the proliferation of HL-60 cells in a dose-dependent manner by 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium bromide (MTT) assay and the IC50 value was 5.67 microM. Flow cytometric analysis revealed that treatment with realgar resulted in increased percentages of apoptotic cells in a dose-dependent manner. On the other hand, membrane lipid peroxidation level, lactate dehydrogenase (LDH) leakage and membrane surface topography alterations were investigated to assess the membrane toxicity induced by realgar. Treatment with realgar at different concentrations accelerated membrane lipid peroxidation, potentiated LDH leakage, which was consistent with enhanced disorganization of membrane surface observed by atomic force microscopy (AFM). These results suggested that such membrane toxicity induced by realgar might play an important role in the process of apoptotic induction and could be considered as one of mechanisms underlying the cytotoxicity of realgar.

  16. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  17. A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.

    Science.gov (United States)

    Huang, Weigang; Wang, Xiaoyang; Endo-Streeter, Stuart; Barrett, Matthew; Waybright, Jarod; Wohlfeld, Christian; Hajicek, Nicole; Harden, T Kendall; Sondek, John; Zhang, Qisheng

    2018-02-02

    A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-β3 by Gαq Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Acidic mist reduces foliar membrane-associated calcium and impairs stomatal responsiveness in red spruce

    Energy Technology Data Exchange (ETDEWEB)

    Borer, C. H.; DeHayes, D. H. [University of Vermont, Rubinstein School of Environment and Natural Resources, Burlington, VT (United States); Schaberg, P. G. [USDA Forest Service, Northeastern Research Station, South Burlington, VT (United States)

    2005-06-01

    The possibility of impairment of stomatal responsiveness due to acidic mist-induced depletion of foliar membrane calcium (mCa) was investigated by exposing red spruce seedlings to either pH 3.0 or pH 5.0 mist treatments for one growing season. Foliar nutrition was assessed following each treatment, and declines in stomatal conductance and net photosynthesis were measured on current year shoots following stem excision. Seedlings subjected to pH 3.0 acidic mist treatment had reduced mCa, and exhibited impaired stomatal function, including a smaller maximum aperture, slower closure, increased lag time between stomatal closure and photosynthetic decline following experimental water stress, relative to seedling treated with pH 5.0 acidic mist. The evidence supports the hypothesis that anthropogenetically caused depletion of mCa may disrupt physiological processes that depend on foliar Ca, in the process reducing the plants ability to respond adaptively to environmental stresses. 69 refs., 1 tab., 1 fig.

  19. Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shiflett, Shelly L; Ward, Diane McVey; Huynh, Dinh; Vaughn, Michael B; Simmons, Jennifer C; Kaplan, Jerry

    2004-03-19

    We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.

  20. The plasma membrane-associated NADH oxidase (ECTO-NOX) of mouse skin responds to blue light

    Science.gov (United States)

    Morre, D. James; Morre, Dorothy M.

    2003-01-01

    NADH oxidases of the external plasma membrane surface (ECTO-NOX proteins) are characterized by oscillations in activity with a regular period length of 24 min. Explants of mouse skin exhibit the oscillatory activity as estimated from the decrease in A(340) suggesting that individual ECTO-NOX molecules must somehow be induced to function synchronously. Transfer of explants of mouse skin from darkness to blue light (495 nm, 2 min, 50 micromol m(-1) s(-1)) resulted in initiation of a new activity maximum (entrainment) with a midpoint 36 min after light exposure followed by maxima every 24 min thereafter. Addition of melatonin resulted in a new maximum 24 min after melatonin addition. The findings suggest that the ECTO-NOX proteins play a central role in the entrainment of the biological clock both by light and by melatonin.

  1. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  2. Membrane-associated glucocorticoid activity Is necessary for modulation of long-term memory via chromatin modification

    NARCIS (Netherlands)

    Roozendaal, Benno; Hernandez, Angelina; Cabrera, Sara M.; Hagewoud, Roelina; Malvaez, Melissa; Stefanko, Daniel P.; Haettig, Jakob; Wood, Marcelo A.

    2010-01-01

    Glucocorticoid hormones enhance the consolidation of long-term memory of emotionally arousing training experiences. This memory enhancement requires activation of the cAMP-dependent kinase pathway and the subsequent phosphorylation of cAMP response-element binding (CREB) protein. Here, we

  3. Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Aβ and insulin degradation

    Directory of Open Access Journals (Sweden)

    Castaño Eduardo M

    2008-12-01

    Full Text Available Abstract Background Insulin degrading enzyme (IDE is implicated in the regulation of amyloid β (Aβ steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD. Although IDE sub-cellular localization has been well studied, the compartments relevant to Aβ degradation remain to be determined. Results Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Aβ and its distribution (DRMs vs. non-DRMs and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-β-cyclodextrin (MβCD, endogenous Aβ accumulated in the extracellular space and exogenous Aβ proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Aβ degradation in vitro. Conclusion Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Aβ accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Aβ degrading proteases away from DRMs may impair the physiological turn-over of Aβ in vivo deserves further investigation in light of therapeutic strategies based on enhancing Aβ proteolysis in which DRM protease-targeting may need to be taken into account.

  4. Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

    Science.gov (United States)

    Adewoye, L O; Worobec, E A

    2000-08-08

    The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

  5. Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain

    OpenAIRE

    Hansen, Majbritt; Le Nours, Jérôme; Johansson, Eva; Antal, Torben; Ullrich, Alexandra; Löffler, Monika; Larsen, Sine

    2004-01-01

    The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have sho...

  6. The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation.

    Science.gov (United States)

    D'Alessio, Flavia; Hentze, Matthias W; Muckenthaler, Martina U

    2012-11-01

    The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP-SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes. We explore protein-protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells. Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120-139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS. Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  7. Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice.

    Science.gov (United States)

    Wu, Bin; Zhang, Baocai; Dai, Yan; Zhang, Lei; Shang-Guan, Keke; Peng, Yonggang; Zhou, Yihua; Zhu, Zhen

    2012-08-01

    Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana).

  8. Protein and DNA technologies for functional expression of membrane-associated cytochromes P450 in bacterial cell factories

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario

    450 engineering guidelines and serves as platform to improve performance of microbial cells, thereby boosting recombinant production of complex plant P450-derived biochemicals. The knowledge generated, could guide future reconstruction of functional plant metabolic pathways leading to high valuable...... potential as medicines, fuels or food for humans. Plants conquered different environments thereby developing adaptation strategies based on the biosynthesis of a myriad of compounds. Unfortunately they are present in small amounts in plants and are too complex and to produce by organic chemical synthesis......The heavy dependence and massive consumption of fossil fuels by humans is changing our environment very rapidly. Some of the side effects of industrial activity include the pollution of the natural resources we rely on, and the reduction of biodiversity. Some chemicals found in nature exhibit great...

  9. The ABCG family of membrane-associated transporters: you don't have to be big to be mighty.

    Science.gov (United States)

    Kerr, Ian D; Haider, Ameena J; Gelissen, Ingrid C

    2011-12-01

    Along with many other mammalian ATP-binding cassette (ABC) transporters, members of the ABCG group are involved in the regulated transport of hydrophobic compounds across cellular membranes. In humans, five ABCG family members have been identified, encoding proteins ranging from 638 to 678 amino acids in length. All five have been the subject of intensive investigation to better understand their physiological roles, expression patterns, interactions with substrates and inhibitors, and regulation at both the transcript and protein level. The principal substrates for at least four of the ABCG proteins are endogenous and dietary lipids, with ABCG1 implicated in particular in the export of cholesterol, and ABCG5 and G8 forming a functional heterodimer responsible for plant sterol elimination from the body. ABCG2 has a much broader substrate specificity and its ability to transport numerous diverse pharmaceuticals has implications for the absorption, distribution, metabolism, excretion and toxicity (ADMETOx) profile of these compounds. ABCG2 is one of at least three so-called multidrug resistant ABC transporters expressed in humans, and its activity is associated with decreased efficacy of anti-cancer agents in several carcinomas. In addition to its role in cancer, ABCG2 also plays a role in the normal physiological transport of urate and haem, the implications of which are described. We summarize here data on all five human ABCG transporters and provide a current perspective on their roles in human health and disease. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  10. Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria

    Directory of Open Access Journals (Sweden)

    Aravind L

    2003-08-01

    Full Text Available Abstract Background A great diversity of multi-pass membrane receptors, typically with 7 transmembrane (TM helices, is observed in the eukaryote crown group. So far, they are relatively rare in the prokaryotes, and are restricted to the well-characterized sensory rhodopsins of various phototropic prokaryotes. Results Utilizing the currently available wealth of prokaryotic genomic sequences, we set up a computational screen to identify putative 7 (TM and other multi-pass membrane receptors in prokaryotes. As a result of this procedure we were able to recover two widespread families of 7 TM receptors in bacteria that are distantly related to the eukaryotic 7 TM receptors and prokaryotic rhodopsins. Using sequence profile analysis, we were able to establish that the first members of these receptor families contain one of two distinct N-terminal extracellular globular domains, which are predicted to bind ligands such as carbohydrates. In their intracellular portions they contain fusions to a variety of signaling domains, which suggest that they are likely to transduce signals via cyclic AMP, cyclic diguanylate, histidine phosphorylation, dephosphorylation, and through direct interactions with DNA. The second family of bacterial 7 TM receptors possesses an α-helical extracellular domain, and is predicted to transduce a signal via an intracellular HD hydrolase domain. Based on comparative analysis of gene neighborhoods, this receptor is predicted to function as a regulator of the diacylglycerol-kinase-dependent glycerolipid pathway. Additionally, our procedure also recovered other types of putative prokaryotic multi-pass membrane associated receptor domains. Of these, we characterized two widespread, evolutionarily mobile multi-TM domains that are fused to a variety of C-terminal intracellular signaling domains. One of these typified by the Gram-positive LytS protein is predicted to be a potential sensor of murein derivatives, whereas the other one typified by the Escherichia coli UhpB protein is predicted to function as sensor of conformational changes occurring in associated membrane proteins Conclusions We present evidence for considerable variety in the types of uncharacterized surface receptors in bacteria, and reconstruct the evolutionary processes that model their diversity. The identification of novel receptor families in prokaryotes is likely to aid in the experimental analysis of signal transduction and environmental responses of several bacteria, including pathogens such as Leptospira, Treponema, Corynebacterium, Coxiella, Bacillus anthracis and Cytophaga.

  11. Interaction of the indole class of vacuolar H+-ATPase inhibitors with lipid bilayers

    NARCIS (Netherlands)

    Fernandes, F.; Loura, L.; Koehorst, R.B.M.; Dixon, N.; Kee, T.P.; Hemminga, M.A.; Prieto, M.

    2006-01-01

    The selective inhibitor of osteoclastic V-ATPase (2Z,4E)-5-(5,6-dichloro-2-indolyi)-2-methoxy-N-(1,2,2,6,6-pentamethylpip eridin-4-yl)-2,4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study

  12. Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids

    DEFF Research Database (Denmark)

    Valls, L A; Winther, Jakob R.; Stevens, T H

    1990-01-01

    The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations alt...

  13. Improving the Processing Quality of Existing Cultivars by Suppressing the Vacuolar Acid Invertase Gene

    Science.gov (United States)

    Storing potato tubers at low temperatures is highly advantageous in that it prevents sprouting, minimizes disease losses and increases the marketing window. Unfortunately, cold storage of existing cultivars causes an unacceptable accumulation of reducing sugars, a phenomenon referred to as cold-indu...

  14. Developing cold-chipping potato varieties by silencing the vacuolar invertase gene

    Science.gov (United States)

    Accumulation of reducing sugars during cold storage is a persistent and costly problem for the potato processing industry. High temperature processing of potato tubers with elevated amounts of reducing sugars results in potato chips, fries and other products that are unacceptable to consumers becaus...

  15. Suppression of the vacuolar invertase gene delays senescent sweetening in chipping potatoes

    Science.gov (United States)

    Background: Potato chip processors require potato tubers that meet quality specifications for fried chip color, and color depends largely upon tuber sugar contents. At later times in storage, potatoes accumulate sucrose, glucose and fructose. This developmental process, senescent sweetening, manifes...

  16. Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato

    Science.gov (United States)

    Storing potato (Solanum tuberosum) tubers at cold temperatures prevents sprouting and minimizes losses due to disease. Unfortunately, cold storage triggers an accumulation of reducing sugars, a phenomenon referred to as cold-induced sweetening (CIS). High-temperature processing of potato tubers wit...

  17. Covalent structures of potato tuber lipases (patatins) and implications for vacuolar import

    DEFF Research Database (Denmark)

    Welinder, Karen Gjesing; Jørgensen, Malene

    Proteome data of potato (Solanum tuberosum) tuber juice and of purified potato tuber vacuoles indicated that mature patatins may perhaps lack a C-terminal (ct) propeptide. We have confirmed this by complete mass spectrometric sequencing of a number of patatin variants as well as their N-linked co......Proteome data of potato (Solanum tuberosum) tuber juice and of purified potato tuber vacuoles indicated that mature patatins may perhaps lack a C-terminal (ct) propeptide. We have confirmed this by complete mass spectrometric sequencing of a number of patatin variants as well as their N......-linked complex-type glycans from the starch-rich cultivar Kuras. For this cultivar full length patatin cDNAs have also been sequenced, as the patatin locus is highly polymorphous. It is well-known that patatins are located in the vacuoles of potato tubers. Furthermore, the complex glycan structures show...

  18. Covalent structures of potato tuber lipases (patatins) and implications for vacuolar import

    DEFF Research Database (Denmark)

    Welinder, Karen Gjesing; Jørgensen, Malene

    2009-01-01

    Proteome data of potato (Solanum tuberosum) tuber juice and of purified potato tuber vacuoles indicated that mature patatins may perhaps lack a C-terminal propeptide. We have confirmed this by complete mass spectrometric sequencing of a number of patatin variants as well as their N-linked complex......-type glycans from the starch-rich cultivar Kuras. For this cultivar full-length patatin cDNAs have also been sequenced, as the patatin locus is highly polymorphous. It is well known that patatins are located in the vacuoles of potato tubers. Furthermore, the complex glycan structures show that the path is via...

  19. Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology.

    OpenAIRE

    Bonangelino, C J; Catlett, N L; Weisman, L S

    1997-01-01

    During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuola...

  20. The study of vacuolar-type ATPases by single particle electron microscopy1

    National Research Council Canada - National Science Library

    Zhao, Jianhua; Rubinstein, John L

    2014-01-01

    .... Electron microscopy (EM) techniques, especially single particle electron cryomicroscopy (cryo-EM) and negative-stain EM, have provided extensive insight into the structure and function of these protein complexes...

  1. Regulation of the synthesis and assembly of the plant vacuolar H sup + -ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.

    1992-01-01

    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  2. The class C Vps complex functions at multiple stages of the vacuolar transport pathway.

    Science.gov (United States)

    Peterson, M R; Emr, S D

    2001-07-01

    The Class C Vps complex, consisting of Vps11, Vps16, Vps18, and Vps33, is required for SNARE-mediated membrane fusion at the lysosome-like yeast vacuole. However, Class C vps mutants display more severe and pleiotropic phenotypes than mutants specifically defective in endosome-to-vacuole transport, suggesting that there are additional functions for the Class C Vps complex. A SNARE double mutant which is defective for both Golgi-to-endosome and endosome-to-vacuole trafficking replicates many of the phenotypes observed in Class C vps mutants. We show that genetic interactions exist between Class C vps alleles and alleles of the Class D vps group, which are defective in the docking and fusion of Golgi-derived vesicles at the endosome. Moreover, the Class D protein Vac1 was found to physically bind to the Class C Vps complex through a direct association with Vps11. Finally, using a random mutagenic screen, a temperature-conditional allele which shares many of the phenotypes of mutants which are selectively defective in Golgi-to-endosome trafficking was isolated (vps11-3ts). Collectively, these results indicate that the Class C Vps complex plays essential roles in the processes of membrane docking and fusion at both the Golgi-to-endosome and endosome-to-vacuole stages of transport.

  3. A tomato vacuolar invertase inhibitor mediates sucrose metabolism an influences fruit ripening

    Science.gov (United States)

    Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening and there is evidence that it influences various aspects of ripening, alth...

  4. Stress regulated members of the plant organic cation transporter family are localized to the vacuolar membrane

    Directory of Open Access Journals (Sweden)

    Koch Wolfgang

    2008-07-01

    Full Text Available Abstract Background In Arabidopsis six genes group into the gene family of the organic cation transporters (OCTs. In animals the members of the OCT-family are mostly characterized as polyspecific transporters involved in the homeostasis of solutes, the transport of monoamine neurotransmitters and the transport of choline and carnitine. In plants little is known about function, localisation and regulation of this gene family. Only one protein has been characterized as a carnitine transporter at the plasma membrane so far. Findings We localized the five uncharacterized members of the Arabidopsis OCT family, designated OCT2-OCT6, via GFP fusions and protoplast transformation to the tonoplast. Expression analysis with RNA Gel Blots showed a distinct, organ-specific expression pattern of the individual genes. With reporter gene fusion of four members we analyzed the tissue specific distribution of OCT2, 3, 4, and 6. In experiments with salt, drought and cold stress, we could show that AtOCT4, 5 and 6 are up-regulated during drought stress, AtOCT3 and 5 during cold stress and AtOCT 5 and 6 during salt stress treatments. Conclusion Localisation of the proteins at the tonoplast and regulation of the gene expression under stress conditions suggests a specific role for the transporters in plant adaptation to environmental stress.

  5. Vacuolar Pathology in the Median Eminence of the Hypothalamus Following Hyponatremia

    OpenAIRE

    Levine, Seymour; Saltzman, Arthur; Ginsberg, Stephen D.

    2011-01-01

    The median eminence of the hypothalamus is an important conduit by which neurosecretory hormones from hypothalamic nuclei are delivered to the pars nervosa (neural lobe) of the pituitary en route to the bloodstream. Dilutional hyponatremia was produced in adult rats to determine the effect on the morphology of the median eminence of the hypothalamus. Hyponatremia was caused by reducing electrolyte and organic osmolyte reserves in order to block the excretion of water through delivery of the n...

  6. Vacuolar pathology in the median eminence of the hypothalamus after hyponatremia.

    Science.gov (United States)

    Levine, Seymour; Saltzman, Arthur; Ginsberg, Stephen D

    2011-02-01

    The median eminence of the hypothalamus is an important conduit by which neurosecretory hormones from hypothalamic nuclei are delivered to the pars nervosa (neural lobe) of the pituitary en route to the bloodstream. Dilutional hyponatremia was produced in adult rats to determine the effect on the morphology of the median eminence of the hypothalamus. Hyponatremia was caused by reducing electrolyte and organic osmolyte reserves to block the excretion of water through delivery of the nephrotoxin mercuric chloride (HgCl2). Histological examination of the brain 1 day after a hyponatremic insult revealed vacuolation within the median eminence of the hypothalamus. No other lesions were found in other parts of the brain after hyponatremia. The hyponatremic lesion consisted of a band of closely packed vacuoles that crossed the floor of the third ventricle. Vacuoles associated with hyponatremia were predominantly in the subependymal, fiber, reticular, and palisade layers of the median eminence. Vacuolation was not observed in the tanycyte layer of the median eminence. This study indicates that the median eminence is a potentially vulnerable site in human hyponatremic conditions that should be evaluated further in relevant animal models.

  7. SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

    NARCIS (Netherlands)

    Baster, P.; Robert, S.; Kleine-Vehn, J.; Vanneste, S.; Kania, U.; Grunewald, W.; Rybel, de B.P.M.; Beeckman, T.; Friml, J.

    2013-01-01

    The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels.

  8. [Acid vacuolar invertase in hibernating and germinating seeds of the horse chestnut].

    Science.gov (United States)

    Obrucheva, N V; Litiagina, S V

    2009-01-01

    A high water content is maintained in the tissues of the axial organs of horse chestnut seeds after the fruit is shed and down to the time the seeds germinate. The plant cell vacuoles, features of whose metabolism can influence the cells' preparation to initiate growth in germination, are preserved. It was shown that the activity of acid invertase and its capacity to digest both sucrose and raphinose remain stable throughout the period of hibernation and the transition to germination, as do the molecular weight of its subunits (63 and 65 kDa) and multimer (500 to 550 kDa). The activity of the enzyme increases when the seeds swell under optimal conditions for germination; this is associated with the synthesis of new molecules of the enzyme in long-lived mRNA matrices. The storability of the enzyme in the vacuoles of hibernating seeds, together with the increase in its activity when seeds coming out of hibernation swell, ensures the rapid hydrolysis of sucrose issuing from the seeds' cotyledons, thus leading to increased osmotic pressure and, as a result, the beginning of cell elongation, i.e., germination.

  9. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45......% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied...... by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  10. Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

    NARCIS (Netherlands)

    Verweij, W.; Spelt, C.E.; Bliek, M.; de Vries, M.; Wit, N.; Faraco, M.; Koes, R.; Quattrocchio, F.

    2016-01-01

    The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) fromArabidopsis thalianaand associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein

  11. Avian vacuolar myelinopathy: a newly recognized fatal neurologic disease of eagles, waterfowl, and other birds

    Science.gov (United States)

    Fischer, John R.; Lewis, L.A.; Augspurger, T.; Rocke, T.E.

    2002-01-01

    Wildlife biologists and health specialists have been frustrated by a long list of negative findings in their AVM investigations, however studies continue to provide pieces of information to aid the determination of the cause and its source. Available data indicated that AVM may have been present since at least 1990, occurs in at least five states, has been documented during October through April at sites of wintering populations of birds where the exposure apparently occurs, and has killed at least 90 bald eagles. Birds with AVM have difficulty or inability to fly, swim, walk, or perch, but there has been resolution of clinical signs in some affected coots. The list of affected species continues to grow, but remains confined to wild avians, including bald eagle, American coot, great horned owl, killdeer, Canada goose, mallard, ring-necked duck and bufflehead. The effects of the AVM agent on mammals, including human beings, are unknown. A neurotoxicant of manmade or natural origin is the suspected cause of AVM because no infectious disease agents, such as viruses, bacteria, parasites and prions, have been found, and the lesion and epizootiology of AVM resemble those of toxicoses. Additionally it is documented, experimentally, that exposure to raptors can occur through ingestion of infected coots. Collaborative studies will continue in the effort to identify the cause of AVM, its geographic distribution, and the range of species susceptibility. Hopefully, this information can be used to identify measures that might be taken to reduce the impact of AVM on the wildlife resource. Multiple agencies, institutions, and individuals must rely on each other's expertise in the multidisciplinary approach to this problem, persevere in their efforts and take advantage of serendipity that presents itself during investigations of this newly recognized cause of wild bird mortality.

  12. Endocytosis and vacuolar degradation of the yeast cell surface glucose sensors Rgt2 and Snf3.

    Science.gov (United States)

    Roy, Adhiraj; Kim, Jeong-Ho

    2014-03-07

    Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations.

  13. Environmental Factors Influencing Blooms of a Neurotoxic Stigonematalan Cyanobacterium Responsible for Avian Vacuolar Myelinopathy

    Science.gov (United States)

    2013-01-01

    feeding trials did not exhibit clinical or histological AVM symptoms (Lewis-Weiss et al. 2004, Fischer et al. 2006; Haynie 2008). AVM researchers have...Ti, Nikon Corp., Melville, NY USA, 11747-3064). For species with large entire leaves (Nymphaea) or blades (Vallisneria), three small sections were...dissected, using a razor blade , and aligned on a slide for viewing. When examining each slide, the screener recorded the sample’s geographic information

  14. Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways.

    Science.gov (United States)

    Jahnke, Martin; Trowsdale, John; Kelly, Adrian P

    2012-03-02

    HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

  15. Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis.

    Science.gov (United States)

    Barreras, Máximo; Abdian, Patricia L; Ielpi, Luis

    2004-03-01

    Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-beta-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.

  16. TNF induction of EL4 hyposensitivity to lysis by recombinant (soluble) and membrane-associated TNFs: TNF binding, internalization, and degradation.

    Science.gov (United States)

    Fishman, M; Costlow, M

    1994-04-01

    EL4 mouse thymoma cells sensitive to TNF-mediated lysis only in the presence of cycloheximide (S-EL4) or in the presence or absence of cycloheximide (N-EL4) were used in these experiments. Murine tumor cell line (S-EL4) sensitivity to TNF cytotoxicity is augmented when cycloheximide is added together with TNF or when cycloheximide is added 1 hr before or after TNF. No enhanced sensitivity is observed when target cells are incubated with cycloheximide 2-4 hr before or after the addition of TNF. In the absence of cycloheximide, S-EL4 cells preexposed to murine TNF are less susceptible to lysis by TNF and TNF receptor-conjugated TNF but are lysed by integral membrane TNF. TNF-induced hyposensitivity is partially reversed by actinomycin D or by culturing the preexposed cells for 4 hr prior to TNF lytic assay. TNF preincubation of N- and S-EL4 cells results in an immediate decrease in 125I-TNF binding due to TNF receptor occupancy. Recovery of TNF-R occupancy and TNF internalization were subsequently noted.

  17. Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes.

    Science.gov (United States)

    Favery, Bruno; Chelysheva, Liudmila A; Lebris, Manuel; Jammes, Fabien; Marmagne, Anne; De Almeida-Engler, Janice; Lecomte, Philippe; Vaury, Chantal; Arkowitz, Robert A; Abad, Pierre

    2004-09-01

    Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.

  18. Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells.

    Science.gov (United States)

    Xu, Jian; Teng, Da; Jiang, Fei; Zhang, Yuewei; El-Ashram, Saeed A; Wang, Hui; Sun, Zhenhong; He, Jinyan; Shen, Junjun; Wu, Wenxue; Li, Jinxiang

    2015-02-01

    Mycoplasma gallisepticum can infect a wide variety of birds including the commercial poultry. M. gallisepticum MGA_0676 is a putative lipoprotein, which is similar to bacterial thermostable nucleases. But the possible pathogenic effect of M. gallisepticum MGA_0676 has not been investigated so far. In the present study, we cloned the MGA_0676 gene after deletion of the amino-terminal signal sequence and mutagenesis of the Mycoplasma TGA tryptophan codons to TGG and expressed recombinant MGA_0676 protein in Escherichia coli. We identified and characterized MGA_0676 as a Ca(2+)-dependent cytotoxic nuclease of M. gallisepticum with a staphylococcal nuclease (SNc) region that displays the hallmarks of nucleases. Membrane protein immunoblot analysis and immunogold electron microscopy revealed that MGA_0676 locates on the membrane surface of M. gallisepticum. Furthermore, apoptosis assay using annexin V-FITC and propidium iodide (annexin V/PI) indicated that MGA_0676 played significant roles in apoptosis induction and pathological damages in chicken cells. Moreover, confocal microscopy showed that MGA_0676 localizes in the nuclei of host cells. Besides, after the SNc region was deleted, MGA_0676 lost its ability of nuclear localization, nuclease activity, and cytotoxicity, which revealed that the SNc region is essential for nuclear translocation and induction of apoptosis in chicken cells. The above results suggest that MGA_0676 is an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M. gallisepticum.

  19. Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme

    Directory of Open Access Journals (Sweden)

    Karla Ramirez-Estrada

    2017-06-01

    Full Text Available Sterol glycosyltransferases (SGTs catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4. On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

  20. Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B(1)4 beta-glucosidase with cellodextrinase and cyanoglycosidase activities.

    Science.gov (United States)

    Wulff-Strobel, C R; Wilson, D B

    1995-01-01

    Prevotella ruminicola B(1)4 is a gram-negative, anaerobic gastrointestinal bacterium. A 2.4-kbp chromosomal fragment from P. ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5 alpha and sequenced. CdxA activity was found predominantly in the membrane fraction of both P. ruminicola and E. coli, but P. ruminicola localized the protein extracellularly while E. coli did not. The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mumol of glucose released min-1 mg of protein-1) and p-nitrophenyl-beta-D-glucoside (3.54 mumol min-1 mg of protein-1). Significant activity (70% of p-nitrophenyl-beta-D-glucoside activity) was also detected on arbutin and prunasin. Less activity was obtained with cellobiose, amygdalin, or gentiobiose. CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4-beta-glucosidase (EC 3.2.1.74) which also is able to attack beta-1,6 linkages. Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B. Henrissat, Biochem. J. 280:309-316, 1991). On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265. This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants. PMID:7592339

  1. Flow-alignment of bicellar lipid mixtures: orientations of probe molecules and membrane-associated biomacromolecules in lipid membranes studied with polarized light

    KAUST Repository

    Kogan, Maxim

    2011-01-01

    Bicelles are excellent membrane-mimicking hosts for a dynamic and structural study of solutes with NMR, but the magnetic fields required for their alignment are hard to apply to optical conditions. Here we demonstrate that bicellar mixtures can be aligned by shear forces in a Couette flow cell, to provide orientation of membrane-bound retinoic acid, pyrene and cytochrome c (cyt c) protein, conveniently studied with linear dichroism spectroscopy. © 2011 The Royal Society of Chemistry.

  2. The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans1[W

    Science.gov (United States)

    Bozkurt, Tolga O.; Richardson, Annis; Dagdas, Yasin F.; Mongrand, Sébastien; Kamoun, Sophien; Raffaele, Sylvain

    2014-01-01

    Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen. PMID:24808104

  3. R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed.

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Liang, Yingjian; Hasegawa, Haruki; Nycz, Deborah C; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C

    2012-07-01

    The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  4. Identification by affinity chromatography of boar sperm membrane-associated proteins bound to immobilized porcine zona pellucida. Mapping of the phosphorylethanolamine-binding region of spermadhesin AWN.

    Science.gov (United States)

    Ensslin, M; Calvete, J J; Thole, H H; Sierralta, W D; Adermann, K; Sanz, L; Töpfer-Petersen, E

    1995-12-01

    We have identified boar sperm membrane components recovered by affinity chromatography on a porcine zona pellucida affinity column. The major zona pellucida-bound proteins were spermadhesins AWN and AQN-3, the heparin-binding protein pAIF, and a homolog of the mouse milk fat globule membrane protein. All these proteins are phospholipid-binding proteins peripherally associated with the plasma membrane. Our data suggest that coating proteins tightly bound to the external lipid bilayer may act as major zona pellucida-binding molecules. Using a synthetic peptide approach we show that the regions of spermadhesin AWN comprising residues 6-12 and 104-108 possess affinity for phosphorylethanolamine. These two amino acid sequences are in close proximity in the predicted structural model for AWN, and in opposite location to its carbohydrate-recognition domain. Taken together, our data provide further evidence for the possible involvement of members of the porcine spermadhesin protein family in gamete interaction and suggest a model for the ultrastructural disposition of functional domains of spermadhesin AWN bound to the sperm surface.

  5. Brittle Culm15 Encodes a Membrane-Associated Chitinase-Like Protein Required for Cellulose Biosynthesis in Rice1[C][W][OA

    Science.gov (United States)

    Wu, Bin; Zhang, Baocai; Dai, Yan; Zhang, Lei; Shang-Guan, Keke; Peng, Yonggang; Zhou, Yihua; Zhu, Zhen

    2012-01-01

    Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana). PMID:22665444

  6. The odyssey of Hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities.

    Directory of Open Access Journals (Sweden)

    Claudia Campanella

    Full Text Available BACKGROUND: In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. PRINCIPAL FINDINGS: We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. CONCLUSIONS/SIGNIFICANCE: We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

  7. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    Science.gov (United States)

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes.IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. Copyright © 2017 American Society for Microbiology.

  8. Malaria Parasite CLAG3, a Protein Linked to Nutrient Channels, Participates in High Molecular Weight Membrane-Associated Complexes in the Infected Erythrocyte.

    Directory of Open Access Journals (Sweden)

    Kayvan Zainabadi

    Full Text Available Malaria infected erythrocytes show increased permeability to a number of solutes important for parasite growth as mediated by the Plasmodial Surface Anion Channel (PSAC. The P. falciparum clag3 genes have recently been identified as key determinants of PSAC, though exactly how they contribute to channel function and whether additional host/parasite proteins are required remain unknown. To begin to answer these questions, I have taken a biochemical approach. Here I have used an epitope-tagged CLAG3 parasite to perform co-immunoprecipitation experiments using membrane fractions of infected erythrocytes. Native PAGE and mass spectrometry studies reveal that CLAG3 participate in at least three different high molecular weight complexes: a ~720kDa complex consisting of CLAG3, RHOPH2 and RHOPH3; a ~620kDa complex consisting of CLAG3 and RHOPH2; and a ~480kDa complex composed solely of CLAG3. Importantly, these complexes can be found throughout the parasite lifecycle but are absent in untransfected controls. Extracellular biotin labeling and protease susceptibility studies localize the 480kDa complex to the erythrocyte membrane. This complex, likely composed of a homo-oligomer of 160kDa CLAG3, may represent a functional subunit, possibly the pore, of PSAC.

  9. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

    DEFF Research Database (Denmark)

    Jensen, R B; Lykke-Andersen, K; Frandsen, G I

    2000-01-01

    Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C...

  10. Expression of specific genes involved in Cd uptake, translocation, vacuolar compartmentalisation and recycling in Populus alba Villafranca clone.

    Science.gov (United States)

    Romè, Chiara; Huang, Xin-Yuan; Danku, John; Salt, David E; Sebastiani, Luca

    2016-09-01

    Cadmium (Cd) is a heavy metal toxic to humans and its occurrence in soils represents a significant environmental problem. Poplar trees may provide one possible option to help remove Cd contamination from soil. However, before this is practicable, the ability of poplar to accumulate Cd needs to be enhanced. A better understanding of the genes involved in Cd accumulation in poplar would help to achieve this goal. Here, we monitored the expression of genes known to be involved in Cd uptake, accumulation and translocation from other species, in order to provide information on their potential role in Cd accumulation in poplar. Cd concentration in poplar was significantly higher in roots than in stem and leaves in Cd treated plants. Expression of the poplar homologues of IRT1, NRAMP and OPT3 was initially increased after exposure to Cd but reduced after longer term Cd exposure. Exposure to Cd also influenced the accumulation of Fe, Ca, Cu, Mg and Mn in poplar. In particular, Cd treated plants had a higher concentration of Fe, Ca, Cu, and Mg in leaves and stem compared to control plants after one day and one week of experiment; while in roots after one month Cd treated plants had a lower concentration of Mn, Fe, Cu, Co, and Mg. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis

    NARCIS (Netherlands)

    Subroto, T; de Vries, H; Schuringa, JJ; Soedjanaatmadja, UMS; Hofsteenge, J; Jekel, PA; Beintema, JJ

    2001-01-01

    The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a beta-1,3-glucanase. The content and

  12. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

    Science.gov (United States)

    Kyöstilä, Kaisa; Syrjä, Pernilla; Jagannathan, Vidhya; Chandrasekar, Gayathri; Jokinen, Tarja S; Seppälä, Eija H; Becker, Doreen; Drögemüller, Michaela; Dietschi, Elisabeth; Drögemüller, Cord; Lang, Johann; Steffen, Frank; Rohdin, Cecilia; Jäderlund, Karin H; Lappalainen, Anu K; Hahn, Kerstin; Wohlsein, Peter; Baumgärtner, Wolfgang; Henke, Diana; Oevermann, Anna; Kere, Juha; Lohi, Hannes; Leeb, Tosso

    2015-04-01

    Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  13. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion.

    Science.gov (United States)

    Sato, T K; Rehling, P; Peterson, M R; Emr, S D

    2000-09-01

    In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport. In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3. Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole. The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3. Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion. Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing. Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion.

  14. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transfer...

  15. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    National Research Council Canada - National Science Library

    Appelhagen, I; Nordholt, N; Seidel, T; Spelt, K; Koes, R; Quattrochio, F; Sagasser, M; Weisshaar, B

    2015-01-01

    ...) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants...

  16. A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease

    Science.gov (United States)

    Kyöstilä, Kaisa; Syrjä, Pernilla; Jagannathan, Vidhya; Chandrasekar, Gayathri; Jokinen, Tarja S.; Seppälä, Eija H.; Becker, Doreen; Drögemüller, Michaela; Dietschi, Elisabeth; Drögemüller, Cord; Lang, Johann; Steffen, Frank; Rohdin, Cecilia; Jäderlund, Karin H.; Lappalainen, Anu K.; Hahn, Kerstin; Wohlsein, Peter; Baumgärtner, Wolfgang; Henke, Diana; Oevermann, Anna; Kere, Juha; Lohi, Hannes; Leeb, Tosso

    2015-01-01

    Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes. PMID:25875846

  17. MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides and Glycoside Malonates in Medicago truncatula[C][W][OA

    Science.gov (United States)

    Zhao, Jian; Huhman, David; Shadle, Gail; He, Xian-Zhi; Sumner, Lloyd W.; Tang, Yuhong; Dixon, Richard A.

    2011-01-01

    The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3′-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage. PMID:21467581

  18. Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

    NARCIS (Netherlands)

    Heijden, van der M.W.; Carette, J.E.; Reinhoud, P.J.; Haegi, A.; Bol, J.F.

    2001-01-01

    Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and

  19. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

    Directory of Open Access Journals (Sweden)

    Kaisa Kyöstilä

    2015-04-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  20. The impact of medium acidity on the chronological life span of Saccharomyces cerevisiae - lipids, signaling cascades, mitochondrial and vacuolar functions.

    Science.gov (United States)

    Yucel, Esra B; Eraslan, Serpil; Ulgen, Kutlu O

    2014-02-01

    Because of its multifactorial nature, aging is one of the most complicated cell phenomena known. A systems biology approach, which aims to understand the organism as a whole rather than concentrate on the behaviors of individual genes, thus comprises a seamless tool for investigating the aging machinery, which arises mainly as a result of degeneration of the collaboration between signaling and regulatory pathways. In the present study, the effects of medium buffering on the chronological life span are investigated via transcriptome analyses and subsequent integration of the data obtained with the chronological aging network of yeast. The comparative inquiry of transcriptome data of young and old cells grown in buffered and unbuffered media reveals new roles for pH control (e.g. the re-organization of lipid metabolism and intracellular signaling cascades) that have beneficial consequences on chronological longevity. Integration of the transcriptome data onto the aging network, as well as validation experiments, suggest that Snf1p is a possible intermediate player in the interjunction of sphingolipid and ergosterol metabolisms with extracellular pH control with respect to regulation of the chronological life span. Consequently, a more detailed insight of the chronological aging mechanism of yeast is obtained. The results of the present study provide a solid basis for further research focusing on uncovering the agents that affect aging and age-related diseases in humans. © 2014 FEBS.

  1. Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

    Czech Academy of Sciences Publication Activity Database

    Petrezsélyová, Silvia; Domínguez, A.; Herynková, Pavla; Macias, J. F.; Sychrová, Hana

    2013-01-01

    Roč. 30, č. 10 (2013), s. 395-402 ISSN 0749-503X R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) IAA500110801 Grant - others:Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR(CZ) M200110901 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : heterologous expression * CCC family * alkali-metal-cation tolerance * Saccharomyces cerevisiae * ion homeostasis Subject RIV: EE - Microbiology, Virology Impact factor: 1.742, year: 2013

  2. Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-?-Acids

    NARCIS (Netherlands)

    Hazelwood, L.A.; Walsh, M.C.; Pronk, J.T.; Daran, J.M.

    2009-01-01

    The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop -acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso--acids have hitherto been restricted to lactic acid bacteria.

  3. The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

    Science.gov (United States)

    Martins, Viviana; Carneiro, Filipa; Conde, Carlos; Sottomayor, Mariana; Gerós, Hernâni

    2017-12-01

    The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca2+, and Na+ in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

  4. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products

    Science.gov (United States)

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to ...

  5. Opt2 mediates the exposure of phospholipids during cellular adaptation to altered lipid asymmetry.

    Science.gov (United States)

    Yamauchi, Saori; Obara, Keisuke; Uchibori, Kenya; Kamimura, Akiko; Azumi, Kaoru; Kihara, Akio

    2015-01-01

    Plasma membrane lipid asymmetry is important for various membrane-associated functions and is regulated by membrane proteins termed flippases and floppases. The Rim101 pathway senses altered lipid asymmetry in the yeast plasma membrane. The mutant lem3Δ cells, in which lipid asymmetry is disturbed owing to the inactivation of the plasma membrane flippases, showed a severe growth defect when the Rim101 pathway was impaired. To identify factors involved in the Rim101-pathway-dependent adaptation to altered lipid asymmetry, we performed DNA microarray analysis and found that Opt2 induced by the Rim101 pathway plays an important role in the adaptation to altered lipid asymmetry. Biochemical investigation of Opt2 revealed its localization to the plasma membrane and the Golgi, and provided several lines of evidence for the Opt2-mediated exposure of phospholipids. In addition, Opt2 was found to be required for the maintenance of vacuolar morphology and polarized cell growth. These results suggest that Opt2 is a novel factor involved in cell homeostasis by regulating lipid asymmetry. © 2015. Published by The Company of Biologists Ltd.

  6. Differential activity of plasma and vacuolar membrane transporters contributes to genotypic differences in salinity tolerance in a Halophyte Species, Chenopodium quinoa.

    Science.gov (United States)

    Bonales-Alatorre, Edgar; Pottosin, Igor; Shabala, Lana; Chen, Zhong-Hua; Zeng, Fanrong; Jacobsen, Sven-Erik; Shabala, Sergey

    2013-04-29

    Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

  7. Failure to Detect the Neurotoxin Beta-n-methylamino-l-alanine in Samples Collected during an Avian Vacuolar Myelinopathy (AVM) Epornitic in J. Strom Thurmond Lake

    Science.gov (United States)

    2015-08-01

    species present (e.g., cyanobacteria, diatoms , green alga) are recorded. To confirm the presence or absence of H. aetokthonos based on microscopy, PCR...have evolved a mechanism to detoxify and/or depurate this compound, resulting in nondetection. Researchers have also hypothesized that a metabolite...of the Aquatic Nuisance Species Research Program (ANSRP), Dr. Linda Nelson (601-634-2656) (Linda.S.Nelson@usace. army.mil). This technical note should

  8. Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

    National Research Council Canada - National Science Library

    MCKENZIE, MARIAN J; CHEN, RONAN K. Y; HARRIS, JOHN C; ASHWORTH, MATTHEW J; BRUMMELL, DAVID A

    2013-01-01

    .... During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose...

  9. Regulation of the synthesis and assembly of the plant vacuolar H{sup +}-ATPase. Progress report, [April 1, 1991--March 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.

    1992-04-01

    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  10. Involvement of the Leaf-Specific Multidrug and Toxic Compound Extrusion (MATE) Transporter Nt-JAT2 in Vacuolar Sequestration of Nicotine in Nicotiana tabacum

    Science.gov (United States)

    Shitan, Nobukazu; Minami, Shota; Morita, Masahiko; Hayashida, Minaho; Ito, Shingo; Takanashi, Kojiro; Omote, Hiroshi; Moriyama, Yoshinori; Sugiyama, Akifumi; Goossens, Alain; Moriyasu, Masataka; Yazaki, Kazufumi

    2014-01-01

    Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues. PMID:25268729

  11. Resistance to inhibitors of cholinesterase (Ric-8A and Gαi contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps34 activity.

    Directory of Open Access Journals (Sweden)

    Cedric Boularan

    Full Text Available Resistance to inhibitors of cholinesterase (Ric-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.

  12. Resistance to inhibitors of cholinesterase (Ric)-8A and Gαi contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity.

    Science.gov (United States)

    Boularan, Cedric; Kamenyeva, Olena; Cho, Hyeseon; Kehrl, John H

    2014-01-01

    Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.

  13. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in trangenic plants with increased vacuolar sodium accumulation

    National Research Council Canada - National Science Library

    Hong-Xia Zhang; Joanna N Hodson; John P Williams; Eduardo Blumwald

    2001-01-01

      Transgenic Brassica napus plants overexpressing AtNHX1, a vaculor Na+/H+ antiport form Arabidopsis thaliana, were able to grow, flower, and produce seeds in the presence of 200 mM sodium chloride...

  14. A Vacuolar β-Glucosidase Homolog That Possesses Glucose-Conjugated Abscisic Acid Hydrolyzing Activity Plays an Important Role in Osmotic Stress Responses in Arabidopsis[W

    Science.gov (United States)

    Xu, Zheng-Yi; Lee, Kwang Hee; Dong, Ting; Jeong, Jae Cheol; Jin, Jing Bo; Kanno, Yuri; Kim, Dae Heon; Kim, Soo Youn; Seo, Mitsunori; Bressan, Ray A.; Yun, Dae-Jin; Hwang, Inhwan

    2012-01-01

    The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses. PMID:22582100

  15. A vacuolar β-glucosidase homolog that possesses glucose-conjugated abscisic acid hydrolyzing activity plays an important role in osmotic stress responses in Arabidopsis.

    Science.gov (United States)

    Xu, Zheng-Yi; Lee, Kwang Hee; Dong, Ting; Jeong, Jae Cheol; Jin, Jing Bo; Kanno, Yuri; Kim, Dae Heon; Kim, Soo Youn; Seo, Mitsunori; Bressan, Ray A; Yun, Dae-Jin; Hwang, Inhwan

    2012-05-01

    The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses.

  16. Nicotine affects bone resorption and suppresses the expression of cathepsin K, MMP-9 and vacuolar-type H(+-ATPase d2 and actin organization in osteoclasts.

    Directory of Open Access Journals (Sweden)

    Hideki Tanaka

    Full Text Available Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10(-5, 10(-4, or 10(-3 M nicotine and/or 50 µM α-bungarotoxin (btx, an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1-5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei, and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

  17. Differential Activity of Plasma and Vacuolar Membrane Transporters Contributes to Genotypic Differences in Salinity Tolerance in a Halophyte Species, Chenopodium quinoa

    Directory of Open Access Journals (Sweden)

    Edgar Bonales-Alatorre

    2013-04-01

    Full Text Available Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd., a facultative C3 halophyte species, can efficiently control the activity of slow (SV and fast (FV tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013 Plant Physiology. This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i a higher rate of Na+ exclusion from leaf mesophyll; (ii maintenance of low cytosolic Na+ levels; (iii better K+ retention in the leaf mesophyll; (iv a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

  18. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    OpenAIRE

    Behrooz Darbani; Mohammed Saddik Motawia; Carl Erik Olsen; Nour-Eldin, Hussam H.; Birger Lindberg Møller; Fred Rook

    2016-01-01

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expresse...

  19. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  20. Differential activity of Plasma and Vacuolar Membrane Transporters contributes to Genotypic Differences in Salinity Tolerance in a Halophyte Species, Chenopodium quinoa

    DEFF Research Database (Denmark)

    Bonales-Alatorre, Edgar; Pottosin, Igor; Shabala, Lana

    2013-01-01

    Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow...... (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two...... quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa...

  1. ALTERNATE FOOD-CHAIN TRANSFER OF THE TOXIN LINKED TO AVIAN VACUOLAR MYELINOPATHY AND IMPLICATIONS FOR THE ENDANGERED FLORIDA SNAIL KITE (ROSTRHAMUS SOCIABILIS)

    National Research Council Canada - National Science Library

    Dodd, Shelley R; Haynie, Rebecca S; Williams, Susan M; Wilde, Susan B

    2016-01-01

    .... that grows epiphytically on submerged aquatic vegetation (SAV). The toxin accumulates, likely in the gastrointestinal tract of waterbirds that consume SAV, and birds of prey are exposed when feeding on the moribund waterbirds...

  2. Cold-induced sweetening, sugar ends, stem-end chip defect and acrylamide can be controlled effectively by silencing of the potato vacuolar invertase gene

    Science.gov (United States)

    Potato (Solanum tuberosum) is the third most important food crop in the world. Processing quality is one of the most important traits for many potato cultivars. Accumulations of reducing sugars in whole tubers as a result of cold-induced sweetening during low temperature storage, or at the tuber ste...

  3. Congregation of orthopoxvirus virions in cytoplasmic A-type inclusions is mediated by interactions of a bridging protein (A26p) with a matrix protein (ATIp) and a virion membrane-associated protein (A27p).

    Science.gov (United States)

    Howard, Amanda R; Weisberg, Andrea S; Moss, Bernard

    2010-08-01

    Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.

  4. Gclust Server: 11424 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available ATMAPR4 (ARABIDOPSIS THALIANA MEMBRANE-ASSOCIATED PROGESTERONE BINDING PROTEIN 4); heme binding / transition...ATMAPR4 (ARABIDOPSIS THALIANA MEMBRANE-ASSOCIATED PROGESTERONE BINDING PROTEIN 4); heme binding / transition

  5. Gclust Server: 46886 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available to intracellular membrane-associated calcium-independent phospholipase A2 gamma ; no annotation 2 1.00e-60...to intracellular membrane-associated calcium-independent phospholipase A2 gamma ; no annotation Number

  6. EST Table: BP116132 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available ref|XP_001660485.1| calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask...) [Aedes aegypti] gb|EAT38133.1| calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask...otein kinase membrane-associated guanylate kinase (cask) [Tribolium castaneum] BP116132 brP- ...

  7. EST Table: BP117264 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available dent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes aegypti] gb|EAT38133.1| calciu...m/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes aegypti] 10/...kinase membrane-associated guanylate kinase (cask) [Tribolium castaneum] BP116132 ce-- ...

  8. EST Table: DC557000 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available dulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes aegypti] gb|EAT3813...3.1| calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes a...ne protein kinase membrane-associated guanylate kinase (cask) [Tribolium castaneum] BP116132 wd-- ...

  9. EST Table: DC556439 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available dulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes aegypti] gb|EAT3813...3.1| calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes a...ne protein kinase membrane-associated guanylate kinase (cask) [Tribolium castaneum] BP116132 wd-- ...

  10. Reference: 193 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ous electrophysiological studies as the slow vacuolar channel. Slow vacuolar chan...scisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies

  11. PH4 of petunia is an R2R3-MYB protein that activates vacuolar acidification through interactions with Basic-Helix-Loop transcription factors of the anthocyanin pathway.

    NARCIS (Netherlands)

    Quattrocchio, F.M.; Verweij, C.W.; Kroon, A.R.; Spelt, C.E.; Mol, J.N.M.; Koes, R.E.

    2006-01-01

    The Petunia hybrids genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color,

  12. Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): a Y210C mutation causes either altered protein handling or altered protein function of N-acetylgalactosamine 4-sulfatase at multiple points in the vacuolar network.

    Science.gov (United States)

    Bradford, Tessa M; Litjens, Tom; Parkinson, Emma J; Hopwood, John J; Brooks, Doug A

    2002-04-16

    The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.

  13. EST Table: FS924605 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available dulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes aegypti] gb|EAT3813...3.1| calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Aedes a...f|XP_972920.2| PREDICTED: similar to calcium/calmodulin-dependent serine protein kinase membrane-associated guanylate kinase (cask) [Tribolium castaneum] BP116132 fwgP ...

  14. Genotyping of polymorphic effectors of Toxoplasma gondii isolates from China

    Directory of Open Access Journals (Sweden)

    Weisheng Cheng

    2017-11-01

    Full Text Available Abstract Background Toxoplasma gondii is an opportunistic protozoan apicomplexan and obligate intracellular parasite that infects a wide range of animals and humans. Rhoptry proteins 5 (ROP5, ROP16, ROP18 and dense granules 15 (GRA15 are the important effectors secreted by T. gondii which link to the strain virulence for mice and modulate the host’s response to the parasite. Little has been known about these molecules as well as GRA3 in type Chinese 1 strains that show polymorphism among strains of archetypical genotypes. This study examined the genetic diversity of these effectors and its correlated virulence in mice among T. gondii isolates from China. Results Twenty-one isolates from stray cats were detected, of which 15 belong to Chinese 1, and 6 to ToxoDB #205. Wh6 isolate, a Chinese 1 strain, has an avirulent phenotype. PCR-RFLP results of ROP5 and ROP18 presented few variations among the strains. Genotyping of GRA15 and ROP16 revealed that all the strains belong to type II allele except Xz7 which carries type I allele. ROP16 amino acid alignment at 503 locus demonstrated that 17 isolates are featured as type I or type III (ROP16I/III, and the other 4 as type II (ROP16II. The strains investigated may be divided into four groups based on GRA3 amino acid alignment, and all isolates of type Chinese 1 belong to the μ-1 allele except Wh6 which is identical to type II strain. Conclusions PCR-RFLP and sequence alignment analyses of ROP5, ROP16, ROP18, GRA3, and GRA15 in T. gondii revealed that strains with the same genotype may have variations in some of their key genes. GRA3 variation exhibited by Wh6 strain may be associated with the difference in phenotype and pathogenesis.

  15. Differential expression in Phanerochaete chrysosporium of membrane- associated proteins relevant to lignin degradation

    Science.gov (United States)

    Semarjit Shary; Alexander N. Kapich; Ellen A. Panisko; Jon K. Magnuson; Daniel Cullen; Kenneth E. Hammel

    2008-01-01

    Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew...

  16. Yeast Interacting Proteins Database: YJR091C, YOR317W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...NAs encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes

  17. Yeast Interacting Proteins Database: YJR091C, YLR059C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes... mRNAs encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes

  18. NCBI nr-aa BLAST: CBRC-DMEL-04-0042 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available ike protein [Geobacter uraniumreducens Rf4] gb|ABQ26219.1| Uncharacterized membrane-associated protein-like protein [Geobacter uraniumreducens Rf4] YP_001230792.1 4.3 28% ... ...CBRC-DMEL-04-0042 ref|YP_001230792.1| Uncharacterized membrane-associated protein-l

  19. Yeast Interacting Proteins Database: YJR091C, YHR026W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...s with mRNAs encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes

  20. Arabidopsis CDS blastp result: AK242307 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242307 J075194A02 At1g78920.1 68414.m09201 vacuolar-type H+-translocating inorganic... pyrophosphatase (AVPL1) identical to vacuolar-type H+-translocating inorganic pyrophosphatase GI:6901676 from [Arabidopsis thaliana] 5e-41 ...

  1. Arabidopsis CDS blastp result: AK070310 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070310 J023047F16 At1g16780.1 vacuolar-type H+-translocating inorganic pyrophosph...atase, putative similar to vacuolar-type H+-translocating inorganic pyrophosphatase GI:6901676 from [Arabidopsis thaliana] 0.0 ...

  2. Arabidopsis CDS blastp result: AK242307 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242307 J075194A02 At1g16780.1 68414.m02016 vacuolar-type H+-translocating inorganic... pyrophosphatase, putative similar to vacuolar-type H+-translocating inorganic pyrophosphatase GI:6901676 from [Arabidopsis thaliana] 2e-40 ...

  3. Yeast Interacting Proteins Database: YDL226C, YJL151C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available s bait as prey (0) YJL151C SNA3 Integral membrane protein localized to vacuolar intralumenal vesicles, computational...intralumenal vesicles, computational analysis of large-scale protein-protein interaction data suggests a pos... gene name SNA3 Prey description Integral membrane protein localized to vacuolar

  4. EST Table: BP126482 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 214#WBGene00016643#locus:vps-45#yeast vacuolar protein sorting protein 33 (SLP1)#status:Partially_confirmed#...2324|ref|XP_970273.1| PREDICTED: similar to Vacuolar protein sorting-associated protein 45 (mVps45) [Tribolium castaneum] BP126820 psV3 ...

  5. EST Table: BP126820 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available #CE04214#WBGene00016643#locus:vps-45#yeast vacuolar protein sorting protein 33 (SLP1)#status:Partially_confi...gi|91092324|ref|XP_970273.1| PREDICTED: similar to Vacuolar protein sorting-associated protein 45 (mVps45) [Tribolium castaneum] BP126820 psV3 ...

  6. Gclust Server: 90378 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available Sequences - 1044 vacuolar-type H+-translocating inorganic pyrophosphatase 1 1.00e+00 0.0 11.11 0.0 0.0 0...Representative annotation vacuolar-type H+-translocating inorganic pyrophosphatase Number of Sequences 1 Homologs

  7. Yeast Interacting Proteins Database: YHR114W, YDR422C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available substrate specificity; vacuolar protein containing KIS (Kinase-Interacting Sequence) and ASC (Association w...strate specificity; vacuolar protein containing KIS (Kinase-Interacting Sequence) and ASC (Association with ...e 4 CuraGen (0 or 1) 0 S. Fields (0 or 1) 0 Association (0 or 1,YPD) 0 Complex (0

  8. A functional connection of Dictyostelium paracaspase with the ...

    Indian Academy of Sciences (India)

    Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the ...

  9. Dicty_cDB: Contig-U09253-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available RecName: Full=Class E vacuolar protein-sorting machiner... 50 6e-05 AK315914_1( AK315914 |pid:none) Homo...RecName: Full=Class E vacuolar protein-sorting machiner... 49 1e-04 CR859070_1( CR859070 |pid:none) Pongo...RecName: Full=Class E vacuolar protein-sorting machiner... 47 4e-04 X62852_1( X62852 |pid:none) H.sapiens...RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.001 FJ629586_1( FJ629586 |pid:none) Synthetic...protein-sorting machiner... 45 0.002 (Q5BBL4) RecName: Full=Class E vacuolar protein-sorting machiner... 45 0

  10. Dicty_cDB: Contig-U13241-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available protein-sorting machiner... 46 0.003 (Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 46 0...RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.007 BX537102_4( BX537102 |pid:none) Zebrafish...RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.007 AB168387_1( AB168387 |pid:none) Macaca...RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.009 BC085613_1( BC085613 |pid:none) Homo...RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.011 ( P85001 ) RecName: Full=Centrosomal

  11. Dicty_cDB: Contig-U15646-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available RecName: Full=Class E vacuolar protein-sorting machiner... 54 2e-05 CR382131_1211( CR382131 |pid:none)...RecName: Full=Class E vacuolar protein-sorting machiner... 50 3e-04 ( O13154 ) RecName: Full=Protein kinase...RecName: Full=Class E vacuolar protein-sorting machiner... 49 5e-04 (A3LYI0) RecName: Full=Stress response...RecName: Full=Class E vacuolar protein-sorting machiner... 49 0.001 ( P39743 ) RecName: Full=Reduced viability...RecName: Full=Class E vacuolar protein-sorting machiner... 48 0.002 AK129064_1( AK129064 |pid:none) Mus

  12. Dicty_cDB: Contig-U12287-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available RecName: Full=Class E vacuolar protein-sorting machiner... 53 2e-05 ( Q99963 ) RecName: Full=Endophilin-A3;...RecName: Full=Class E vacuolar protein-sorting machiner... 50 1e-04 AF067617_4( AF067617 |pid:none) Caenorhabditis...RecName: Full=Class E vacuolar protein-sorting machiner... 46 0.003 AM392656_1( AM392656 |pid:none) Synthetic...RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.010 AF182199_1( AF182199 |pid:none) Homo...RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.010 AL392107_2( AL392107 |pid:none) Human

  13. Dicty_cDB: Contig-U12117-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available RecName: Full=Class E vacuolar protein-sorting machiner... 48 5e-04 FN392321_512( FN392321 |pid:none)...RecName: Full=Class E vacuolar protein-sorting machiner... 47 0.001 BC090430_1( BC090430 |pid:none) Danio...RecName: Full=Class E vacuolar protein-sorting machiner... 46 0.002 AK160794_1( AK160794 |pid:none) Mus...RecName: Full=Class E vacuolar protein-sorting machiner... 42 0.023 AK291025_1( AK291025 |pid:none) Homo...RecName: Full=Class E vacuolar protein-sorting machiner... 42 0.030 A46243( A46243 )epidermal growth

  14. Dicty_cDB: Contig-U08836-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q6BNP6) RecName: Full=Class E vacuolar protein-sorting machiner... 43 0.009 BC124012_1( BC124012 |pid:none)...(Q6C2N2) RecName: Full=Class E vacuolar protein-sorting machiner... 37 0.64 AP007164_582( AP007164 |pid:none)...(A4RF61) RecName: Full=Class E vacuolar protein-sorting machiner... 37 0.84 V01170_1( V01170 |pid:none) Y73 sarcoma...(Q5KFQ8) RecName: Full=Class E vacuolar protein-sorting machiner... 37 0.84 AM270061_75( AM270061 |pid:none)...(Q4P5J4) RecName: Full=Class E vacuolar protein-sorting machiner... 33 9.3 JQ1321( JQ1321 ;S18974) protein-tyrosine

  15. Dicty_cDB: Contig-U15202-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 47 4e-04 BC166180_1( BC166180 |pid:none)...(Q4WHP5) RecName: Full=Class E vacuolar protein-sorting machiner... 47 5e-04 BC082699_1( BC082699 |pid:none)...(Q0CJU8) RecName: Full=Class E vacuolar protein-sorting machiner... 47 6e-04 (A0JNJ1) RecName: Full=SH3 and cysteine-rich...(Q5BBL4) RecName: Full=Class E vacuolar protein-sorting machiner... 47 6e-04 ( P97306 ) RecName: Full=SH3 and...(Q4P5J4) RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.001 DQ214657_1( DQ214657 |pid:none)

  16. Dicty_cDB: Contig-U14179-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q7S6J4) RecName: Full=Class E vacuolar protein-sorting machiner... 42 0.037 FN357528_10( FN357528 |pid:none)...(Q4P5J4) RecName: Full=Class E vacuolar protein-sorting machiner... 41 0.11 (Q8K1I7) RecName: Full=WAS/WASL-interacting...protein-sorting machiner... 41 0.11 (Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 41...(Q6C2N2) RecName: Full=Class E vacuolar protein-sorting machiner... 39 0.32 M57290_3( M57290 |pid:none) Chicken...(Q6CVA8) RecName: Full=Class E vacuolar protein-sorting machiner... 39 0.32 ( P25911 ) RecName: Full=Tyrosine-protein

  17. Dicty_cDB: Contig-U12140-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q7S6J4) RecName: Full=Class E vacuolar protein-sorting machiner... 48 0.001 CU633895_130( CU633895 |pid:none)...(Q5BBL4) RecName: Full=Class E vacuolar protein-sorting machiner... 46 0.004 EF575994_1( EF575994 |pid:none)...(Q0CJU8) RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.014 AB173827_1( AB173827 |pid:none)...protein-sorting machiner... 43 0.032 (Q5KFQ8) RecName: Full=Class E vacuolar protein-sorting machiner... 43...(Q6C2N2) RecName: Full=Class E vacuolar protein-sorting machiner... 42 0.042 AK302278_1( AK302278 |pid:none)

  18. Dicty_cDB: Contig-U04139-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q7S6J4) RecName: Full=Class E vacuolar protein-sorting machiner... 45 0.002 AF067617_4( AF067617 |pid:none)...(Q4P5J4) RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.005 AY398419_1( AY398419 |pid:none)...(A4RF61) RecName: Full=Class E vacuolar protein-sorting machiner... 44 0.005 BC166180_1( BC166180 |pid:none)...(Q75DS3) RecName: Full=Class E vacuolar protein-sorting machiner... 42 0.015 AL732325_2( AL732325 |pid:none)...(Q5KFQ8) RecName: Full=Class E vacuolar protein-sorting machiner... 41 0.025 AE017341_341( AE017341 |pid:none)

  19. Dicty_cDB: Contig-U11224-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q5A895) RecName: Full=Class E vacuolar protein-sorting machiner... 55 5e-06 AB024330_1( AB024330 |pid:none)...(Q6BNP6) RecName: Full=Class E vacuolar protein-sorting machiner... 54 1e-05 AK122480_1( AK122480 |pid:none)...(Q75DS3) RecName: Full=Class E vacuolar protein-sorting machiner... 54 1e-05 EF134956_1( EF134956 |pid:none)...(Q6C2N2) RecName: Full=Class E vacuolar protein-sorting machiner... 54 2e-05 AB037472_1( AB037472 |pid:none)...(Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 52 6e-05 AF203899_1( AF203899 |pid:none)

  20. Dicty_cDB: Contig-U05629-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available (Q5KFQ8) RecName: Full=Class E vacuolar protein-sorting machiner... 53 3e-06 X16316_1( X16316 |pid:none) Human...protein-sorting machiner... 53 4e-06 (A4RF61) RecName: Full=Class E vacuolar protein-sorting machiner... 53...(Q6FN49) RecName: Full=Class E vacuolar protein-sorting machiner... 53 4e-06 BC085566_1( BC085566 |pid:none)...) RecName: Full=Class E vacuolar protein-sorting machiner... 52 9e-06 ( O75886 ) RecName: Full=Signal...(Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 50 2e-05 AE016816_470( AE016816 |pid:none)

  1. Dicty_cDB: Contig-U14963-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available protein-sorting machiner... 52 1e-04 (Q0CJU8) RecName: Full=Class E vacuolar protein-sorting machiner... 51...(Q4WHP5) RecName: Full=Class E vacuolar protein-sorting machiner... 51 2e-04 FN392320_986( FN392320 |pid:none)...protein-sorting machiner... 47 0.002 (Q1E878) RecName: Full=Class E vacuolar protein-sorting machiner... 47...(Q6FN49) RecName: Full=Class E vacuolar protein-sorting machiner... 47 0.004 BC085566_1( BC085566 |pid:none)...(A4RF61) RecName: Full=Class E vacuolar protein-sorting machiner... 46 0.006 AL117204_38( AL117204 |pid:none)

  2. SwissProt search result: AK120333 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120333 J013059C19 (P97690) Structural maintenance of chromosome 3 (Chondroitin sulfate proteoglycan... 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 2e-18 ...

  3. SwissProt search result: AK242597 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242597 J090013N08 (P97690) Structural maintenance of chromosome 3 (Chondroitin sulfate proteoglycan... 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 1e-18 ...

  4. SwissProt search result: AK065733 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065733 J013038C11 (P97690) Structural maintenance of chromosome 3 (Chondroitin sulfate proteoglycan... 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 4e-18 ...

  5. SwissProt search result: AK103514 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103514 J033131G15 (P97690) Structural maintenance of chromosome 3 (Chondroitin sulfate proteoglycan... 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 7e-32 ...

  6. Yeast Interacting Proteins Database: YJR091C, YKL113C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...olved in localizing the Arp2/3 complex to mitochondria; overexpression causes increased sensitivity to benom

  7. Developmental biology: vasculogenesis is a wreck without RECK.

    Science.gov (United States)

    Welm, Bryan; Mott, Joni; Werb, Zena

    2002-03-19

    The unique membrane-associated inhibitor of matrix metalloproteinases, RECK, is required for vascular maturation during embryogenesis. The phenotype of a loss of function mutation of RECK shows the importance of pericellular proteolysis in development.

  8. Yeast Interacting Proteins Database: YJR091C, YEL013W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...ed proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes increased sens

  9. Yeast Interacting Proteins Database: YJR091C, YOR265W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...ns; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes increased sensitivity t

  10. SwissProt search result: AK065733 [KOME

    Lifescience Database Archive (English)

    Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 4e-18 ...

  11. SwissProt search result: AK120333 [KOME

    Lifescience Database Archive (English)

    Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 2e-18 ...

  12. SwissProt search result: AK242597 [KOME

    Lifescience Database Archive (English)

    Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 5e-19 ...

  13. Arabidopsis CDS blastp result: AK105135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105135 001-102-A05 At2g27170.1 structural maintenance of chromosomes (SMC) family protein similar to basem...ent membrane-associated chondroitin proteoglycan Bamacan [Rattus norvegicus] GI:178

  14. SwissProt search result: AK110012 [KOME

    Lifescience Database Archive (English)

    Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 1e-14 ...

  15. SwissProt search result: AK103514 [KOME

    Lifescience Database Archive (English)

    Full Text Available lfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 1e-33 ...

  16. SwissProt search result: AK105135 [KOME

    Lifescience Database Archive (English)

    Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 3e-61 ...

  17. SwissProt search result: AK064293 [KOME

    Lifescience Database Archive (English)

    Full Text Available ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) (Mad member-interacting protein 1) SMC3_MOUSE 2e-15 ...

  18. Yeast Interacting Proteins Database: YJR091C, YMR067C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...olved in localizing the Arp2/3 complex to mitochondria; overexpression causes increased sensitivity to benom

  19. Yeast Interacting Proteins Database: YJR091C, YKL076C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...Arp2/3 complex to mitochondria; overexpression causes increased sensitivity to benomyl Rows with this bait a

  20. Yeast Interacting Proteins Database: YJR091C, YDL147W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpression causes...mplex to mitochondria; overexpression causes increased sensitivity to benomyl Rows with this bait as bait Ro

  1. NADH→NAD+ Transhydrogenation in Adult Ascaris suum Mitochondria

    National Research Council Canada - National Science Library

    Andrew Holowiecki; Carmen F. Fioravanti

    2015-01-01

    Although lacking an NADPH→NAD+ transhydrogenase system, the essentially energetically anaerobic mitochondria of the adult intestinal nematode Ascaris suum display an inner membrane-associated NADH→NAD...

  2. Cholesterol, sphingolipids, and glycolipids: What do we know about their role in raft-like membranes?

    DEFF Research Database (Denmark)

    Rog, T.; Vattulainen, I.

    2014-01-01

    Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units with pote......Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units...

  3. PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress

    Science.gov (United States)

    Ding, Jun; Holzwarth, Garrett; Bradford, C. Samuel; Cooley, Ben; Yoshinaga, Allen S.; Patton-Vogt, Jana; Abeliovich, Hagai; Penner, Michael H.; Bakalinsky, Alan T.

    2017-01-01

    In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity. PMID:26051671

  4. PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress.

    Science.gov (United States)

    Ding, Jun; Holzwarth, Garrett; Bradford, C Samuel; Cooley, Ben; Yoshinaga, Allen S; Patton-Vogt, Jana; Abeliovich, Hagai; Penner, Michael H; Bakalinsky, Alan T

    2015-10-01

    In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.

  5. CLUTO - A Clustering Toolkit

    Science.gov (United States)

    2002-04-23

    CNTF CNTFR CX43 cyclinA...Brm CCO1 CCO2 cellubrevin cjun CNTF CNTFR CX43 cyclinA cyclinB EGF FABP G67I8086 G67I86 GAD65 GAD67 GAP43 GFAP GMFb GRa1 GRa2 GRa3 GRa4 GRa5 GRb1 GRb2...te r 4 5HT1b 5HT2 5HT3 ACHE actin aFGF BDNF bFGF Brm CCO1 CCO2 cellubrevin cjun CNTF CNTFR CX43 cyclinA cyclinB EGF FABP G67I8086 G67I86 GAD65

  6. Polyphosphate and Acidocalcisomes

    Science.gov (United States)

    Lander, Noelia; Cordeiro, Ciro; Huang, Guozhong; Docampo, Roberto

    2016-01-01

    Inorganic polyphosphate (polyP) accumulates in acidocalcisomes, acidic calcium stores that have been found from bacteria to human cells. Proton pumps, such as the vacuolar proton pyrophosphatase (V-H+-PPase, or VP1), the vacuolar proton ATPase (V-H+-ATPase), or both, maintain their acidity. A vacuolar transporter chaperone complex (VTC) is involved in the synthesis and translocation of polyP to these organelles in several eukaryotes, such as yeast, trypanosomatids, Apicomplexan, and algae. Studies in trypanosomatids have revealed the role of polyP and acidocalcisomes in osmoregulation and calcium signaling. PMID:26862180

  7. Does autophagy have a license to kill mammalian cells?

    NARCIS (Netherlands)

    Scarlatti, F.; Granata, R.; Meijer, A. J.; Codogno, P.

    2009-01-01

    Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the

  8. Hev b 11, a peculiar class I chitinase?

    NARCIS (Netherlands)

    Beintema, J. J.

    2007-01-01

    The recently identified rubber allergen Hev b 11, which is a class I chitinase, may be a cytosolic (C-serum) protein. This is a rather unique feature, as all other known plant class I chitinases are vacuolar proteins.

  9. Sequence Classification: 891979 [

    Lifescience Database Archive (English)

    Full Text Available nown function, component of the Swr1p complex that incorporates Htz1p into chromatin; required for vacuolar protein sorting; Vps71p || http://www.ncbi.nlm.nih.gov/protein/6323600 ...

  10. [Processing and targeting of the thiol protease aleurain

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1993-01-01

    Progress is reported in characterizing the processing steps and intracellular destination of aleurain including identification of vacuolar targeting determinants, purification from barley leaf tissue to homogeneity, and comparisons of structures and Michaelis enzyme kinetics of proaleurain and procathepsin H.

  11. [Processing and targeting of the thiol protease aleurain]. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1993-04-01

    Progress is reported in characterizing the processing steps and intracellular destination of aleurain including identification of vacuolar targeting determinants, purification from barley leaf tissue to homogeneity, and comparisons of structures and Michaelis enzyme kinetics of proaleurain and procathepsin H.

  12. THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    S. V. Isayenkov

    2013-06-01

    Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

  13. Sequence Classification: 893456 [

    Lifescience Database Archive (English)

    Full Text Available cles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes to the vacuole, involved in endocytosis; Did4p || http://www.ncbi.nlm.nih.gov/protein/9755336 ...

  14. Yeast Interacting Proteins Database: YKL002W, YDL165W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes...ins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes t

  15. Yeast Interacting Proteins Database: YKL002W, YLR423C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthes... into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes to t

  16. Sequence Classification: 892097 [

    Lifescience Database Archive (English)

    Full Text Available into the multivesicular body pathway to the lysosomal/vacuolar lumen; cytoplasmic protein recruited to endosomal membranes; Vps20p || http://www.ncbi.nlm.nih.gov/protein/6323723 ...

  17. Insights into the genome of large sulfur bacteria revealed by analysis of single filaments

    DEFF Research Database (Denmark)

    Mussmann, Marc; Hu, Fen Z.; Richter, Michael

    2007-01-01

    . We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable...... Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur...

  18. The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue.

    Science.gov (United States)

    Yoshida, Kumi; Kawachi, Miki; Mori, Mihoko; Maeshima, Masayoshi; Kondo, Maki; Nishimura, Mikio; Kondo, Tadao

    2005-03-01

    The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.

  19. Calcium Signals from the Vacuole

    Directory of Open Access Journals (Sweden)

    Gerald Schönknecht

    2013-10-01

    Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

  20. Extracellular localization of catalase is associated with the transformed state of malignant cells.

    Science.gov (United States)

    Böhm, Britta; Heinzelmann, Sonja; Motz, Manfred; Bauer, Georg

    2015-12-01

    Oncogenic transformation is dependent on activated membrane-associated NADPH oxidase (NOX). However, the resultant extracellular superoxide anions are also driving the NO/peroxynitrite and the HOCl pathway, which eliminates NOX-expressing transformed cells through selective apoptosis induction. Tumor progression is dependent on dominant interference with intercellular apoptosis-inducing ROS signaling through membrane-associated catalase, which decomposes H2O2 and peroxynitrite and oxidizes NO. Particularly, the decomposition of extracellular peroxynitrite strictly requires membrane-associated catalase. We utilized small interfering RNA (siRNA)-mediated knockdown of catalase and neutralizing antibodies directed against the enzyme in combination with challenging H2O2 or peroxynitrite to determine activity and localization of catalase in cells from three distinct steps of multistage oncogenesis. Nontransformed cells did not generate extracellular superoxide anions and only showed intracellular catalase activity. Transformed cells showed superoxide anion-dependent intercellular apoptosis-inducing ROS signaling in the presence of suboptimal catalase activity in their membrane. Tumor cells exhibited tight control of intercellular apoptosis-inducing ROS signaling through a high local concentration of membrane-associated catalase. These data demonstrate that translocation of catalase to the outside of the cell membrane is already associated with the transformation step. A strong local increase in the concentration of membrane-associated catalase is achieved during tumor progression and is controlled by tumor cell-derived H2O2 and by transglutaminase.

  1. Use of a subunit feline leukemia virus vaccine in exotic cats.

    Science.gov (United States)

    Citino, S B

    1988-04-01

    Three adult bengal tigers, 2 immature white tigers, and 3 adult servals were vaccinated IM with three 1-ml doses of a subunit FeLV vaccine with dosage interval guidelines of the manufacturer. All cats had increased antibody titers to FeLV gp 70 capsular antigen and feline oncornavirus cell membrane-associated antigen during the vaccination trial. Three weeks after the third vaccination, 7 of the 8 cats had gp70 antibody titers greater than 0.2 (optical density), and all 8 cats had feline oncornavirus cell membrane-associated antigen antibody titers greater than 1:8.

  2. Discovery of the breast cancer gene BASE using a molecular approach to enrich for genes encoding membrane and secreted proteins

    OpenAIRE

    Egland, Kristi A.; Vincent, James J.; Strausberg, Robert; Lee, Byungkook; Pastan, Ira

    2003-01-01

    To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones ...

  3. Regulation of transport processes across the tonoplast membrane

    Directory of Open Access Journals (Sweden)

    Oliver eTrentmann

    2014-09-01

    Full Text Available In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g. due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation.

  4. Shigella subverts the host recycling compartment to rupture its vacuole.

    Science.gov (United States)

    Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

    2014-10-08

    Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

    Science.gov (United States)

    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  6. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Zhang

    2010-06-01

    Full Text Available Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H(+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma(- phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca(2+ and H(+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

  7. Characterization of a Novel Prevacuolar Compartment in Neurospora crassa.

    Science.gov (United States)

    Bowman, Barry J; Draskovic, Marija; Schnittker, Robert R; El-Mellouki, Tarik; Plamann, Michael D; Sánchez-León, Eddy; Riquelme, Meritxell; Bowman, Emma Jean

    2015-12-01

    Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

    Directory of Open Access Journals (Sweden)

    Kannan Alpadi

    2012-01-01

    Full Text Available SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a, Q(b, and Q(c from target (t-SNAREs and one (R from the vesicular (v-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a SNARE, leaving behind a Q(bcR subcomplex. This subcomplex serves as an acceptor for a Q(a SNARE from the opposite membrane, leading to Q(a-Q(bcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bcR cis-complex and the formation of the Q(a-Q(bcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex. This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

  9. Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion

    Science.gov (United States)

    Alpadi, Kannan; Kulkarni, Aditya; Comte, Veronique; Reinhardt, Monique; Schmidt, Andrea; Namjoshi, Sarita; Mayer, Andreas; Peters, Christopher

    2012-01-01

    SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Qa, Qb, and Qc) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Qa-QbcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the QbcR cis-complex and the formation of the Qa-QbcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology. PMID:22272185

  10. Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens

    DEFF Research Database (Denmark)

    Rosenberg, Carina Agerbo; De Craeye, S.; Jongert, E.

    2009-01-01

    Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complicat......Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological...... complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic...

  11. SwissProt search result: AK110012 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110012 002-159-G11 (Q9UQE7) Structural maintenance of chromosome 3 (Chondroitin s...ulfate proteoglycan 6) (Chromosome-associated polypeptide) (hCAP) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_HUMAN 2e-14 ...

  12. SwissProt search result: AK110012 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110012 002-159-G11 (P97690) Structural maintenance of chromosome 3 (Chondroitin s...ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 5e-14 ...

  13. SwissProt search result: AK242597 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242597 J090013N08 (Q9UQE7) Structural maintenance of chromosome 3 (Chondroitin su...lfate proteoglycan 6) (Chromosome-associated polypeptide) (hCAP) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_HUMAN 1e-18 ...

  14. SwissProt search result: AK103514 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103514 J033131G15 (Q9UQE7) Structural maintenance of chromosome 3 (Chondroitin su...lfate proteoglycan 6) (Chromosome-associated polypeptide) (hCAP) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_HUMAN 2e-33 ...

  15. SwissProt search result: AK105135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105135 001-102-A05 (P97690) Structural maintenance of chromosome 3 (Chondroitin s...ulfate proteoglycan 6) (Chromosome segregation protein SmcD) (Bamacan) (Basement membrane-associated chondroitin proteoglycan) SMC3_RAT 3e-50 ...

  16. Galectin-9: From cell biology to complex disease dynamics

    Indian Academy of Sciences (India)

    2016-07-16

    Jul 16, 2016 ... was seen during spontaneous abortion. Variants of membrane-associated Gal-9 (Ecalectin, hUAT) is also seen, which differs only in few amino acids (Heusschen et al. 2013). Further, splicing of the linker encoding exons has been shown to influence galectin-9 valency and function. (Heusschen et al. 2013) ...

  17. Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

    DEFF Research Database (Denmark)

    Rumessen, Jüri Johs.; Vanderwinden, Jean-Marie; Rasmussen, Helle

    2009-01-01

    and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells......, and close contacts (lamina, and peg and socket...

  18. Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

    NARCIS (Netherlands)

    Ulrich, AS; Glabe, CG; Hoekstra, D

    1998-01-01

    Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal

  19. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis

    DEFF Research Database (Denmark)

    Kallipolitis, B H; Ingmer, H

    2001-01-01

    Environmental sensing by two-component signal transduction systems is likely to play a role for growth and survival of Listeria monocytogenes both during transmission in food products and within a host organism. Two-component systems typically consist of a membrane-associated sensor histidine...

  20. The actin homologue MreB organizes the bacterial cell membrane

    NARCIS (Netherlands)

    Strahl, H.; Burmann, F.; Hamoen, L.W.

    2014-01-01

    The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

  1. Effect of monoclonal antibodies on phagocytosis and killing of Toxoplasma gondii by normal macrophages.

    OpenAIRE

    Hauser, W E; Remington, J S

    1981-01-01

    Treatment of intact toxoplasma tachyzoites with individual mouse monoclonal antibodies to toxoplasma which are directed against individual membrane-associated antigenic determinants facilitated the phagocytosis of toxoplasma and also prepared the toxoplasma for intracellular destruction by nonelicited mouse peritoneal macrophages. In instances in which the organisms survived intracellularly, their multiplication was significantly reduced. Such monoclonal antibodies should be useful in further...

  2. Polyphosphate nanoparticles on the platelet surface trigger contact system activation.

    Science.gov (United States)

    Verhoef, Johan J F; Barendrecht, Arjan D; Nickel, Katrin F; Dijkxhoorn, Kim; Kenne, Ellinor; Labberton, Linda; McCarty, Owen J T; Schiffelers, Raymond; Heijnen, Harry F; Hendrickx, Antoni P; Schellekens, Huub; Fens, Marcel H; de Maat, Steven; Renné, Thomas; Maas, Coen

    2017-03-23

    Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII. © 2017 by The American Society of Hematology.

  3. Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

    NARCIS (Netherlands)

    Kronenburg, N.A.E.; Bont, de J.A.M.; Fischer, L.

    2001-01-01

    The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were

  4. Vibrio chromosome-specific families

    DEFF Research Database (Denmark)

    Lukjancenko, Oksana; Ussery, David

    2014-01-01

    many membrane-associated activities, such as ion channels, transmembrane transporters, and electron transport chain proteins. Thus, it appears that whilst there are many "housekeeping systems" encoded in chromosome 1, there are far fewer core functions found in chromosome 2. However, the presence...

  5. The native architecture of a photosynthetic membrane

    NARCIS (Netherlands)

    Bahatyrova, S.; Frese, R.N.; Siebert, C. Alistair; Olsen, John D.; van der Werf, Kees; van Grondelle, Rienk; Niederman, Robert A.; Bulough, Per A.; Otto, Cornelis; Hunter, C. Neil

    2004-01-01

    In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membraneassociated chlorophyll–protein complexes. Although the detailed structure of each complex has been determined1–4, the

  6. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    DEFF Research Database (Denmark)

    Botkjaer, Kenneth A; Kwok, Hang Fai; Terp, Mikkel G

    2016-01-01

    The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential the...

  7. Exchange of catenins in cadherin-catenin complex

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Troyanovsky, Regina B; Laur, Oscar Y

    2003-01-01

    beta-Catenin is an intracellular multifunctional protein. In complex with the transmembrane adhesive receptor E-cadherin, it becomes plasma membrane-associated and mediates intercellular adhesion. A cytosolic pool of beta-catenin interacts with DNA-binding proteins and participates in signal tran...

  8. Isoprenoid biosynthesis in Archaea - Biochemical and evolutionary implications

    NARCIS (Netherlands)

    Matsumi, R.; Atomi, H.; Driessen, A.J.M.; Oost, van der J.

    2011-01-01

    Isoprenoids are indispensable for all types of cellular life in the Archaea, Bacteria, and Eucarya. These membrane-associated molecules are involved in a wide variety of vital biological functions, ranging from compartmentalization and stability, to protection and energy-transduction. In Archaea,

  9. Isoprenoid biosynthesis in Archaea - Biochemical and evolutionary implications

    NARCIS (Netherlands)

    Matsumi, Rie; Atomi, Haruyuki; Driessen, Arnold J. M.; van der Oost, John

    Isoprenoids are indispensable for all types of cellular life in the Archaea, Bacteria, and Eucarya. These membrane-associated molecules are involved in a wide variety of vital biological functions, ranging from compartmentalization and stability, to protection and energy-transduction. In Archaea,

  10. RGM is a repulsive guidance molecule for retinal axons

    DEFF Research Database (Denmark)

    Monnier, Philippe P; Sierra, Ana; Macchi, Paolo

    2002-01-01

    the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology...

  11. Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo

    NARCIS (Netherlands)

    Holtmaat, Anthony J D G; Hermens, W.T.J.M.C.; Sonnemans, M.A.F.; Giger, Roman J; Van Leeuwen, F W; Kaplitt, M G; Oestreicher, A B; Gispen, Willem Hendrik; Verhaagen, J

    1997-01-01

    B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established

  12. Production of dehydroamino acid-containing peptides by Lactococcus lactis

    NARCIS (Netherlands)

    Rink, Rick; Wierenga, Jenny; Kuipers, Anneke; Kluskens, Leon D.; Driessen, Arnold J.M.; Kuipers, Oscar P.; Moll, Gert N.

    2007-01-01

    Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In

  13. Photointeraction between substituted benzoquinones and heterocyclic compounds

    Science.gov (United States)

    Porkhun, V. I.; Aristova, Yu. V.; Sharkevich, I. V.

    2017-07-01

    It is shown that mechanisms of photoreactions, investigated using the effects of chemically-induced dynamic nuclear polarization (CIDNP), can serve as models of primary electron transfer during photosynthesis from sensitizers (chlorophyll or porphyrin) to acceptors (membrane-associated quinone in media with different polarities).

  14. A unique mechanism for protein processing and degradation in Arabidopsis thaliana

    Science.gov (United States)

    Rojo, Enrique; Zouhar, Jan; Carter, Clay; Kovaleva, Valentina; Raikhel, Natasha V.

    2003-01-01

    Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-γ (VPEγ) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEγ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpeγ mutants but not in WT plants, suggesting a unique role of VPEγ in protein processing and degradation in Arabidopsis. PMID:12773619

  15. Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

    Science.gov (United States)

    van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

    2015-04-01

    Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Proteome analysis of potato juice and tuber vacuoles from cv. Kuras

    DEFF Research Database (Denmark)

    Jørgensen, Malene

    from the vacuoles of mature potato tuber. A comprehensive investigation of the vacuolar proteome and a detailed analysis of the identified proteins and their possible roles in vacuolar function were carried out. A primary requirement of any proteomic analysis of an organelle is purity of the isolated......-grown potato tubers cv. Kuras were used for isolation of vacuoles in order to do an organelle based proteome analysis and to gain a better fundament for understanding the vacuolar targeting mechanisms in plants.   In this study complementary methods were used to identify high and low abundance proteins derived...... organelle. A method was developed for isolation of highly purified intact vacuoles. The proteome analysis of the purified vacuoles involved separation of native soluble proteins by gel filtration with Superdex 200 into nine fractions. Each fraction was analyzed in two ways, by SDS-PAGE followed by protein...

  17. The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis.

    Science.gov (United States)

    Coonrod, Emily M; Stevens, Tom H

    2010-12-01

    In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

  18. The yeast VPS genes affect telomere length regulation.

    Science.gov (United States)

    Rog, Ofer; Smolikov, Sarit; Krauskopf, Anat; Kupiec, Martin

    2005-01-01

    Eukaryotic cells invest a large proportion of their genome in maintaining telomere length homeostasis. Among the 173 non-essential yeast genes found to affect telomere length, a large proportion is involved in vacuolar traffic. When mutated, these vacuolar protein-sorting (VPS) genes lead to telomeres shorter than those observed in the wild type. Using genetic analysis, we characterized the pathway by which VPS15, VPS34, VPS22, VPS23 and VPS28 affect the telomeres. Our results indicate that these VPS genes affect telomere length through a single pathway and that this effect requires the activity of telomerase and the Ku heterodimer, but not the activity of Tel1p or Rif2p. We present models to explain the link between vacuolar traffic and telomere length homeostasis.

  19. Yeast translation elongation factor-1A binds vacuole-localized Rho1p to facilitate membrane integrity through F-actin remodeling.

    Science.gov (United States)

    Bodman, James A R; Yang, Yang; Logan, Michael R; Eitzen, Gary

    2015-02-20

    Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Hedman, Jamie M; Eggleston, Matthew D; Attryde, Amanda L; Marshall, Pamela A

    2007-10-01

    Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.