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Sample records for er membrane proteins

  1. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.

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    Manford, Andrew G; Stefan, Christopher J; Yuan, Helen L; Macgurn, Jason A; Emr, Scott D

    2012-12-11

    Endoplasmic reticulum-plasma membrane (ER-PM) junctions are conserved structures defined as regions of the ER that tightly associate with the plasma membrane. However, little is known about the mechanisms that tether these organelles together and why such connections are maintained. Using a quantitative proteomic approach, we identified three families of ER-PM tethering proteins in yeast: Ist2 (related to mammalian TMEM16 ion channels), the tricalbins (Tcb1/2/3, orthologs of the extended synaptotagmins), and Scs2 and Scs22 (vesicle-associated membrane protein-associated proteins). Loss of all six tethering proteins results in the separation of the ER from the PM and the accumulation of cytoplasmic ER. Importantly, we find that phosphoinositide signaling is misregulated at the PM, and the unfolded protein response is constitutively activated in the ER in cells lacking ER-PM tether proteins. These results reveal critical roles for ER-PM contacts in cell signaling, organelle morphology, and ER function. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

    Science.gov (United States)

    Barz, W P; Walter, P

    1999-04-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

  3. A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

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    Sujoy Lahiri

    2014-10-01

    Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

  4. A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis.

    Science.gov (United States)

    Joshi, Amit S; Huang, Xiaofang; Choudhary, Vineet; Levine, Tim P; Hu, Junjie; Prinz, William A

    2016-11-21

    Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

  5. An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

    DEFF Research Database (Denmark)

    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco

    2014-01-01

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary...... phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic...... and explain how cells switch neutral lipid metabolism from storage to consumption....

  6. An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

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    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

    2014-01-16

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

    Directory of Open Access Journals (Sweden)

    Daniel F. Markgraf

    2014-01-01

    Full Text Available Eukaryotic cells store neutral lipids such as triacylglycerol (TAG in lipid droplets (LDs. Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER. We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG. During LD breakdown in early exponential phase, an ER membrane protein (Ice2p facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.

  8. Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

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    Kasaras Alexis

    2012-04-01

    Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

  9. ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

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    Melisa C Monteleone

    Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

  10. The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

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    Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

    2007-05-01

    The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

  11. Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network.

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    Wang, Songyu; Tukachinsky, Hanna; Romano, Fabian B; Rapoport, Tom A

    2016-09-13

    In higher eukaryotes, the endoplasmic reticulum (ER) contains a network of membrane tubules, which transitions into sheets during mitosis. Network formation involves curvature-stabilizing proteins, including the reticulons (Rtns), as well as the membrane-fusing GTPase atlastin (ATL) and the lunapark protein (Lnp). Here, we have analyzed how these proteins cooperate. ATL is needed to not only form, but also maintain, the ER network. Maintenance requires a balance between ATL and Rtn, as too little ATL activity or too high Rtn4a concentrations cause ER fragmentation. Lnp only affects the abundance of three-way junctions and tubules. We suggest a model in which ATL-mediated fusion counteracts the instability of free tubule ends. ATL tethers and fuses tubules stabilized by the Rtns, and transiently sits in newly formed three-way junctions. Lnp subsequently moves into the junctional sheets and forms oligomers. Lnp is inactivated by mitotic phosphorylation, which contributes to the tubule-to-sheet conversion of the ER.

  12. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

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    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © 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.

  13. The ER in 3-D: a multifunctional dynamic membrane network

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    Friedman, Jonathan R.; Voeltz, Gia K.

    2011-01-01

    The endoplasmic reticulum (ER) is a large, singular, membrane-bound organelle that has an elaborate 3-D structure with a diversity of structural domains. It contains regions that are flat and cisternal, ones that are highly curved and tubular, and others adapted to form contact with nearly every other organelle and with the plasma membrane. ER 3-D structure is determined by both integral ER membrane proteins and by interactions with the cytoskeleton. Here, we describe some of the factors that...

  14. Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability.

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    Fausto Arellano-Carbajal

    2011-03-01

    Full Text Available Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²(+ transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²(+ response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²(+ channels, also fails to disrupt Ca²(+ responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²(+ channel α1 subunit, recapitulate the Ca²(+ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or

  15. The ER in 3D: a multifunctional dynamic membrane network.

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    Friedman, Jonathan R; Voeltz, Gia K

    2011-12-01

    The endoplasmic reticulum (ER) is a large, singular, membrane-bound organelle that has an elaborate 3D structure with a diversity of structural domains. It contains regions that are flat and cisternal, ones that are highly curved and tubular, and others adapted to form contacts with nearly every other organelle and with the plasma membrane. The 3D structure of the ER is determined by both integral ER membrane proteins and by interactions with the cytoskeleton. In this review, we describe some of the factors that are known to regulate ER structure and discuss how this structural organization and the dynamic nature of the ER membrane network allow it to perform its many different functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: the early history of ERAD.

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    Needham, Patrick G; Brodsky, Jeffrey L

    2013-11-01

    All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation. What was less clear was how secreted and integral membrane proteins, which in eukaryotes enter the endoplasmic reticulum (ER), were subject to quality control decisions. In this review, we highlight early studies that ultimately led to the discovery that secreted and integral membrane proteins also utilize several components that constitute the cytoplasmic quality control machinery. This component of the cellular quality control pathway is known as ER associated degradation, or ERAD. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (DeltaPsim). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of DeltaPsim. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

  18. The pepper Bs4C proteins are localized to the endoplasmic reticulum (ER) membrane and confer disease resistance to bacterial blight in transgenic rice.

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    Wang, Jun; Zeng, Xuan; Tian, Dongsheng; Yang, Xiaobei; Wang, Lanlan; Yin, Zhongchao

    2018-03-30

    Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strain-specific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99 A (pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. © 2018 TEMASEK LIFE SCIENCES LABORATORY. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

  19. Autophagosomal membranes assemble at ER-plasma membrane contact sites.

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    Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

    2017-01-01

    The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

  20. Herp enhances ER-associated protein degradation by recruiting ubiquilins

    International Nuclear Information System (INIS)

    Kim, Tae-Yeon; Kim, Eunmin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2008-01-01

    ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates

  1. Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

    OpenAIRE

    Wang, Songyu; Tukachinsky, Hanna; Romano, Fabian B; Rapoport, Tom A

    2016-01-01

    eLife digest The endoplasmic reticulum is a compartment within the cells of plants, animals and other eukaryotes. This compartment plays a number of roles within cells, for example, serving as the site where many proteins and fat molecules are built. Most often the endoplasmic reticulum exists as a network of thin tubules. However, this shape changes during the lifetime of a single cell, and the endoplasmic reticulum converts into flattened structures known as sheets when the cell divides. Th...

  2. ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.

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    Costello, Joseph L; Castro, Inês G; Hacker, Christian; Schrader, Tina A; Metz, Jeremy; Zeuschner, Dagmar; Azadi, Afsoon S; Godinho, Luis F; Costina, Victor; Findeisen, Peter; Manner, Andreas; Islinger, Markus; Schrader, Michael

    2017-02-01

    Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering. © 2017 Costello et al.

  3. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    OpenAIRE

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit t...

  4. Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline.

    Science.gov (United States)

    Gaspar, Maria L; Chang, Yu-Fang; Jesch, Stephen A; Aregullin, Manuel; Henry, Susan A

    2017-11-10

    In the yeast Saccharomyces cerevisiae , the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p ( scs2 Δ) and a mutant, OPI1FFAT , lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A Hands-On Approach to Teaching Protein Translation & Translocation into the ER

    Science.gov (United States)

    LaBonte, Michelle L.

    2013-01-01

    The process of protein translation and translocation into the endoplasmic reticulum (ER) can often be challenging for introductory college biology students to visualize. To help them understand how proteins become oriented in the ER membrane, I developed a hands-on activity in which students use Play-Doh to simulate the process of protein…

  6. Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

    Science.gov (United States)

    Guo, Jinya; Miao, Yansong; Cai, Yi

    2017-01-01

    Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

  7. Characterization of the C-terminal ER membrane anchor of PTP1B

    International Nuclear Information System (INIS)

    Anderie, Ines; Schulz, Irene; Schmid, Andreas

    2007-01-01

    The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure

  8. Membrane trafficking: ER export encounters dualism.

    Science.gov (United States)

    Barlowe, Charles

    2015-02-16

    Cytoplasmic coat protein complexes perform central roles in sorting protein constituents within the endomembrane system. A new study reveals that the COPII coat operates through dual recognition of signals in a sorting receptor and its bound cargo to promote efficient export from the endoplasmic reticulum. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis.

    Science.gov (United States)

    Nascimbeni, Anna Chiara; Giordano, Francesca; Dupont, Nicolas; Grasso, Daniel; Vaccaro, Maria I; Codogno, Patrice; Morel, Etienne

    2017-07-14

    The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER-plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER-plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. © 2017 The Authors.

  10. Synaptotagmin SYTA forms ER-plasma membrane junctions that are recruited to plasmodesmata for plant virus movement.

    Science.gov (United States)

    Levy, Amit; Zheng, Judy Y; Lazarowitz, Sondra G

    2015-08-03

    Metazoan synaptotagmins are Ca(2+) sensors that regulate exocytosis and endocytosis in various cell types, notably in nerve and neuroendocrine cells [1, 2]. Recently, the structurally related extended synaptotagmins were shown to tether the cortical ER to the plasma membrane in human and yeast cells to maintain ER morphology and stabilize ER-plasma membrane (ER-PM) contact sites for intracellular lipid and Ca(2+) signaling [3, 4]. The Arabidopsis synaptotagmin SYTA regulates endocytosis and the ability of plant virus movement proteins (MPs) to alter plasmodesmata to promote virus cell-to-cell transport [5, 6]. Yet how MPs modify plasmodesmata, the cellular functions of SYTA and how these aid MP activity, and the proteins essential to form plant cell ER-PM contact sites remain unknown. We addressed these questions using an Arabidopsis SYTA knockdown line syta-1 and a Tobamovirus movement protein MP(TVCV) [5, 7]. We report here that SYTA localized to ER-PM contact sites. These sites were depleted and the ER network collapsed in syta-1, and both reformed upon rescue with SYTA. MP(TVCV) accumulation in plasmodesmata, but not secretory trafficking, was also inhibited in syta-1. During infection, MP(TVCV) recruited SYTA to plasmodesmata, and SYTA and the cortical ER were subsequently remodeled to form viral replication sites adjacent to plasmodesmata in which MP(TVCV) and SYTA directly interacted caged within ER membrane. SYTA also accumulated in plasmodesmata active in MP(TVCV) transport. Our findings show that SYTA is essential to form ER-PM contact sites and suggest that MPs interact with SYTA to recruit these sites to alter plasmodesmata for virus cell-to-cell movement. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Effect of the unfolded protein response on ER protein export: a potential new mechanism to relieve ER stress.

    Science.gov (United States)

    Shaheen, Alaa

    2018-05-05

    The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.

  12. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    Science.gov (United States)

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PMID:21857022

  13. Aberrant accumulation of the diabetes autoantigen GAD65 in Golgi membranes in conditions of ER stress and autoimmunity

    DEFF Research Database (Denmark)

    Phelps, Edward A; Cianciaruso, Chiara; Michael, Iacovos P

    2016-01-01

    Pancreatic islet beta cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in beta cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes GABA......, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes...... release from stressed and/or damaged beta cells, triggering autoimmunity....

  14. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

    2017-01-01

    of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

  15. ER stress proteins in autoimmune and inflammatory diseases

    Directory of Open Access Journals (Sweden)

    Daisuke eMorito

    2012-03-01

    Full Text Available Over the past two decades, heat shock proteins (HSPs have been implicated in inflammatory responses and autoimmunity. HSPs were originally believed to maintain protein quality control in the cytosol. However, they also exist extracellularly and appear to act as inflammatory factors. Recently, a growing body of evidence suggested that the other class of stress proteins such as, endoplasmic reticulum (ER stress proteins, which originally act as protein quality control factors in the secretory pathway and are induced by ER stress in inflammatory lesions, also participate in inflammation and autoimmunity. The immunoglobulin heavy-chain binding protein (Bip/glucose-regulated protein 78 (Grp78, homocysteine-induced ER protein (Herp, calnexin, calreticulin, glucose-regulated protein 94 (Grp94/gp96, oxygen-regulated protein 150 (ORP150 and heat shock protein 47 (Hsp47/Serpin H1, which are expressed not only in the ER but also occasionally at the cell surface play pathophysiological roles in autoimmune and inflammatory diseases as pro- or anti-inflammatory factors. Here we describe the accumulating evidence of the participation of ER stress proteins in autoimmunity and inflammation and discuss the critical differences between the two classes of stress proteins.

  16. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly ...

  17. Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

    Science.gov (United States)

    Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

    2015-11-01

    The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

  18. Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

    Directory of Open Access Journals (Sweden)

    Ivan Kulich

    2014-04-01

    Full Text Available Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER to vacuole (and also, apoplast transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast, exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA and salicylic acid (SA degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

  19. Nanodisc-solubilized membrane protein library reflects the membrane proteome.

    Science.gov (United States)

    Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

    2013-05-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

  20. Nanodisc-solubilized membrane protein library reflects the membrane proteome

    OpenAIRE

    Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

    2013-01-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

  1. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  2. The ER membrane insertase Get1/2 is required for efficient mitophagy in yeast.

    Science.gov (United States)

    Onishi, Mashun; Nagumo, Sachiyo; Iwashita, Shohei; Okamoto, Koji

    2018-05-10

    Mitophagy is an evolutionarily conserved autophagy pathway that selectively eliminates mitochondria to control mitochondrial quality and quantity. Although mitophagy is thought to be crucial for cellular homeostasis, how this catabolic process is regulated remains largely unknown. Here we demonstrate that mitophagy during prolonged respiratory growth is strongly impaired in yeast cells lacking Get1/2, a transmembrane complex mediating insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane. Under the same conditions, loss of Get1/2 caused only slight defects in other types of selective and bulk autophagy. In addition, mitophagy and other autophagy-related processes are mostly normal in cells lacking Get3, a cytosolic ATP-driven chaperone that promotes delivery of TA proteins to the Get1/2 complex. We also found that Get1/2-deficient cells exhibited wildtype-like induction and mitochondrial localization of Atg32, a protein essential for mitophagy. Notably, Get1/2 is important for Atg32-independent, ectopically promoted mitophagy. Together, we propose that Get1/2-dependent TA protein(s) and/or the Get1/2 complex itself may act specifically in mitophagy. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

    Science.gov (United States)

    Carrasco, Silvia; Meyer, Tobias

    2011-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

  4. Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release.

    Science.gov (United States)

    van Kuppeveld, F J; Hoenderop, J G; Smeets, R L; Willems, P H; Dijkman, H B; Galama, J M; Melchers, W J

    1997-01-01

    Digital-imaging microscopy was performed to study the effect of Coxsackie B3 virus infection on the cytosolic free Ca2+ concentration and the Ca2+ content of the endoplasmic reticulum (ER). During the course of infection a gradual increase in the cytosolic free Ca2+ concentration was observed, due to the influx of extracellular Ca2+. The Ca2+ content of the ER decreased in time with kinetics inversely proportional to those of viral protein synthesis. Individual expression of protein 2B was sufficient to induce the influx of extracellular Ca2+ and to release Ca2+ from ER stores. Analysis of mutant 2B proteins showed that both a cationic amphipathic alpha-helix and a second hydrophobic domain in 2B were required for these activities. Consistent with a presumed ability of protein 2B to increase membrane permeability, viruses carrying a mutant 2B protein exhibited a defect in virus release. We propose that 2B gradually enhances membrane permeability, thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny. PMID:9218794

  5. Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2018-01-01

    of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane......The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein...... recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast....

  6. Novel Tripod Amphiphiles for Membrane Protein Analysis

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Kruse, Andrew C; Gotfryd, Kamil

    2013-01-01

    Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution...

  7. Proteins and Peptides in Biomimetic Polymeric Membranes

    DEFF Research Database (Denmark)

    Perez, Alfredo Gonzalez

    2013-01-01

    This chapter discusses recent advances and the main advantages of block copolymers for functional membrane protein reconstitution in biomimetic polymeric membranes. A rational approach to the reconstitution of membrane proteins in a functional form can be addressed by a more holistic view by using...... other kind of nonbiological amphiphilic molecules. An interesting possibility could be the use of self-assembled proteins in a lipid-free membrane mimicking the capside of some viruses. The membrane proteins that have been more actively used in combination with block copolymer membranes are gramicidin...

  8. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  9. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  10. Er:YAG delamination of immersed biological membranes using sealed flexible hollow waveguides

    Science.gov (United States)

    Sagi-Dolev, A. M.; Dror, Jacob; Inberg, Alexandra; Ferencz, J. R.; Croitoru, Nathan I.

    1996-04-01

    The radiation of Er-YAG laser ((lambda) equals 2.94 micrometer) gives selective interaction with tissues. The extinction in soft tissues is only a few micrometers and in hard tissues is of the order of hundreds of micrometers. This makes this type of laser very suitable for treatments in dentistry, orthopedy, or ophthalmology. Because the usual silica fibers are not transmitting the radiation at lambda equals 2.94 micrometer of this laser, many applications cannot be presently performed. Fused silica hollow fibers for Er-YAG radiation were developed in our laboratory and several possible applications in dentistry, orthopedy and ophthalmology were indicated. Hole opening and implantation preparation of teeth were experimented, using Er-YAG laser and hollow plastic waveguide delivery systems. Hole drilling in cow bones was demonstrated for applications in orthopedy. A new procedure of delivering Er-YAG radiation on fibrotic membranes of inner eggshell as a model of the membranes in eyes was developed employing silica hollow waveguides of 0.5 and 0.7 mm ID or a plastic waveguide of 1.0 mm ID. For this purpose waveguides with sealed distal tip were employed to enable us to approach the delivery system through liquid media near to the membrane. This experiment demonstrates the possibility of surgical applications in vitectomy in ophthalmology using Er-YAG laser and silica hollow waveguides.

  11. Analysis of Protein-Membrane Interactions

    DEFF Research Database (Denmark)

    Kemmer, Gerdi Christine

    Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions....... Discovered interactions were then probed on the level of the membrane using liposome-based assays. In the second part, a transmembrane protein was investigated. Assays to probe activity of the plasma membrane ATPase (Arabidopsis thaliana H+ -ATPase isoform 2 (AHA2)) in single liposomes using both giant...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...

  12. Yeast Interacting Proteins Database: YGL145W, YNL258C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ripheral membrane protein required for Golgi-to-ER retrograde traffic; component ... membrane protein required for Golgi-to-ER retrograde traffic; component of the ER target site that interact

  13. Yeast Interacting Proteins Database: YNL258C, YGL145W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YNL258C DSL1 Peripheral membrane protein required for Golgi-to-ER retrograde traffi...t description Peripheral membrane protein required for Golgi-to-ER retrograde traffic; component of the ER t

  14. Rab7a modulates ER stress and ER morphology.

    Science.gov (United States)

    Mateus, Duarte; Marini, Elettra Sara; Progida, Cinzia; Bakke, Oddmund

    2018-05-01

    The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Kinetics and Thermodynamics of Membrane Protein Folding

    Directory of Open Access Journals (Sweden)

    Ernesto A. Roman

    2014-03-01

    Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

  16. Membrane alterations induced by nonstructural proteins of human norovirus.

    Directory of Open Access Journals (Sweden)

    Sylvie Y Doerflinger

    2017-10-01

    Full Text Available Human noroviruses (huNoV are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4 variants. The viral nonstructural (NS proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV. Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER which included single membrane vesicles (SMVs, double membrane vesicles (DMVs and multi membrane vesicles (MMVs. In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and

  17. Peripheral Protein Unfolding Drives Membrane Bending.

    Science.gov (United States)

    Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

    2018-06-20

    Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

  18. No Evidence for Spontaneous Lipid Transfer at ER-PM Membrane Contact Sites.

    Science.gov (United States)

    Merklinger, Elisa; Schloetel, Jan-Gero; Spitta, Luis; Thiele, Christoph; Lang, Thorsten

    2016-04-01

    Non-vesicular lipid transport steps play a crucial role in lipid trafficking and potentially include spontaneous exchange. Since membrane contact facilitates this lipid transfer, it is most likely to occur at membrane contact sites (MCS). However, to date it is unknown whether closely attached biological membranes exchange lipids spontaneously. We have set up a system for studying the exchange of lipids at MCS formed between the endoplasmic reticulum (ER) and the plasma membrane. Contact sites were stably anchored and the lipids cholesterol and phosphatidylcholine (PC) were not capable of transferring spontaneously into the opposed bilayer. We conclude that physical contact between two associated biological membranes is not sufficient for transfer of the lipids PC and cholesterol.

  19. Biomimetic devices functionalized by membrane channel proteins

    Science.gov (United States)

    Schmidt, Jacob

    2004-03-01

    We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

  20. Isomeric Detergent Comparison for Membrane Protein Stability

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

    2016-01-01

    and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

  1. The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

    Science.gov (United States)

    Peiró, Ana; Martínez-Gil, Luis; Tamborero, Silvia; Pallás, Vicente

    2014-01-01

    ABSTRACT Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport

  2. Detection of proteins on blot transfer membranes.

    Science.gov (United States)

    Sasse, Joachim; Gallagher, Sean R

    2003-11-01

    In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

  3. Overcoming barriers to membrane protein structure determination.

    Science.gov (United States)

    Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

    2011-04-01

    After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

  4. Lipid Directed Intrinsic Membrane Protein Segregation

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Thompson, James R.; Helix Nielsen, Claus

    2013-01-01

    We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily h...

  5. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles....

  6. Controlling the shape of membrane protein polyhedra

    Science.gov (United States)

    Li, Di; Kahraman, Osman; Haselwandter, Christoph A.

    2017-03-01

    Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.

  7. Characterising antimicrobial protein-membrane complexes

    International Nuclear Information System (INIS)

    Xun, Gloria; Dingley, Andrew; Tremouilhac, Pierre

    2009-01-01

    Full text: Antimicrobial proteins (AMPs) are host defence molecules that protect organisms from microbial infection. A number of hypotheses for AMP activity have been proposed which involve protein membrane interactions. However, there is a paucity of information describing AMP-membrane complexes in detail. The aim of this project is to characterise the interactions of amoebapore-A (APA-1) with membrane models using primarily solution-state NMR spectroscopy. APA-1 is an AMP which is regulated by a pH-dependent dimerisation event. Based on the atomic resolution solution structure of monomeric APA-1, it is proposed that this dimerisation is a prerequisite for ring-like hexameric pore formation. Due to the cytotoxicity of APA-1, we have developed a cell-free system to produce this protein. To facilitate our studies, we have adapted the cell-free system to isotope label APA-1. 13 C /15 N -enriched APA-1 sample was achieved and we have begun characterising APA-1 dimerisation and membrane interactions using NMR spectroscopy and other biochemical/biophysical methods. Neutron reflectometry is a surface-sensitive technique and therefore represents an ideal technique to probe how APA-1 interacts with membranes at the molecular level under different physiological conditions. Using Platypus, the pH-induced APA-1-membrane interactions should be detectable as an increase of the amount of protein adsorbed at the membrane surface and changes in the membrane properties. Specifically, detailed information of the structure and dimensions of the protein-membrane complex, the position and amount of the protein in the membrane, and the perturbation of the membrane phospholipids on protein incorporation can be extracted from the neutron reflectometry measurement. Such information will enable critical assessment of current proposed mechanisms of AMP activity in bacterial membranes and complement our NMR studies

  8. Membrane shape modulates transmembrane protein distribution.

    Science.gov (United States)

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

    2014-01-27

    Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Functional dynamics of cell surface membrane proteins.

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Biophysical EPR Studies Applied to Membrane Proteins

    Science.gov (United States)

    Sahu, Indra D; Lorigan, Gary A

    2015-01-01

    Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented. PMID:26855825

  11. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    Science.gov (United States)

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  12. Electron microscopy of cyanobacterial membrane proteins

    NARCIS (Netherlands)

    Folea, Ioana Mihaela

    2008-01-01

    The main focus of this thesis is photosynthetic protein complexes, and their organization within the membrane of cyanobacteria. In cyanobacteria large proteins catalyze the light reactions of photosynthesis. One of the key proteins is photosystem II. We have found for the first time by electron

  13. Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

    Science.gov (United States)

    Sun, Bingyun; Hood, Leroy

    2014-06-06

    The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

  14. Revolutionizing membrane protein overexpression in bacteria

    NARCIS (Netherlands)

    Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickstrom, David; Slotboom, Dirk Jan; de Gier, Jan-Willem; Wickström, David

    The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory,

  15. Protein profiles of hatchery egg shell membrane.

    Science.gov (United States)

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  16. ER stress-induced protein, VIGG, disturbs plant cation homeostasis, which is correlated with growth retardation and robustness to ER stress

    International Nuclear Information System (INIS)

    Katoh, Hironori; Fujita, Keiko; Takuhara, Yuki; Ogawa, Atsushi; Suzuki, Shunji

    2011-01-01

    Highlights: → VIGG is an ER stress-induced protein in plant. → We examine the characteristics of VIGG-overexpressing Arabidopsis plants. → VIGG-overexpressing plants reveal growth retardation and robustness to ER stress. → VIGG disturbs cation homeostasis in plant. -- Abstract: VIGG is a putative endoplasmic reticulum (ER) resident protein induced by virus infection and ER stress, and is correlated with fruit quality in grapevine. The present study was undertaken to determine the biological function of VIGG in grapevine. Experiments using fluorescent protein-VIGG fusion protein demonstrated that VIGG is localized in ER and the ER targeting sequence is in the N-terminus. The overexpression of VIGG in Arabidopsis plant led to growth retardation. The rosette leaves of VIGG-overexpressing plants were smaller than those of the control plants and rolled at 42 days after seeding. VIGG-overexpressing plants revealed robustness to ER stress as well as the low expression of ER stress marker proteins, such as the luminal binding proteins. These characteristics of VIGG-overexpressing plants were supported by a microarray experiment that demonstrated the disruption of genes related to ER stress response and flowering, as well as cation mobility, in the plants. Finally, cation homeostasis in the plants was disturbed by the overexpression of VIGG. Taken together, these results suggest that VIGG may disturb cation homeostasis in plant, which is correlated with the robustness to ER stress and growth retardation.

  17. ER stress-induced protein, VIGG, disturbs plant cation homeostasis, which is correlated with growth retardation and robustness to ER stress

    Energy Technology Data Exchange (ETDEWEB)

    Katoh, Hironori; Fujita, Keiko; Takuhara, Yuki [Laboratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu, Yamanashi 400-0005 (Japan); Ogawa, Atsushi [Department of Biological Production, Akita Prefectural University, Shimosinjyou-nakano 241-438, Akita 010-0195 (Japan); Suzuki, Shunji, E-mail: suzukis@yamanashi.ac.jp [Laboratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, University of Yamanashi, Kofu, Yamanashi 400-0005 (Japan)

    2011-02-18

    Highlights: {yields} VIGG is an ER stress-induced protein in plant. {yields} We examine the characteristics of VIGG-overexpressing Arabidopsis plants. {yields} VIGG-overexpressing plants reveal growth retardation and robustness to ER stress. {yields} VIGG disturbs cation homeostasis in plant. -- Abstract: VIGG is a putative endoplasmic reticulum (ER) resident protein induced by virus infection and ER stress, and is correlated with fruit quality in grapevine. The present study was undertaken to determine the biological function of VIGG in grapevine. Experiments using fluorescent protein-VIGG fusion protein demonstrated that VIGG is localized in ER and the ER targeting sequence is in the N-terminus. The overexpression of VIGG in Arabidopsis plant led to growth retardation. The rosette leaves of VIGG-overexpressing plants were smaller than those of the control plants and rolled at 42 days after seeding. VIGG-overexpressing plants revealed robustness to ER stress as well as the low expression of ER stress marker proteins, such as the luminal binding proteins. These characteristics of VIGG-overexpressing plants were supported by a microarray experiment that demonstrated the disruption of genes related to ER stress response and flowering, as well as cation mobility, in the plants. Finally, cation homeostasis in the plants was disturbed by the overexpression of VIGG. Taken together, these results suggest that VIGG may disturb cation homeostasis in plant, which is correlated with the robustness to ER stress and growth retardation.

  18. Sphingolipid topology and the dynamic organization and function of membrane proteins.

    Science.gov (United States)

    van Meer, Gerrit; Hoetzl, Sandra

    2010-05-03

    When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    Czech Academy of Sciences Publication Activity Database

    Javanainen, M.; Martinez-Seara, Hector; Metzler, R.; Vattulainen, I.

    2017-01-01

    Roč. 8, č. 17 (2017), s. 4308-4313 ISSN 1948-7185 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : giant unilamellar vesicles * single-molecule tracking * lipid bilayer membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 9.353, year: 2016

  20. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

    Directory of Open Access Journals (Sweden)

    Soromani Christina

    2012-12-01

    Full Text Available Abstract Background Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER. It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p. Little is known about the biogenesis and regulation of individual Sec complex subunits. Results We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. Conclusions We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5

  1. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Efficient preparation and analysis of membrane and membrane protein systems

    Czech Academy of Sciences Publication Activity Database

    Javanainen, M.; Martinez-Seara, Hector

    2016-01-01

    Roč. 1858, č. 10 (2016), s. 2468-2482 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : tools and software * membrane building * protein insertion * molecular dynamics * lipid bilayer Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016

  3. Overcoming barriers to membrane protein structure determination

    NARCIS (Netherlands)

    Bill, Roslyn M.; Henderson, Peter J. F.; Iwata, So; Kunji, Edmund R. S.; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G.; Vogel, Horst

    After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new

  4. Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

    Science.gov (United States)

    Wong, Louise H; Levine, Tim P

    2016-04-15

    Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

  5. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

    Science.gov (United States)

    Pan, Yuan; Laird, Joseph G; Yamaguchi, David M; Baker, Sheila A

    2015-06-01

    Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.

  6. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    Science.gov (United States)

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  7. Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

    International Nuclear Information System (INIS)

    Lehto, Markku; Hynynen, Riikka; Karjalainen, Katja; Kuismanen, Esa; Hyvaerinen, Kati; Olkkonen, Vesa M.

    2005-01-01

    The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P 2 and PI(3,4,5)P 3 . A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein

  8. Major Intrinsic Proteins in Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus

    2010-01-01

    or as sensor devices based on e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix....../separation technology, a unique class of membrane transport proteins is especially interesting the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells...... internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 109 molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other...

  9. Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

    Science.gov (United States)

    Sreeja, S; Thampan, Raghava Varman

    2004-04-01

    An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

  10. Membranes and mammalian glycolipid transferring proteins.

    Science.gov (United States)

    Tuuf, Jessica; Mattjus, Peter

    2014-02-01

    Glycolipids are synthesized in and on various organelles throughout the cell. Their trafficking inside the cell is complex and involves both vesicular and protein-mediated machineries. Most important for the bulk lipid transport is the vesicular system, however, lipids moved by transfer proteins are also becoming more characterized. Here we review the latest advances in the glycolipid transfer protein (GLTP) and the phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) field, from a membrane point of view. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Dietary fatty acids and membrane protein function.

    Science.gov (United States)

    Murphy, M G

    1990-02-01

    In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

  12. The role of phosphatidylinositol-transfer proteins at membrane contact sites.

    Science.gov (United States)

    Selitrennik, Michael; Lev, Sima

    2016-04-15

    Phosphatidylinositol-transfer proteins (PITPs) have been initially identified as soluble factors that accelerate the monomeric exchange of either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayersin vitro They are highly conserved in eukaryotes and have been implicated in different cellular processes, including vesicular trafficking, signal transduction, and lipid metabolism. Recent studies suggest that PITPs function at membrane contact sites (MCSs) to facilitate the transport of PI from its synthesis site at the endoplasmic reticulum (ER) to various membrane compartments. In this review, we describe the underlying mechanism of PITPs targeting to MCSs, discuss their cellular roles and potential mode of action. © 2016 Authors; published by Portland Press Limited.

  13. Detergent-Mediated Reconstitution of Membrane Proteins

    NARCIS (Netherlands)

    Knol, J; Sjollema, K.A; Poolman, B.

    1998-01-01

    The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed

  14. TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

    Science.gov (United States)

    Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

    2012-06-01

    Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

  15. Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

    Science.gov (United States)

    Ceriotti, A; Colman, A

    1988-03-01

    We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.

  16. Characterization of membrane association of Rinderpest virus matrix protein

    International Nuclear Information System (INIS)

    Subhashri, R.; Shaila, M.S.

    2007-01-01

    Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

  17. Endoplasmic Reticulum (ER Stress and Endocrine Disorders

    Directory of Open Access Journals (Sweden)

    Daisuke Ariyasu

    2017-02-01

    Full Text Available The endoplasmic reticulum (ER is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR, which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI, Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2 are discussed in this article.

  18. Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

    Science.gov (United States)

    Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

    2017-01-01

    The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

  19. Water Transport Mediated by Other Membrane Proteins.

    Science.gov (United States)

    Huang, Boyue; Wang, Hongkai; Yang, Baoxue

    2017-01-01

    Water transport through membrane is so intricate that there are still some debates. (Aquaporins) AQPs are entirely accepted to allow water transmembrane movement depending on osmotic gradient. Cotransporters and uniporters , however, are also concerned in water homeotatsis. Urea transporter B (UT-B) has a single-channel water permeability that is similar to AQP1. Cystic fibrosis transmembrane conductance regulator (CFTR ) was initially thought as a water channel but now not believed to transport water directly. By cotranporters, water is transported by water osmosis coupling with substrates, which explains how water is transported across the isolated small intestine. This chapter provides information about water transport mediated by other membrane proteins except AQPs .

  20. Cryo-electron microscopy of membrane proteins.

    Science.gov (United States)

    Goldie, Kenneth N; Abeyrathne, Priyanka; Kebbel, Fabian; Chami, Mohamed; Ringler, Philippe; Stahlberg, Henning

    2014-01-01

    Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

  1. Protein permeation through an electrically tunable membrane

    International Nuclear Information System (INIS)

    Jou, Ining A; Melnikov, Dmitriy V; Gracheva, Maria E

    2016-01-01

    Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein’s dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane–electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions. (paper)

  2. Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

    Directory of Open Access Journals (Sweden)

    Szu-Chia Hsieh

    Full Text Available The envelope (E of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

  3. Chaperone-Mediated Sec61 Channel Gating during ER Import of Small Precursor Proteins Overcomes Sec61 Inhibitor-Reinforced Energy Barrier

    Directory of Open Access Journals (Sweden)

    Sarah Haßdenteufel

    2018-05-01

    Full Text Available Summary: Protein transport into the mammalian endoplasmic reticulum (ER is mediated by the heterotrimeric Sec61 channel. The signal recognition particle (SRP and TRC systems and Sec62 have all been characterized as membrane-targeting components for small presecretory proteins, whereas Sec63 and the lumenal chaperone BiP act as auxiliary translocation components. Here, we report the transport requirements of two natural, small presecretory proteins and engineered variants using semipermeabilized human cells after the depletion of specific ER components. Our results suggest that hSnd2, Sec62, and SRP and TRC receptor each provide alternative targeting pathways for short secretory proteins and define rules of engagement for the actions of Sec63 and BiP during their membrane translocation. We find that the Sec62/Sec63 complex plus BiP can facilitate Sec61 channel opening, thereby allowing precursors that have weak signal peptides or other inhibitory features to translocate. A Sec61 inhibitor can mimic the effect of BiP depletion on Sec61 gating, suggesting that they both act at the same essential membrane translocation step. : Protein transport into the human endoplasmic reticulum (ER is mediated by the heterotrimeric Sec61 channel. Haßdenteufel et al. map the determinants for requirement of different targeting pathways and different auxiliary components of the Sec61 channel in ER import of short presecretory proteins. Different characteristics of precursor polypeptides dictate the engagement of each component. Keywords: endoplasmic reticulum, protein targeting and translocation, Sec61 channel gating, Sec62, Sec63, BiP, CAM741, signal peptide, mature region, cluster of positive charges

  4. An ER-directed fusion protein comprising a bacterial subtilisin ...

    African Journals Online (AJOL)

    nausch

    subtilase tag was fused to human interleukin 6 (IL6) and transiently expressed in Nicotiana ..... MP, tobacco mosaic virus (TMV) movement protein; TVCV-3'-NTR, TVCV-3' untranslated .... on the degradation pattern of heterologous proteins.

  5. Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study

    DEFF Research Database (Denmark)

    Sadaf, Aiman; Du, Yang; Santillan, Claudia

    2017-01-01

    The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein...... alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6...

  6. Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

    2016-09-01

    The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

  7. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  8. Pre-emptive Quality Control Protects the ER from Protein Overload via the Proximity of ERAD Components and SRP

    Directory of Open Access Journals (Sweden)

    Hisae Kadowaki

    2015-11-01

    Full Text Available Cells possess ER quality control systems to adapt to ER stress and maintain their function. ER-stress-induced pre-emptive quality control (ER pQC selectively degrades ER proteins via translocational attenuation during ER stress. However, the molecular mechanism underlying this process remains unclear. Here, we find that most newly synthesized endogenous transthyretin proteins are rerouted to the cytosol without cleavage of the signal peptide, resulting in proteasomal degradation in hepatocytes during ER stress. Derlin family proteins (Derlins, which are ER-associated degradation components, reroute specific ER proteins, but not ER chaperones, from the translocon to the proteasome through interactions with the signal recognition particle (SRP. Moreover, the cytosolic chaperone Bag6 and the AAA-ATPase p97 contribute to the degradation of ER pQC substrates. These findings demonstrate that Derlins-mediated substrate-specific rerouting and Bag6- and p97-mediated effective degradation contribute to the maintenance of ER homeostasis without the need for translocation.

  9. Serial Millisecond Crystallography of Membrane Proteins.

    Science.gov (United States)

    Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

    2016-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.

  10. Peripheral myelin protein 22 alters membrane architecture

    Science.gov (United States)

    Mittendorf, Kathleen F.; Marinko, Justin T.; Hampton, Cheri M.; Ke, Zunlong; Hadziselimovic, Arina; Schlebach, Jonathan P.; Law, Cheryl L.; Li, Jun; Wright, Elizabeth R.; Sanders, Charles R.; Ohi, Melanie D.

    2017-01-01

    Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin. PMID:28695207

  11. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

    OpenAIRE

    Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette; Chae, Pil Seok

    2016-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

  12. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation.

    Science.gov (United States)

    Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

    2016-10-04

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

  13. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

    Science.gov (United States)

    Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette

    2017-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research. PMID:27711401

  14. Macrolide Resistance Mediated by a Bifidobacterium breve Membrane Protein

    OpenAIRE

    Margolles, Abelardo; Moreno, José Antonio; van Sinderen, Douwe; de los Reyes-Gavilán, Clara G.

    2005-01-01

    A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.

  15. Hamster endogenous retrovirus (HaER) - distinct properties of structural proteins and DNA polymerase

    International Nuclear Information System (INIS)

    Goldschmied-Reouven, A.; Yaniv, A.

    1983-01-01

    The structural proteins as well as some features of the RNA-dependent DNA polymerase of the hamster endogenous retrovirus (HaER) were examined. The polypeptide pattern of this virus is substantially different from that of other known retroviruses in containing major polypeptides with molecular weights of 68000, 59000, 27000, 24000 daltons. Double antibody competitive radioimmunoassays showed that the HaER particles do not share any detectable antigenic relatedness with the murine viruses' p30, but manifest a considerable relatedness with the feline leukemia virus p27 and a slight cross-reactivity with the rat virus major protein. The RNA-dependent DNA polymerase of HaER virus has a molecular size of approximately 73000 daltons and in contrast to other mammalian retroviruses shows no significant preference for Mn 2+ over Mg 2+ . Apart from the lack of antigenic relatedness between the HaER virus proteins and the p30 protein of murine viruses, there is also no antigenic relatedness between HaER and murine viruses insofar as their DNA polymerase is concerned. (Author)

  16. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

    International Nuclear Information System (INIS)

    Apostolou, Andria; Shen Yuxian; Liang Yan; Luo Jun; Fang Shengyun

    2008-01-01

    The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR

  17. Xanthophylls as modulators of membrane protein function.

    Science.gov (United States)

    Ruban, Alexander V; Johnson, Matthew P

    2010-12-01

    This review discusses the structural aspect of the role of photosynthetic antenna xanthophylls. It argues that xanthophyll hydrophobicity/polarity could explain the reason for xanthophyll variety and help to understand their recently emerging function--control of membrane organization and the work of membrane proteins. The structure of a xanthophyll molecule is discussed in relation to other amphiphilic compounds like lipids, detergents, etc. Xanthophyll composition of membrane proteins, the role of their variety in protein function are discussed using as an example for the major light harvesting antenna complex of photosystem II, LHCII, from higher plants. A new empirical parameter, hydrophobicity parameter (H-parameter), has been introduced as an effective measure of the hydrophobicity of the xanthophyll complement of LHCII from different xanthophyll biosynthesis mutants of Arabidopsis. Photosystem II quantum efficiency was found to correlate well with the H-parameter of LHCII xanthophylls. PSII down-regulation by non-photochemical chlorophyll fluorescence quenching, NPQ, had optimum corresponding to the wild-type xanthophyll composition, where lutein occupies intrinsic sites, L1 and L2. Xanthophyll polarity/hydrophobicity alteration by the activity of the xanthophyll cycle explains the allosteric character of NPQ regulation, memory of illumination history and the hysteretic nature of the relationship between the triggering factor, ΔpH, and the energy dissipation process. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Disruption of the mitochondria-associated ER membrane (MAM) plays a central role in palmitic acid-induced insulin resistance.

    Science.gov (United States)

    Shinjo, Satoko; Jiang, Shuying; Nameta, Masaaki; Suzuki, Tomohiro; Kanai, Mai; Nomura, Yuta; Goda, Nobuhito

    2017-10-01

    The mitochondria-associated ER membrane (MAM) is a specialized subdomain of ER that physically connects with mitochondria. Although disruption of inter-organellar crosstalk via the MAM impairs cellular homeostasis, its pathological significance in insulin resistance in type 2 diabetes mellitus remains unclear. Here, we reveal the importance of reduced MAM formation in the induction of fatty acid-evoked insulin resistance in hepatocytes. Palmitic acid (PA) repressed insulin-stimulated Akt phosphorylation in HepG2 cells within 12h. Treatment with an inhibitor of the ER stress response failed to restore PA-mediated suppression of Akt activation. Mitochondrial reactive oxygen species (ROS) production did not increase in PA-treated cells. Even short-term exposure (3h) to PA reduced the calcium flux from ER to mitochondria, followed by a significant decrease in MAM contact area, suggesting that PA suppressed the functional interaction between ER and mitochondria. Forced expression of mitofusin-2, a critical component of the MAM, partially restored MAM contact area and ameliorated the PA-elicited suppression of insulin sensitivity with Ser473 phosphorylation of Akt selectively improved. These results suggest that loss of proximity between ER and mitochondria, but not perturbation of homeostasis in the two organelles individually, plays crucial roles in PA-evoked Akt inactivation in hepatic insulin resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins.

    Directory of Open Access Journals (Sweden)

    Louise Madsen

    Full Text Available The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.

  20. Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

    Science.gov (United States)

    Abeyrathne, Priyanka D; Lam, Joseph S

    2007-04-01

    A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

  1. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Buck, Teresa M; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L; Needham, Patrick G; Kleyman, Thomas R; Brodsky, Jeffrey L

    2015-06-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. Copyright © 2015 the American Physiological Society.

  2. Die HECT-Ligase Hul5, eine neue Komponente der ER-assoziierten Proteindegradation

    OpenAIRE

    Kohlmann, Sonja

    2007-01-01

    Die meisten sekretorischen Proteine der eukaryontischen Zellen erreichen durch das endoplasmatische Retikulum (ER) den sekretorischen Signalweg. Sie gelangen vom Zytoplasma durch einen Kanal in der ER-Membran in das ER, wo sie ihre native Konformation erhalten. Das ER enthält ein strenges Qualitätskontrollsystem, welches fehlgefaltete Proteine erkennt, im ER zurückhält und letztendlich der ER-assoziierten Degradation (ERAD) zuführt. Die ER-Qualitätskontrolle und die ER-assoziierte Degradation...

  3. Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

    Science.gov (United States)

    Cheng, Chi-Yuan; Han, Songi

    2013-01-01

    Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

  4. Oxygen permeation properties of dense Bi1.5Er0.5O3-Ag cermet membranes

    NARCIS (Netherlands)

    ten Elshof, Johan E.; Nguyen, N.Q.; den Otter, M.W.; Bouwmeester, Henricus J.M.

    1997-01-01

    Oxygen permeation experiments were performed on dense mixed-conducting ceramic-metal composite membranes (thickness 0.2 to 2 mm) Bi1.5Er0.5O3-Ag with 10.0, 27.8, and 40.0 volume percent (v/o) silver, respectively, in the temperature range 873 to 993 K and oxygen partial pressure range 10–3.5 to 1

  5. Membrane Proteins : The Key Players of a Cancer Cell

    NARCIS (Netherlands)

    Kampen, Kim R.

    Membrane proteins are involved in the prognosis of the most common forms of cancer. Membrane proteins are the hallmark of a cancer cell. The overexpressed membrane receptors are becoming increasingly important in cancer cell therapy. Current renewing therapy approaches based on receptor

  6. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    Science.gov (United States)

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  7. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  8. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    Science.gov (United States)

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

    Science.gov (United States)

    Saher, Gesine; Quintes, Susanne; Möbius, Wiebke; Wehr, Michael C; Krämer-Albers, Eva-Maria; Brügger, Britta; Nave, Klaus-Armin

    2009-05-13

    Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

  10. Production of membrane proteins without cells or detergents.

    Science.gov (United States)

    Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

    2011-04-30

    The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope.

    Science.gov (United States)

    Blackstone, Craig

    2017-12-04

    Efficient targeting of newly synthesized membrane proteins from the endoplasmic reticulum to the inner nuclear membrane depends on nucleotide hydrolysis. A new study shows that this dependence reflects critical actions of the atlastin family of GTPases in maintaining the morphology of the endoplasmic reticulum network. Published by Elsevier Ltd.

  12. Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

    Science.gov (United States)

    Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

    2018-02-20

    Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

  13. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    Science.gov (United States)

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  14. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    Science.gov (United States)

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Endoplasmic reticulum protein targeting of phospholamban: a common role for an N-terminal di-arginine motif in ER retention?

    Directory of Open Access Journals (Sweden)

    Parveen Sharma

    2010-07-01

    Full Text Available Phospholamban (PLN is an effective inhibitor of the sarco(endoplasmic reticulum Ca(2+-ATPase, which transports Ca(2+ into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation.Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RDelta14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the beta-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins.We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.

  16. Challenges in the Development of Functional Assays of Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Sophie Demarche

    2012-11-01

    Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  17. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    Directory of Open Access Journals (Sweden)

    Rebecca F Alford

    2015-09-01

    Full Text Available Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1 prediction of free energy changes upon mutation; (2 high-resolution structural refinement; (3 protein-protein docking; and (4 assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

  18. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    Science.gov (United States)

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  19. Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

    Science.gov (United States)

    Quon, Evan; Beh, Christopher T.

    2015-01-01

    Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

  20. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  1. Cytoskeletal Components Define Protein Location to Membrane Microdomains*

    Science.gov (United States)

    Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

    2015-01-01

    The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

  2. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    Science.gov (United States)

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  3. Studying Membrane Protein Structure and Function Using Nanodiscs

    DEFF Research Database (Denmark)

    Huda, Pie

    The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

  4. Static light scattering to characterize membrane proteins in detergent solution

    NARCIS (Netherlands)

    Slotboom, Dirk Jan; Duurkens, Ria H.; Olieman, Kees; Erkens, Guus B.

    2008-01-01

    Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium

  5. Membrane interaction of retroviral Gag proteins

    Directory of Open Access Journals (Sweden)

    Robert Alfred Dick

    2014-04-01

    Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

  6. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  7. NMR structural studies of peptides and proteins in membranes

    Energy Technology Data Exchange (ETDEWEB)

    Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

    1994-12-31

    The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.

  8. Disturbed vesicular trafficking of membrane proteins in prion disease.

    Science.gov (United States)

    Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

    2013-01-01

    The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

  9. NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis.

    Science.gov (United States)

    Widenmaier, Scott B; Snyder, Nicole A; Nguyen, Truc B; Arduini, Alessandro; Lee, Grace Y; Arruda, Ana Paula; Saksi, Jani; Bartelt, Alexander; Hotamisligil, Gökhan S

    2017-11-16

    Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol. Copyright © 2017. Published by Elsevier Inc.

  10. Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rana, Rohini R; Gotfryd, Kamil

    2013-01-01

    The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo ...

  11. Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study.

    Science.gov (United States)

    Chae, Pil Seok; Rana, Rohini R; Gotfryd, Kamil; Rasmussen, Søren G F; Kruse, Andrew C; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J; Gether, Ulrik; Banerjee, Surajit; Byrne, Bernadette; Lee, John K; Gellman, Samuel H

    2013-03-21

    The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. (Science, 2012, 337, 473).

  12. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation

    DEFF Research Database (Denmark)

    Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea

    2016-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared...

  13. Influence of ionizing radiation on the plasma membrane proteins

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1992-01-01

    The effect of ionizing radiation on the meat cattle thymocytes plasma membranes was studied. Using fluorescence quenching technique the effect of irradiation of proteins conformation was investigated. The influence of ionizing radiation on the plasma membranes was shown to be followed by changes of the protein structure-dynamic organization

  14. Identification and characterization of stable membrane protein complexes

    NARCIS (Netherlands)

    Spelbrink, R.E.J.

    2007-01-01

    Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

  15. The outer membrane protein assembly machinery of Neisseria meningitidis

    NARCIS (Netherlands)

    Volokhina, E.B.|info:eu-repo/dai/nl/304837202

    2009-01-01

    Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

  16. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  17. The role of antioxidant-protein interactions in biological membrane

    International Nuclear Information System (INIS)

    McGillivray, Duncan J; Singh, Rachna; Melton, Laurence D.; Worcester, David L.; Gilbert, Elliot P.

    2009-01-01

    Full text: Oxidative damage of cellular membranes has been linked to a variety of disease pathologies, including cardiac disease, Alzheimer's and complications due to diabetes. The oxidation of unsaturated and polyunsaturated fatty acid chains found in cellular membranes leads to significant alteration in membrane physical properties, including lipid orientation and membrane permeability, which ultimately affect biological function. Polyphenols are naturally occurring phytochemicals present in a number of fruit and vegetables that are of interest for their anti-oxidative powers. These polyphenols inhibit lipid oxidation in cellular membrane surfaces, although the mechanism of this inhibition is not entirely clear. Moreover, the polyphenols have significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein-polyphenol complexes Significantly, in the presence of casein proteins the oxidation inhibition the polyphenols in the membrane is significantly enhanced (as assessed by Lipid Peroxidation Inhibition Capacity assays). Thus the antioxidant pathway appears to involve these protein/polyphenol complexes, as well as direct antioxidant action by the polyphenol. Here we discuss neutron and x-ray scattering results from phospholipid membranes, looking at the positioning of two examples of polyphenolic antioxidants in phospholipid membranes, quercetin and phloretin, the antioxidants' impact on the membrane organisation, and the interaction between antioxidant and extra-membranous protein. This information sheds light on the mechanism of antioxidant protection in these systems, which may be used to understand biological responses to oxidative stress.

  18. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    Science.gov (United States)

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  19. Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

    2006-01-01

    A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

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

    Science.gov (United States)

    2010-01-01

    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. PMID:20704729

  1. Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response

    DEFF Research Database (Denmark)

    Sehgal, Pankaj; Szalai, Paula; Olesen, Claus

    2017-01-01

    Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA-inhibition induces cell death are incompletely...... extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover...... and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response (UPR). We conclude that ER Ca2+ drainage and sustained UPR activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high...

  2. Overcoming bottlenecks in the membrane protein structural biology pipeline.

    Science.gov (United States)

    Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

    2016-06-15

    Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  3. ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

    Directory of Open Access Journals (Sweden)

    Jarom Heijmans

    2013-04-01

    Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

  4. Folding Membrane Proteins by Deep Transfer Learning

    KAUST Repository

    Wang, Sheng

    2017-08-29

    Computational elucidation of membrane protein (MP) structures is challenging partially due to lack of sufficient solved structures for homology modeling. Here, we describe a high-throughput deep transfer learning method that first predicts MP contacts by learning from non-MPs and then predicts 3D structure models using the predicted contacts as distance restraints. Tested on 510 non-redundant MPs, our method has contact prediction accuracy at least 0.18 better than existing methods, predicts correct folds for 218 MPs, and generates 3D models with root-mean-square deviation (RMSD) less than 4 and 5 Å for 57 and 108 MPs, respectively. A rigorous blind test in the continuous automated model evaluation project shows that our method predicted high-resolution 3D models for two recent test MPs of 210 residues with RMSD ∼2 Å. We estimated that our method could predict correct folds for 1,345–1,871 reviewed human multi-pass MPs including a few hundred new folds, which shall facilitate the discovery of drugs targeting at MPs.

  5. SURVEY REGARDING THE ULTRAFILTRATION OF PROTEINES THROUGH MEMBRANE BASED PROCEDURES

    Directory of Open Access Journals (Sweden)

    CAMELIA HODOSAN

    2008-05-01

    Full Text Available This work is based on examples that emphasize the complexity of the proteins ultrafiltration process, pointing out the first 10-15 minutes of ultrafiltration. The knowledgement of the factors that influence the separation through ultrafiltration of proteins will allow to choose the right type of membrane, the frequent use of the same membrane and the operation in mechanical and chemical conditions adequate to the ultrafiltration system, when it is separated a protein with certain molecular weight.

  6. Yeast Interacting Proteins Database: YNL258C, YKR022C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YNL258C DSL1 Peripheral membrane protein required for Golgi-to-ER retrograde traffi...equired for Golgi-to-ER retrograde traffic; component of the ER target site that interacts with coatomer, th...it ORF YNL258C Bait gene name DSL1 Bait description Peripheral membrane protein r

  7. 3D pressure field in lipid membranes and membrane-protein complexes

    DEFF Research Database (Denmark)

    Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti

    2009-01-01

    We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...... a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane....

  8. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

    2014-01-01

    -ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

  9. Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

    Science.gov (United States)

    Schneiter, Roger; Cole, Charles N

    2010-01-01

    The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

  10. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  11. High throughput platforms for structural genomics of integral membrane proteins.

    Science.gov (United States)

    Mancia, Filippo; Love, James

    2011-08-01

    Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. ApoER2 expression increases Aβ production while decreasing Amyloid Precursor Protein (APP endocytosis: Possible role in the partitioning of APP into lipid rafts and in the regulation of γ-secretase activity

    Directory of Open Access Journals (Sweden)

    Bu Guojun

    2007-07-01

    Full Text Available Abstract Background The generation of the amyloid-β peptide (Aβ through the proteolytic processing of the amyloid precursor protein (APP is a central event in the pathogenesis of Alzheimer's disease (AD. Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing. Results Here, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Aβ production and reduced levels of APP-CTFs. The increased Aβ production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased γ-secretase activity, both of which might contribute to increased Aβ production. Conclusion These findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Aβ production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting β-secretase and γ-secretase mediated amyloidogenic processing and also by incrementing the activity of γ-secretase.

  13. Integral membrane protein structure determination using pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

    2015-04-15

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

  14. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    Science.gov (United States)

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-α silencing and overexpression.

    Science.gov (United States)

    Hooshmand, Somayeh; Ghaderi, Abbas; Yusoff, Khatijah; Thilakavathy, Karuppiah; Rosli, Rozita; Mojtahedi, Zahra

    2014-01-01

    The consequence of Rho GDP dissociation inhibitor alpha (RhoGDIα) activity on migration and invasion of estrogen receptor positive (ER+) and negative (ER-) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDIα and other proteins interacting directly or indirectly with RhoGDIα in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα. The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

  16. Protein-lipid interactions: from membrane domains to cellular networks

    National Research Council Canada - National Science Library

    Tamm, Lukas K

    2005-01-01

    ... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...

  17. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Directory of Open Access Journals (Sweden)

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  18. Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Emilie eGauliard

    2015-02-01

    Full Text Available Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins. Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

  19. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  20. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  1. Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

    NARCIS (Netherlands)

    Pham, T.K.; Sierocinski, P.; Oost, van der J.; Wright, P.C.

    2010-01-01

    A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30 % of the total

  2. Lactococcus lactis as host for overproduction of functional membrane proteins

    NARCIS (Netherlands)

    Kunji, ERS; Slotboom, DJ; Poolman, B

    2003-01-01

    Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled

  3. Mixed-matrix membrane adsorbers for protein separation

    NARCIS (Netherlands)

    Avramescu, M.E.; Borneman, Z.; Wessling, M.

    2003-01-01

    The separation of two similarly sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (Hb) was carried out using a new type of ion-exchange mixed-matrix adsorber membranes. The adsorber membranes were prepared by incorporation of various types of Lewatit ion-exchange resins into an

  4. Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1 in the porcine system

    Directory of Open Access Journals (Sweden)

    Jin Dong-Il

    2011-05-01

    Full Text Available Abstract Background The unfolded protein response (UPR is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER stress conditions. X-box-binding protein-1 (Xbp1 is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1 gene in ER stress using porcine embryonic fibroblast (PEF cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing. Results We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected. Conclusions It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

  5. Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements.

    Science.gov (United States)

    Buxa, Stefanie V; Degola, Francesca; Polizzotto, Rachele; De Marco, Federica; Loschi, Alberto; Kogel, Karl-Heinz; di Toppi, Luigi Sanità; van Bel, Aart J E; Musetti, Rita

    2015-01-01

    Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by 'Candidatus Phytoplasma solani,' the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes.

  6. Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

    Science.gov (United States)

    Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

    2014-10-01

    All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

  7. Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183

    International Nuclear Information System (INIS)

    Larkins, Brian A.

    2003-01-01

    Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactions between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-alpha-zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD alpha-zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15

  8. Organizing membrane-curving proteins: the emerging dynamical picture.

    Science.gov (United States)

    Simunovic, Mijo; Bassereau, Patricia; Voth, Gregory A

    2018-03-30

    Lipid membranes play key roles in cells, such as in trafficking, division, infection, remodeling of organelles, among others. The key step in all these processes is creating membrane curvature, typically under the control of many anchored, adhered or included proteins. However, it has become clear that the membrane itself can mediate the interactions among proteins to produce highly ordered assemblies. Computer simulations are ideally suited to investigate protein organization and the dynamics of membrane remodeling at near-micron scales, something that is extremely challenging to tackle experimentally. We review recent computational efforts in modeling protein-caused membrane deformation mechanisms, specifically focusing on coarse-grained simulations. We highlight work that exposed the membrane-mediated ordering of proteins into lines, meshwork, spirals and other assemblies, in what seems to be a very generic mechanism driven by a combination of short and long-ranged forces. Modulating the mechanical properties of membranes is an underexplored signaling mechanism in various processes deserving of more attention in the near future. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

    Science.gov (United States)

    Ogino, Kazutoyo; Low, Sean E.; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y.; Hirata, Hiromi

    2015-01-01

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753

  10. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  11. Transport proteins of the plant plasma membrane

    Science.gov (United States)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  12. Isolation of Protein Storage Vacuoles and Their Membranes.

    Science.gov (United States)

    Shimada, Tomoo; Hara-Nishimura, Ikuko

    2017-01-01

    Protein-storage vacuoles (PSVs) are specialized vacuoles that sequester large amounts of storage proteins. During seed development, PSVs are formed de novo and/or from preexisting lytic vacuoles. Seed PSVs can be subdivided into four distinct compartments: membrane, globoid, matrix, and crystalloid. In this chapter, we introduce easy methods for isolation of PSVs and their membranes from pumpkin seeds. These methods facilitate the identification and characterization of PSV components.

  13. Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

    International Nuclear Information System (INIS)

    Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

    1987-01-01

    The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

  14. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  15. Phytochemicals perturb membranes and promiscuously alter protein function.

    Science.gov (United States)

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

  16. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

    Science.gov (United States)

    Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

    2018-01-01

    Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

  17. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

    KAUST Repository

    Knodel, Markus

    2018-01-08

    Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

  18. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

    Directory of Open Access Journals (Sweden)

    Markus M. Knodel

    2018-01-01

    Full Text Available Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

  19. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

    Science.gov (United States)

    Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

    2018-01-08

    Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

  20. Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis

    Directory of Open Access Journals (Sweden)

    Bernardo Blanco-Sánchez

    2014-05-01

    Full Text Available Usher syndrome (USH, the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER. Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

  1. Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

    Science.gov (United States)

    Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

    2015-08-04

    The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. The dynamics of plant plasma membrane proteins: PINs and beyond.

    Science.gov (United States)

    Luschnig, Christian; Vert, Grégory

    2014-08-01

    Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

  3. Hunting for low abundant redox proteins in plant plasma membranes.

    Science.gov (United States)

    Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

    2009-04-13

    Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

  4. Structural adaptations of proteins to different biological membranes

    Science.gov (United States)

    Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

    2013-01-01

    To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

  5. G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

    International Nuclear Information System (INIS)

    Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol; Yoo, Hyun-Seung; Ha, Ji Min; Kim, Jin Young; Choi, Jinmi; Zang, Shu Liang; Hou, Xiao; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2006-01-01

    G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus

  6. Current strategies for protein production and purification enabling membrane protein structural biology.

    Science.gov (United States)

    Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

    2016-12-01

    Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

  7. Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP

    2006-12-01

    Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

  8. Codon optimizing for increased membrane protein production

    DEFF Research Database (Denmark)

    Mirzadeh, K.; Toddo, S.; Nørholm, Morten

    2016-01-01

    . As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification...

  9. DNA nanotubes for NMR structure determination of membrane proteins.

    Science.gov (United States)

    Bellot, Gaëtan; McClintock, Mark A; Chou, James J; Shih, William M

    2013-04-01

    Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

  10. Role of rab proteins in epithelial membrane traffic

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Mostov, KE; Hoekstra, D

    2003-01-01

    Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such

  11. Denaturation of membrane proteins and hyperthermic cell killing

    NARCIS (Netherlands)

    Burgman, Paulus Wilhelmus Johannes Jozef

    1993-01-01

    Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

  12. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    NARCIS (Netherlands)

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  13. Biophysical characterization of membrane protein-small molecule interactions

    NARCIS (Netherlands)

    Chen, Dan

    2015-01-01

    Membrane proteins are account for up to two thirds of known druggable targets. Traditionally, new drugs against this class of proteins have been discovered through HTS. However, not all GPCRs are amenable to traditional screening methods. Recently, fragment-based drug discovery (FBDD) has emerged as

  14. The Multifaceted Role of SNARE Proteins in Membrane Fusion.

    Science.gov (United States)

    Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

    2017-01-01

    Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

  15. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    Science.gov (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  16. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  17. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  18. A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

    Science.gov (United States)

    Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

    2011-02-01

    Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Topology of membrane proteins-predictions, limitations and variations.

    Science.gov (United States)

    Tsirigos, Konstantinos D; Govindarajan, Sudha; Bassot, Claudio; Västermark, Åke; Lamb, John; Shu, Nanjiang; Elofsson, Arne

    2017-10-26

    Transmembrane proteins perform a variety of important biological functions necessary for the survival and growth of the cells. Membrane proteins are built up by transmembrane segments that span the lipid bilayer. The segments can either be in the form of hydrophobic alpha-helices or beta-sheets which create a barrel. A fundamental aspect of the structure of transmembrane proteins is the membrane topology, that is, the number of transmembrane segments, their position in the protein sequence and their orientation in the membrane. Along these lines, many predictive algorithms for the prediction of the topology of alpha-helical and beta-barrel transmembrane proteins exist. The newest algorithms obtain an accuracy close to 80% both for alpha-helical and beta-barrel transmembrane proteins. However, lately it has been shown that the simplified picture presented when describing a protein family by its topology is limited. To demonstrate this, we highlight examples where the topology is either not conserved in a protein superfamily or where the structure cannot be described solely by the topology of a protein. The prediction of these non-standard features from sequence alone was not successful until the recent revolutionary progress in 3D-structure prediction of proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Dissection of membrane protein degradation mechanisms by reversible inhibitors

    International Nuclear Information System (INIS)

    Hare, J.F.

    1988-01-01

    The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma membrane polypeptides in response to reversible temperature and lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were radiolabeled and left for 24 h to allow the removal of rapidly degraded proteins. Remaining trichloroacetic acid-precipitable protein was degraded (t 1/2 = 40-68 h) by an apparent first order process 60-86% sensitive to 10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by temperature reduction to 18 degrees C. Thus, membrane proteins are selected for degradation in a time-dependent manner by a system which is sensitive to both 18 degrees C and to lysosomotropic amines. When inhibitory conditions were removed after 40-48 h, degradation of 125I-labeled protein resumed at the same rate as that seen in their absence. Since membrane proteins do not exhibit accelerated degradation after removal of inhibitory conditions, there can be no marking or sorting of those proteins destined for degradation during the 40-h exposure to inhibitory conditions. Exposure to amines or 18 degrees C did not affect the position of two-dimensionally resolved labeled polypeptides. Fractionation of labeled cells on Percoll gradients after 40 h of exposure to low temperature or amines showed that labeled protein remained in the plasma membrane fractions of the gradient although shifted to a slightly lower buoyant density in the presence of amines. These results support the notion that selection of plasma membrane proteins for degradation requires their internalization into acidic vesicles. Lysosomotropic amines and reduced temperature interfere with the selection process by preventing membrane fusion events

  1. Functional discrimination of membrane proteins using machine learning techniques

    Directory of Open Access Journals (Sweden)

    Yabuki Yukimitsu

    2008-03-01

    Full Text Available Abstract Background Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters. Results We observed that the residues Asp, Asn and Tyr are dominant in channels/pores whereas the composition of hydrophobic residues, Phe, Gly, Ile, Leu and Val is high in electrochemical potential-driven transporters. The composition of all the amino acids in primary active transporters lies in between other two classes of proteins. We have utilized different machine learning algorithms, such as, Bayes rule, Logistic function, Neural network, Support vector machine, Decision tree etc. for discriminating these classes of proteins. We observed that most of the algorithms have discriminated them with similar accuracy. The neural network method discriminated the channels/pores, electrochemical potential-driven transporters and active transporters with the 5-fold cross validation accuracy of 64% in a data set of 1718 membrane proteins. The application of amino acid occurrence improved the overall accuracy to 68%. In addition, we have discriminated transporters from other α-helical and β-barrel membrane proteins with the accuracy of 85% using k-nearest neighbor method. The classification of transporters and all other proteins (globular and membrane showed the accuracy of 82%. Conclusion The performance of discrimination with amino acid occurrence is better than that with amino acid composition. We suggest that this method could be effectively used to discriminate transporters from all other globular and membrane proteins, and classify them into channels/pores, electrochemical and active transporters.

  2. 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.

  3. Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

    International Nuclear Information System (INIS)

    Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

    1976-01-01

    Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

  4. Palmitoylation of POTE family proteins for plasma membrane targeting

    International Nuclear Information System (INIS)

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-01-01

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

  5. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

    DEFF Research Database (Denmark)

    Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde

    2012-01-01

    A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps....../periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli....

  6. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

    International Nuclear Information System (INIS)

    Wylie, Benjamin J.; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

    2015-01-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces

  7. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-11-29

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  8. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    International Nuclear Information System (INIS)

    Mahon, A.C.; Hartig, P.R.

    1982-01-01

    3 H-Lysergic acid diethylamide ( 3 H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3 H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays

  9. Structure and Dynamic Properties of Membrane Proteins using NMR

    DEFF Research Database (Denmark)

    Rösner, Heike; Kragelund, Birthe

    2012-01-01

    conformational changes. Their structural and functional decoding is challenging and has imposed demanding experimental development. Solution nuclear magnetic resonance (NMR) spectroscopy is one of the techniques providing the capacity to make a significant difference in the deciphering of the membrane protein...... structure-function paradigm. The method has evolved dramatically during the last decade resulting in a plethora of new experiments leading to a significant increase in the scientific repertoire for studying membrane proteins. Besides solving the three-dimensional structures using state-of-the-art approaches......-populated states, this review seeks to introduce the vast possibilities solution NMR can offer to the study of membrane protein structure-function analyses with special focus on applicability. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012....

  10. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Nühse, Thomas S; Foster, Leonard J

    2003-01-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

  11. Solubilization of lipids and membrane proteins into nanodiscs : Mode of action and applications of SMA copolymers

    NARCIS (Netherlands)

    Scheidelaar, S.

    2016-01-01

    Cell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little

  12. Periplasmic quality control in biogenesis of outer membrane proteins.

    Science.gov (United States)

    Lyu, Zhi Xin; Zhao, Xin Sheng

    2015-04-01

    The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

  13. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    Science.gov (United States)

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition.

    Science.gov (United States)

    Hayat, Maqsood; Khan, Asifullah

    2011-02-21

    Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Role for chlamydial inclusion membrane proteins in inclusion membrane structure and biogenesis.

    Directory of Open Access Journals (Sweden)

    Jeffrey Mital

    Full Text Available The chlamydial inclusion membrane is extensively modified by the insertion of type III secreted effector proteins. These inclusion membrane proteins (Incs are exposed to the cytosol and share a common structural feature of a long, bi-lobed hydrophobic domain but little or no primary amino acid sequence similarity. Based upon secondary structural predictions, over 50 putative inclusion membrane proteins have been identified in Chlamydia trachomatis. Only a limited number of biological functions have been defined and these are not shared between chlamydial species. Here we have ectopically expressed several C. trachomatis Incs in HeLa cells and find that they induce the formation of morphologically distinct membranous vesicular compartments. Formation of these vesicles requires the bi-lobed hydrophobic domain as a minimum. No markers for various cellular organelles were observed in association with these vesicles. Lipid probes were incorporated by the Inc-induced vesicles although the lipids incorporated were dependent upon the specific Inc expressed. Co-expression of Inc pairs indicated that some colocalized in the same vesicle, others partially overlapped, and others did not associate at all. Overall, it appears that Incs may have an intrinsic ability to induce membrane formation and that individual Incs can induce membranous structures with unique properties.

  17. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

    DEFF Research Database (Denmark)

    Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

    2010-01-01

    itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology....

  18. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...... to humans. GPI-APs confer important cellular functions as receptors, enzymes and scaffolding molecules. Specific enzymes and detergent extraction methods combined with separation technologies and mass spectrometry permit proteomic analysis of GPI-APs from plasma membrane preparations to reveal cell...

  19. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC.

    Science.gov (United States)

    Garbarino, Jeanne; Pan, Meihui; Chin, Harvey F; Lund, Frederik W; Maxfield, Frederick R; Breslow, Jan L

    2012-12-01

    STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

  20. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

    Science.gov (United States)

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-07-25

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

  1. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

  2. Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

    Science.gov (United States)

    Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

    2015-12-08

    Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

  3. The tissue-specific Rep8/UBXD6 tethers p97 to the endoplasmic reticulum membrane for degradation of misfolded proteins

    DEFF Research Database (Denmark)

    Madsen, Louise; Kriegenburg, Franziska; Lages Lino Vala, Andrea

    2011-01-01

    is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p......The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded...... protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8...

  4. Investigating ER-Associated Degradation with RNAi Screening - and Searching for Model Proteins to Do It with

    DEFF Research Database (Denmark)

    Jensen, Njal Winther

    Abstract In eukaryotes, secretory proteins are translocated into the endoplasmic reticulum (ER) for folding assistance, acquisition of posttranslational modifications and sorting. Proteins that do not obtain their native conformation are eliminated by ER-associated degradation (ERAD). ERAD...... is a sophisticated pathway that recognizes misfolded proteins and targets them for degradation by the 26S proteasome residing in the cytosol. More than 60 diseases including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have been linked to the ERAD pathway underscoring its crucial role...... for cellular homeostasis. The aim of this thesis has been to gain insight into ERAD. The experimental approach was RNAi screening, which is a fast and efficient method for initial evaluation of a large pool of genes. Since relatively few proteins routinely are used as ERAD substrates, the first goal...

  5. Self-assembling layers created by membrane proteins on gold.

    Science.gov (United States)

    Shah, D S; Thomas, M B; Phillips, S; Cisneros, D A; Le Brun, A P; Holt, S A; Lakey, J H

    2007-06-01

    Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

  6. MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

    Directory of Open Access Journals (Sweden)

    N. G. Zemlianskykh

    2016-10-01

    Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

  7. Modulation of Membrane Protein Lateral Mobility by Polyphosphates and Polyamines

    Science.gov (United States)

    Schindler, Melvin; Koppel, Dennis E.; Sheetz, Michael P.

    1980-03-01

    The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of ``fluorescence redistribution after fusion.'' Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.

  8. Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

    Science.gov (United States)

    Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

  9. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  10. [Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

    Science.gov (United States)

    Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

    2008-01-01

    To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

  11. A model for the generation and interconversion of ER morphologies.

    Science.gov (United States)

    Shemesh, Tom; Klemm, Robin W; Romano, Fabian B; Wang, Songyu; Vaughan, Joshua; Zhuang, Xiaowei; Tukachinsky, Hanna; Kozlov, Michael M; Rapoport, Tom A

    2014-12-09

    The peripheral endoplasmic reticulum (ER) forms different morphologies composed of tubules and sheets. Proteins such as the reticulons shape the ER by stabilizing the high membrane curvature in cross-sections of tubules and sheet edges. Here, we show that membrane curvature along the edge lines is also critical for ER shaping. We describe a theoretical model that explains virtually all observed ER morphologies. The model is based on two types of curvature-stabilizing proteins that generate either straight or negatively curved edge lines (R- and S-type proteins). Dependent on the concentrations of R- and S-type proteins, membrane morphologies can be generated that consist of tubules, sheets, sheet fenestrations, and sheet stacks with helicoidal connections. We propose that reticulons 4a/b are representatives of R-type proteins that favor tubules and outer edges of sheets. Lunapark is an example of S-type proteins that promote junctions between tubules and sheets. In a tubular ER network, lunapark stabilizes three-way junctions, i.e., small triangular sheets with concave edges. The model agrees with experimental observations and explains how curvature-stabilizing proteins determine ER morphology.

  12. Structural investigation of membrane proteins by electron microscopy

    NARCIS (Netherlands)

    Moscicka, Katarzyna Beata

    2009-01-01

    Biological membranes are vital components of all living systems, forming the boundaries of cells and their organelles. They consist of a lipid bilayer and embedded proteins, which are nanomachines that fulfill key functions such as energy conversion, solute transport, secretion, and signal

  13. Toponomics method for the automated quantification of membrane protein translocation.

    Science.gov (United States)

    Domanova, Olga; Borbe, Stefan; Mühlfeld, Stefanie; Becker, Martin; Kubitz, Ralf; Häussinger, Dieter; Berlage, Thomas

    2011-09-19

    Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state. An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results. New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.

  14. Salinity induced changes in cell membrane stability, protein and ...

    African Journals Online (AJOL)

    control), 4.7, 9.4 and 14.1 dS m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability, protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and roots decreased ...

  15. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    Science.gov (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  16. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  17. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Science.gov (United States)

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Stochastic lattice model of synaptic membrane protein domains.

    Science.gov (United States)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  19. Coronavirus infection, ER stress and Apoptosis

    Directory of Open Access Journals (Sweden)

    TO SING eFUNG

    2014-06-01

    Full Text Available The replication of coronavirus, a family of important animal and human pathogens, is closely associated with the cellular membrane compartments, especially the endoplasmic reticulum (ER. Coronavirus infection of cultured cells was previously shown to cause ER stress and induce the unfolded protein response (UPR, a process that aims to restore the ER homeostasis by global translation shutdown and increasing the ER folding capacity. However under prolonged ER stress, UPR can also induce apoptotic cell death. Accumulating evidence from recent studies has shown that induction of ER stress and UPR may constitute a major aspect of coronavirus-host interaction. Activation of the three branches of UPR modulates a wide variety of signaling pathways, such as mitogen-activated protein (MAP kinases activation, autophagy, apoptosis and innate immune response. ER stress and UPR activation may therefore contribute significantly to the viral replication and pathogenesis during coronavirus infection. In this review, we summarize current knowledge on coronavirus-induced ER stress and UPR activation, with emphasis on their cross-talking to apoptotic signaling.

  20. Identification of membrane proteins by tandem mass spectrometry of protein ions

    Science.gov (United States)

    Carroll, Joe; Altman, Matthew C.; Fearnley, Ian M.; Walker, John E.

    2007-01-01

    The most common way of identifying proteins in proteomic analyses is to use short segments of sequence (“tags”) determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning α-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1–4 transmembrane α-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5–18 transmembrane α-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase. PMID:17720804

  1. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  2. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  3. Probing protein-lipid interactions by FRET between membrane fluorophores

    Science.gov (United States)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  4. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    International Nuclear Information System (INIS)

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-01-01

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H 2 O/D 2 O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished

  5. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  6. [Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

    Science.gov (United States)

    Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

    2017-10-01

    In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

  7. Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

    Science.gov (United States)

    Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

    2014-03-01

    Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Tandem malonate-based glucosides (TMGs) for membrane protein structural studies

    DEFF Research Database (Denmark)

    Hussain, Hazrat; Mortensen, Jonas S.; Du, Yang

    2017-01-01

    class of glucoside amphiphiles, designated tandem malonate-based glucosides (TMGs). A few TMG agents proved effective at both stabilizing a range of membrane proteins and extracting proteins from the membrane environment. These favourable characteristics, along with synthetic convenience, indicate...

  9. Novel Xylene-Linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Du, Yang; Scull, Nicola J

    2015-01-01

    Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions...

  10. AlignMe—a membrane protein sequence alignment web server

    Science.gov (United States)

    Stamm, Marcus; Staritzbichler, René; Khafizov, Kamil; Forrest, Lucy R.

    2014-01-01

    We present a web server for pair-wise alignment of membrane protein sequences, using the program AlignMe. The server makes available two operational modes of AlignMe: (i) sequence to sequence alignment, taking two sequences in fasta format as input, combining information about each sequence from multiple sources and producing a pair-wise alignment (PW mode); and (ii) alignment of two multiple sequence alignments to create family-averaged hydropathy profile alignments (HP mode). For the PW sequence alignment mode, four different optimized parameter sets are provided, each suited to pairs of sequences with a specific similarity level. These settings utilize different types of inputs: (position-specific) substitution matrices, secondary structure predictions and transmembrane propensities from transmembrane predictions or hydrophobicity scales. In the second (HP) mode, each input multiple sequence alignment is converted into a hydrophobicity profile averaged over the provided set of sequence homologs; the two profiles are then aligned. The HP mode enables qualitative comparison of transmembrane topologies (and therefore potentially of 3D folds) of two membrane proteins, which can be useful if the proteins have low sequence similarity. In summary, the AlignMe web server provides user-friendly access to a set of tools for analysis and comparison of membrane protein sequences. Access is available at http://www.bioinfo.mpg.de/AlignMe PMID:24753425

  11. The Origin and Early Evolution of Membrane Proteins

    Science.gov (United States)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  12. ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice

    Directory of Open Access Journals (Sweden)

    Frances E. Jones

    2016-02-01

    Full Text Available Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.

  13. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  14. Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

    International Nuclear Information System (INIS)

    Mecham, R.P.; Hinek, A.; Entwistle, R.; Wrenn, D.S.; Griffin, G.L.; Senior, R.M.

    1989-01-01

    Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [ 3 H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. The authors propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment

  15. Alterations in membrane protein-profile during cold treatment of alfalfa

    International Nuclear Information System (INIS)

    Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S.

    1988-01-01

    Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [ 35 S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo

  16. Artificial membranes with selective nanochannels for protein transport

    KAUST Repository

    Sutisna, Burhannudin

    2016-09-05

    A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

  17. Artificial membranes with selective nanochannels for protein transport

    KAUST Repository

    Sutisna, Burhannudin; Polymeropoulos, Georgios; Mygiakis, E.; Musteata, Valentina-Elena; Peinemann, Klaus-Viktor; Smilgies, D. M.; Hadjichristidis, Nikolaos; Nunes, Suzana Pereira

    2016-01-01

    A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

  18. Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

    Science.gov (United States)

    Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

  19. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

    Science.gov (United States)

    Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

    2014-01-01

    Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

  20. Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

    Science.gov (United States)

    Ashok, Yashwanth; Jaakola, Veli-Pekka

    2016-01-01

    Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

  1. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  2. Entry and exit of bacterial outer membrane proteins.

    Science.gov (United States)

    Misra, Rajeev

    2015-08-01

    The sites of new outer membrane protein (OMP) deposition and the fate of pre-existing OMPs are still enigmatic despite numerous concerted efforts. Rassam et al. identified mid-cell regions as the primary entry points for new OMP insertion in clusters, driving the pre-existing OMP clusters towards cell poles for long-term storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Refractive-index-based screening of membrane-protein-mediated transfer across biological membranes.

    Science.gov (United States)

    Brändén, Magnus; Tabaei, Seyed R; Fischer, Gerhard; Neutze, Richard; Höök, Fredrik

    2010-07-07

    Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Biomimetic Membranes for Multi-Redox Center Proteins

    Directory of Open Access Journals (Sweden)

    Renate L. C. Naumann

    2016-03-01

    Full Text Available His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs. An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM. MRPs, such as the cytochrome c oxidase (CcO from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS and surface-enhanced resonance Raman spectroscopy (SERRS, were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs. The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

  5. Evaluating descriptors for the lateral translocation of membrane proteins.

    Science.gov (United States)

    Domanova, Olga; Borbe, Stefan; Mühlfeld, Stefanie; Becker, Martin; Kubitz, Ralf; Häussinger, Dieter; Berlage, Thomas

    2011-01-01

    Microscopic images of tissue sections are used for diagnosis and monitoring of therapy, by analysis of protein patterns correlating to disease states. Spatial protein distribution is influenced by protein translocation between different membrane compartments and quantified by comparison of microscopic images of biological samples. Cholestatic liver diseases are characterized by translocation of transport proteins, and quantification of their dislocation offers new diagnostic options. However, reliable and unbiased tools are lacking. The nowadays used manual method is slow, subjective and error-prone. We have developed a new workflow based on automated image analysis and improved it by the introduction of scale-free descriptors for the translocation quantification. This fast and unbiased method can substitute the manual analysis, and the suggested descriptors perform better than the earlier used statistical variance.

  6. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    International Nuclear Information System (INIS)

    Witt, P.L.; Bownds, M.D.

    1987-01-01

    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

  7. Membrane protein damage and repair: selective loss of a quinone-protein function in chloroplast membranes

    International Nuclear Information System (INIS)

    Kyle, D.J.; Ohad, I.; Arntzen, C.J.

    1984-01-01

    A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q/sub B/), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q/sub B/ apoprotein was demonstrated by a loss of binding sites for [ 14 C]atraazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities. 21 references, 4 figures, 2 tables

  8. Correlation Study of PVDF Membrane Morphology with Protein Adsorption: Quantitative Analysis by FTIR/ATR Technique

    Science.gov (United States)

    Ideris, N.; Ahmad, A. L.; Ooi, B. S.; Low, S. C.

    2018-05-01

    Microporous PVDF membranes were used as protein capture matrices in immunoassays. Because the most common labels in immunoassays were detected based on the colour change, an understanding of how protein concentration varies on different PVDF surfaces was needed. Herein, the correlation between the membrane pore size and protein adsorption was systematically investigated. Five different PVDF membrane morphologies were prepared and FTIR/ATR was employed to accurately quantify the surface protein concentration on membranes with small pore sizes. SigmaPlot® was used to find a suitable curve fit for protein adsorption and membrane pore size, with a high correlation coefficient, R2, of 0.9971.

  9. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  10. Dynamic, electronically switchable surfaces for membrane protein microarrays.

    Science.gov (United States)

    Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

    2006-02-01

    Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

  11. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    Directory of Open Access Journals (Sweden)

    Marciniak Bogumiła C

    2012-05-01

    Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

  12. Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

    Science.gov (United States)

    Perry, Ben D; Rahnert, Jill A; Xie, Yang; Zheng, Bin; Woodworth-Hobbs, Myra E; Price, S Russ

    2018-01-01

    Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

  13. Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

    Directory of Open Access Journals (Sweden)

    Ben D Perry

    Full Text Available Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4. Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides, palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242. Inflammatory indicators of TLR4 activation (IL-6 and TNFα and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

  14. Equilibrium fluctuation relations for voltage coupling in membrane proteins.

    Science.gov (United States)

    Kim, Ilsoo; Warshel, Arieh

    2015-11-01

    A general theoretical framework is developed to account for the effects of an external potential on the energetics of membrane proteins. The framework is based on the free energy relation between two (forward/backward) probability densities, which was recently generalized to non-equilibrium processes, culminating in the work-fluctuation theorem. Starting from the probability densities of the conformational states along the "voltage coupling" reaction coordinate, we investigate several interconnected free energy relations between these two conformational states, considering voltage activation of ion channels. The free energy difference between the two conformational states at zero (depolarization) membrane potential (i.e., known as the chemical component of free energy change in ion channels) is shown to be equivalent to the free energy difference between the two "equilibrium" (resting and activated) conformational states along the one-dimensional voltage couplin reaction coordinate. Furthermore, the requirement that the application of linear response approximation to the free energy functionals of voltage coupling should satisfy the general free energy relations, yields a novel closed-form expression for the gating charge in terms of other basic properties of ion channels. This connection is familiar in statistical mechanics, known as the equilibrium fluctuation-response relation. The theory is illustrated by considering the coupling of a unit charge to the external voltage in the two sites near the surface of membrane, representing the activated and resting states. This is done using a coarse-graining (CG) model of membrane proteins, which includes the membrane, the electrolytes and the electrodes. The CG model yields Marcus-type voltage dependent free energy parabolas for the response of the electrostatic environment (electrolytes etc.) to the transition from the initial to the final configuratinal states, leading to equilibrium free energy difference and free

  15. A critical role of a cellular membrane traffic protein in poliovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    George A Belov

    2008-11-01

    Full Text Available Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA, implicating some components(s of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.

  16. Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

    Science.gov (United States)

    Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

    2017-07-20

    Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  18. Exploring the Spatiotemporal Organization of Membrane Proteins in Living Plant Cells.

    Science.gov (United States)

    Wang, Li; Xue, Yiqun; Xing, Jingjing; Song, Kai; Lin, Jinxing

    2018-04-29

    Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we assess recent progress in the development of labeling and imaging technology for membrane protein analysis. We focus in particular on several single-molecule techniques for quantifying the dynamics and assembly of membrane proteins. Finally, we provide examples of how these new techniques are advancing our understanding of the complex biological functions of membrane proteins.

  19. Evaluating the potential bioactivity of a novel compound ER1626.

    Science.gov (United States)

    Wang, Lijun; Zeng, Yanyan; Wang, Tianling; Liu, Hongyi; Xiao, Hong; Xiang, Hua

    2014-01-01

    ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626. MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis. ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC50 of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells. In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.

  20. Evaluating the potential bioactivity of a novel compound ER1626.

    Directory of Open Access Journals (Sweden)

    Lijun Wang

    Full Text Available ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC50 of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.

  1. Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi; Li, Shimo; Itoh, Masanori; Wang, Miao-xing; Hayakawa, Miki; Islam, Saiful; Tana; Nakagawa, Kiyomi [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Chen, Huayue [Department of Anatomy, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Nakagawa, Toshiyuki, E-mail: tnakagaw@gifu-u.ac.jp [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan)

    2016-05-27

    The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.

  2. Molecular organization in bacterial cell membranes. Specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes

    Energy Technology Data Exchange (ETDEWEB)

    Larraga, V; Munoz, E [Consejo Superior de Investigaciones Cientificas, Madrid (Spain). Instituto de Biologia Celular

    1975-05-01

    The paper reports about an investigation into the question of the specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes. The method of sample preparation is described: Tritium labelling of glycoproteins in protoplasts and membranes, iodination of proteins, trypsin treatment and polyacrylamide gel electrophoresis. The findings suggest an asymmetrical distribution of the glycoproteins in membranes and a weak accessibility to iodine label. A structural model of the plasma membranes of Streptomyces albus is proposed similar to the general 'fluid mosaic' model of Singer and Nicholson.

  3. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Science.gov (United States)

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei

    2013-05-01

    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Copyright © 2013 Wiley Periodicals, Inc.

  4. EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

    Directory of Open Access Journals (Sweden)

    Sarah Appel

    2009-05-01

    Full Text Available Sarah Appel1,2,6, Susanne Vetterkind1,2,6, Ansgar Koplin1,3, Barbara Maertens1,4, Meike Boosen1,5, Ute Preuss11The Institute of Genetics, University of Bonn, Bonn, Germany; 2Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, MA, USA; 3Center for Molecular Biology Heidelberg (ZMBH, Heidelberg, Germany; 4Institute of Biochemistry II, University of Cologne, Cologne, Germany; 5Institute of Pharmacology and Toxicology, University Hospital of Johann Wolfgang Goethe-University Frankfurt am Main, Frankfurt am Main, Germany; 6These authors contributed equally to this work.Abstract: We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1 as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4. Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca2+ stress-inducing agents was significantly decreased after si

  5. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Dufresne, Philippe J.; Thivierge, Karine; Cotton, Sophie; Beauchemin, Chantal; Ide, Christine; Ubalijoro, Eliane; Laliberte, Jean-Francois; Fortin, Marc G.

    2008-01-01

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

  6. The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

    NARCIS (Netherlands)

    Bosch, B.J.

    2004-01-01

    The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

  7. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

    Science.gov (United States)

    Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

  8. INTERSPECIES EXTRAPOLATION AND IN VITRO SCREENING BASED UPON RECOMBINANT AR AND ER PROTEINS

    Science.gov (United States)

    While EDCs have the potential to act via several mechanisms of action, one of the most widely accepted is the ability of environmental chemicals to interact directly with either the estrogen (ER) or androgen receptor (AR). Although we know that the steroid receptors of the lower...

  9. Monitoring Protein Fouling on Polymeric Membranes Using Ultrasonic Frequency-Domain Reflectometry

    Directory of Open Access Journals (Sweden)

    Robin Fong

    2011-08-01

    Full Text Available Novel signal-processing protocols were used to extend the in situ sensitivity of ultrasonic frequency-domain reflectometry (UFDR for real-time monitoring of microfiltration (MF membrane fouling during protein purification. Different commercial membrane materials, with a nominal pore size of 0.2 µm, were challenged using bovine serum albumin (BSA and amylase as model proteins. Fouling induced by these proteins was observed in flat-sheet membrane filtration cells operating in a laminar cross-flow regime. The detection of membrane-associated proteins using UFDR was determined by applying rigorous statistical methodology to reflection spectra of ultrasonic signals obtained during membrane fouling. Data suggest that the total power reflected from membrane surfaces changes in response to protein fouling at concentrations as low as 14 μg/cm2, and results indicate that ultrasonic spectra can be leveraged to detect and monitor protein fouling on commercial MF membranes.

  10. Protein diffusion in plant cell plasma membranes: The cell-wall corral

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

    Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  11. Protein diffusion in plant cell plasma membranes: the cell-wall corral.

    Science.gov (United States)

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  12. Unfolding study of a trimeric membrane protein AcrB.

    Science.gov (United States)

    Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

    2014-07-01

    The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. © 2014 The Protein Society.

  13. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  14. Clusters of proteins in bio-membranes: insights into the roles of interaction potential shapes and of protein diversity

    OpenAIRE

    Meilhac, Nicolas; Destainville, Nicolas

    2011-01-01

    It has recently been proposed that proteins embedded in lipidic bio-membranes can spontaneously self-organize into stable small clusters, or membrane nano-domains, due to the competition between short-range attractive and longer-range repulsive forces between proteins, specific to these systems. In this paper, we carry on our investigation, by Monte Carlo simulations, of different aspects of cluster phases of proteins in bio-membranes. First, we compare different long-range potentials (includ...

  15. Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

    OpenAIRE

    Evan Quon; Christopher T. Beh

    2016-01-01

    Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer...

  16. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

    Science.gov (United States)

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-29

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

  17. The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

    Science.gov (United States)

    Leu, Jiann-Horng; Liu, Kuan-Fu; Chen, Kuan-Yu; Chen, Shu-Hwa; Wang, Yu-Bin; Lin, Chung-Yen; Lo, Chu-Fang

    2015-04-01

    By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Changes in exposed membrane proteins during in vitro capacitation of boar sperm

    International Nuclear Information System (INIS)

    Berger, T.

    1990-01-01

    Exposed plasma membrane proteins were labeled with 125 I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane

  19. BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

    DEFF Research Database (Denmark)

    Madsen, Kenneth Lindegaard; Bhatia, V K; Gether, U

    2010-01-01

    /ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can...

  20. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

    Directory of Open Access Journals (Sweden)

    Stefania ePizzimenti

    2013-09-01

    Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

  2. Ultrananocrystalline Diamond Membranes for Detection of High-Mass Proteins

    Science.gov (United States)

    Kim, H.; Park, J.; Aksamija, Z.; Arbulu, M.; Blick, R. H.

    2016-12-01

    Mechanical resonators realized on the nanoscale by now offer applications in mass sensing of biomolecules with extraordinary sensitivity. The general idea is that perfect mechanical mass sensors should be of extremely small size to achieve zepto- or yoctogram sensitivity in weighing single molecules similar to a classical scale. However, the small effective size and long response time for weighing biomolecules with a cantilever restricts their usefulness as a high-throughput method. Commercial mass spectrometry (MS), on the other hand, such as electrospray ionization and matrix-assisted laser desorption and ionization (MALDI) time of flight (TOF) and their charge-amplifying detectors are the gold standards to which nanomechanical resonators have to live up to. These two methods rely on the ionization and acceleration of biomolecules and the following ion detection after a mass selection step, such as TOF. The principle we describe here for ion detection is based on the conversion of kinetic energy of the biomolecules into thermal excitation of chemical vapor deposition diamond nanomembranes via phonons followed by phonon-mediated detection via field emission of thermally emitted electrons. We fabricate ultrathin diamond membranes with large lateral dimensions for MALDI TOF MS of high-mass proteins. These diamond membranes are realized by straightforward etching methods based on semiconductor processing. With a minimal thickness of 100 nm and cross sections of up to 400 ×400 μ m2 , the membranes offer extreme aspect ratios. Ion detection is demonstrated in MALDI TOF analysis over a broad range from insulin to albumin. The resulting data in detection show much enhanced resolution as compared to existing detectors, which can offer better sensitivity and overall performance in resolving protein masses.

  3. Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

    International Nuclear Information System (INIS)

    Zhou, Donghua H.; Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.; Rienstra, Chad M.

    2012-01-01

    Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

  4. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    International Nuclear Information System (INIS)

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

    2005-01-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

  5. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  6. Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

    Science.gov (United States)

    Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

    2018-01-30

    Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

  7. Nanoscale protein architecture of the kidney glomerular basement membrane

    Science.gov (United States)

    Suleiman, Hani; Zhang, Lei; Roth, Robyn; Heuser, John E; Miner, Jeffrey H; Shaw, Andrey S; Dani, Adish

    2013-01-01

    In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI: http://dx.doi.org/10.7554/eLife.01149.001 PMID:24137544

  8. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  9. Nanoscopic dynamics of bicontinous microemulsions: effect of membrane associated protein.

    Science.gov (United States)

    Sharma, V K; Hayes, Douglas G; Urban, Volker S; O'Neill, Hugh M; Tyagi, M; Mamontov, E

    2017-07-19

    Bicontinous microemulsions (BμE) generally consist of nanodomains formed by surfactant in a mixture of water and oil at nearly equal proportions and are potential candidates for the solubilization and purification of membrane proteins. Here we present the first time report of nanoscopic dynamics of surfactant monolayers within BμEs formed by the anionic surfactant sodium dodecyl sulfate (SDS) measured on the nanosecond to picosecond time scale using quasielastic neutron scattering (QENS). BμEs investigated herein consisted of middle phases isolated from Winsor-III microemulsion systems that were formed by mixing aqueous and oil solutions under optimal conditions. QENS data indicates that surfactants undergo two distinct motions, namely (i) lateral motion along the surface of the oil nanodomains and (ii) localized internal motion. Lateral motion can be described using a continuous diffusion model, from which the lateral diffusion coefficient is obtained. Internal motion of surfactant is described using a model which assumes that a fraction of the surfactants' hydrogens undergoes localized translational diffusion that could be considered confined within a spherical volume. The effect of cytochrome c, an archetypal membrane-associated protein known to strongly partition near the surfactant head groups in BμEs (a trend supported by small-angle X-ray scattering [SAXS] analysis), on the dynamics of BμE has also been investigated. QENS results demonstrated that cytochrome c significantly hindered both the lateral and the internal motions of surfactant. The lateral motion was more strongly affected: a reduction of the lateral diffusion coefficient by 33% was measured. This change is mainly attributable to the strong association of cytochrome c with oppositely charged SDS. In contrast, analysis of SAXS data suggested that thermal fluctuations (for a longer length and slower time scale compared to QENS) were increased upon incorporation of cytochrome c. This study

  10. Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 B139

    Energy Technology Data Exchange (ETDEWEB)

    Brian A. Larkins

    2003-03-21

    Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactions between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-alpha-zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD alpha-zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15

  11. G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

    Science.gov (United States)

    O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

    2012-12-18

    Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

  12. A New Strain Collection for Improved Expression of Outer Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Ina Meuskens

    2017-11-01

    Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

  13. Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

    Science.gov (United States)

    Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

    2018-04-17

    Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  14. Revealing Linear Aggregates of Light Harvesting Antenna Proteins in Photosynthetic Membranes

    OpenAIRE

    He, Yufan; Zeng, Xiaohua; Mukherjee, Saptarshi; Rajapaksha, Suneth; Kaplan, Samuel; Lu, H. Peter

    2010-01-01

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under...

  15. Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Darwish, Tamim A.; Pedersen, Martin Cramer

    2018-01-01

    A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron sca...... solution structure determination of membrane proteins by SANS and subsequent data analysis available to non-specialists. This article is protected by copyright. All rights reserved....

  16. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R

    2012-01-01

    Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show th...... that the new amphiphile, "glyco-diosgenin" (GDN; see figure), confers enhanced stability to a variety of membrane proteins in solution relative to popular conventional detergents, such as dodecylmaltoside (DDM)....

  17. Identification of Salt-Tolerant Sinorhizobium sp Strain BL3 Membrane Proteins Based on Proteomics

    DEFF Research Database (Denmark)

    Tanthanuch, Waraporn; Mohammed, Shabaz; Matthiesen, Rune

    2010-01-01

    functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off-line...... SCX fractionation coupled to nanoLC-MS/MS. These techniques would be useful for further comparative analysis of membrane proteins that function in the response to environmental stress....

  18. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

  19. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  20. Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

    Science.gov (United States)

    Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

    2006-05-19

    Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

  1. Effect of Adsorbed Protein on the Hydraulic Permeability, Membrane and Streaming Potential Values Measured across a Microporous Membrane

    DEFF Research Database (Denmark)

    Benavente, Juana; Jonsson, Gunnar Eigil

    1998-01-01

    permeability decreases strongly when the pH decreases, having its minimum value at the isoelectric point of the protein; the apparent zeta potential values are also dependent on both pH and salt concentration. Differences in the streaming potential coefficient determined for two membranes fouled under......The effect of the adsorption of a protein, bovine serum albumin (BSA), on the membrane potential, flux reduction and streaming potential measured across a microporous polysulphone membrane with different NaCl solutions and pH values is studied. From electrokinetic phenomena, information about...... the electrical properties of the membrane (fixed charge concentration and ionic transport numbers) or the membrane/solute interactions (streaming and zeta potentials) can be obtained. The influence of pH and ionic strength on volume flux and streaming potential values is considered. Results show that hydraulic...

  2. Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone

    International Nuclear Information System (INIS)

    Ozegovic, B.; Dobrovic-Jenik, D.; Milkovic, S.

    1988-01-01

    The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3 H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3 H-aldosterone - kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3 H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3 H-aldosterone peak while the second 3 H-aldosterone peak was eluted with Ve corresponding to Ve of free 3 H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3 H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass. (author)

  3. Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

    Science.gov (United States)

    Dong, Yuanchen; Chen, Shuobing; Zhang, Shijian; Sodroski, Joseph; Yang, Zhongqiang; Liu, Dongsheng; Mao, Youdong

    2018-02-19

    Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Directory of Open Access Journals (Sweden)

    Laulumaa Saara

    2015-01-01

    Full Text Available Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  5. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    Science.gov (United States)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  6. Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

    Science.gov (United States)

    McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

    2015-09-01

    Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by

  7. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang

    2016-01-01

    Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents...

  8. The synthesis of recombinant membrane proteins in yeast for structural studies.

    Science.gov (United States)

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Yasuyuki Suda

    2018-01-01

    Full Text Available A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER. They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

  10. Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.

    Science.gov (United States)

    Yang, Yoosoo; Hong, Yeonsun; Cho, Eunji; Kim, Gi Beom; Kim, In-San

    2018-01-01

    Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.

  11. Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

    Science.gov (United States)

    Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

    2016-01-01

    We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

  12. Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

    Science.gov (United States)

    Harner, Max; Neupert, Walter; Deponte, Marcel

    2011-07-15

    The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

  13. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    Energy Technology Data Exchange (ETDEWEB)

    Cervia, Davide, E-mail: d.cervia@unitus.it [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Catalani, Elisabetta; Belardinelli, Maria Cristina [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Perrotta, Cristiana [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Picchietti, Simona [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Alimenti, Claudio [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy); Casini, Giovanni; Fausto, Anna Maria [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Vallesi, Adriana [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  14. Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress.

    Science.gov (United States)

    Kim, Jinhwan; Choi, Tae Gyu; Ding, Yan; Kim, Yeonghwan; Ha, Kwon Soo; Lee, Kyung Ho; Kang, Insug; Ha, Joohun; Kaufman, Randal J; Lee, Jinhwa; Choe, Wonchae; Kim, Sung Soo

    2008-11-01

    Prolonged accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in ER stress-mediated apoptosis. Cyclophilins are protein chaperones that accelerate the rate of protein folding through their peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, we demonstrated that ER stress activates the expression of the ER-localized cyclophilin B (CypB) gene through a novel ER stress response element. Overexpression of wild-type CypB attenuated ER stress-induced cell death, whereas overexpression of an isomerase activity-defective mutant, CypB/R62A, not only increased Ca(2+) leakage from the ER and ROS generation, but also decreased mitochondrial membrane potential, resulting in cell death following exposure to ER stress-inducing agents. siRNA-mediated inhibition of CypB expression rendered cells more vulnerable to ER stress. Finally, CypB interacted with the ER stress-related chaperones, Bip and Grp94. Taken together, we concluded that CypB performs a crucial function in protecting cells against ER stress via its PPIase activity.

  15. Helicobacter pylori Outer Membrane Protein-Related Pathogenesis

    Directory of Open Access Journals (Sweden)

    Yuichi Matsuo

    2017-03-01

    Full Text Available Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system.

  16. Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

    Science.gov (United States)

    Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

    2017-08-22

    The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

  17. Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

    Directory of Open Access Journals (Sweden)

    Thomas eSimmen

    2014-10-01

    Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

  18. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR) Gene Promoter-Binding Proteins in Estrogen Receptor (ER)-Positive and ER-Depleted Breast Cancer Cells

    International Nuclear Information System (INIS)

    Sarfstein, Rive; Belfiore, Antonino; Werner, Haim

    2010-01-01

    The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immune-precipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells

  19. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR) Gene Promoter-Binding Proteins in Estrogen Receptor (ER)-Positive and ER-Depleted Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Sarfstein, Rive [Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Belfiore, Antonino [Department of Clinical and Experimental Medicine, University Magna Graecia of Catanzaro, Catanzaro 88100 (Italy); Werner, Haim, E-mail: hwerner@post.tau.ac.il [Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel)

    2010-03-25

    The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immune-precipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells.

  20. The role of hydrophobic interactions in positioning of peripheral proteins in membranes

    Directory of Open Access Journals (Sweden)

    Lomize Mikhail A

    2007-06-01

    Full Text Available Abstract Background Three-dimensional (3D structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined. Results We report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM database. Conclusion Positions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our

  1. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

    Science.gov (United States)

    Leser, George P; Lamb, Robert A

    2017-05-01

    Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

  2. A Finite Element Solution of Lateral Periodic Poisson–Boltzmann Model for Membrane Channel Proteins

    Science.gov (United States)

    Xu, Jingjie; Lu, Benzhuo

    2018-01-01

    Membrane channel proteins control the diffusion of ions across biological membranes. They are closely related to the processes of various organizational mechanisms, such as: cardiac impulse, muscle contraction and hormone secretion. Introducing a membrane region into implicit solvation models extends the ability of the Poisson–Boltzmann (PB) equation to handle membrane proteins. The use of lateral periodic boundary conditions can properly simulate the discrete distribution of membrane proteins on the membrane plane and avoid boundary effects, which are caused by the finite box size in the traditional PB calculations. In this work, we: (1) develop a first finite element solver (FEPB) to solve the PB equation with a two-dimensional periodicity for membrane channel proteins, with different numerical treatments of the singular charges distributions in the channel protein; (2) add the membrane as a dielectric slab in the PB model, and use an improved mesh construction method to automatically identify the membrane channel/pore region even with a tilt angle relative to the z-axis; and (3) add a non-polar solvation energy term to complete the estimation of the total solvation energy of a membrane protein. A mesh resolution of about 0.25 Å (cubic grid space)/0.36 Å (tetrahedron edge length) is found to be most accurate in linear finite element calculation of the PB solvation energy. Computational studies are performed on a few exemplary molecules. The results indicate that all factors, the membrane thickness, the length of periodic box, membrane dielectric constant, pore region dielectric constant, and ionic strength, have individually considerable influence on the solvation energy of a channel protein. This demonstrates the necessity to treat all of those effects in the PB model for membrane protein simulations. PMID:29495644

  3. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    Science.gov (United States)

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

  4. A positive feedback-based gene circuit to increase the production of a membrane protein

    Directory of Open Access Journals (Sweden)

    Gennis Robert B

    2010-05-01

    Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

  5. Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

    Science.gov (United States)

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

    2008-10-23

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  6. Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

    Science.gov (United States)

    Jaiswal, Dinesh Kumar; Mishra, Poonam; Subba, Pratigya; Rathi, Divya; Chakraborty, Subhra; Chakraborty, Niranjan

    2014-02-01

    Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.

  7. Effect of membrane protein concentration on binding of 3H-imipramine in human platelets

    International Nuclear Information System (INIS)

    Barkai, A.I.; Kowalik, S.; Baron, M.

    1985-01-01

    Binding of 3 H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing 3 H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The 3 H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies

  8. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

    Science.gov (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

    2009-11-30

    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  9. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    Science.gov (United States)

    Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit

    2012-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. PMID:23165475

  10. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    OpenAIRE

    Chae, Pil Seok; Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit; Byrne, Bernadette; Lee, John K.; Gellman, Samuel H.

    2013-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al.

  11. An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

    NARCIS (Netherlands)

    Stetsenko, Artem; Guskov, Albert

    2017-01-01

    To study integral membrane proteins, one has to extract them from the membrane—the step that is typically achieved by the application of detergents. In this mini-review, we summarize the top 10 detergents used for the structural analysis of membrane proteins based on the published results. The aim

  12. Training-induced changes in membrane transport proteins of human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, C.

    2006-01-01

    Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded...

  13. Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Birkelund, Svend

    1998-01-01

    Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol...

  14. Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

    NARCIS (Netherlands)

    Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

    2010-01-01

    The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

  15. Isolation of monodisperse nanodisc-reconstituted membrane proteins using free flow electrophoresis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Laursen, Tomas; Weber, Gerhard

    2013-01-01

    Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e...

  16. Interaction between La(III) and proteins on the plasma membrane of horseradish

    Science.gov (United States)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  17. Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Jaiswal Dinesh Kumar

    2012-10-01

    Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

  18. Protein transport across and into cell membranes in bacteria and archaea

    NARCIS (Netherlands)

    Yuan, Jijun; Zweers, Jessica C.; van Dijl, Jan Maarten; Dalbey, Ross E.

    In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been

  19. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  20. Marinesco-Sjögren syndrome protein SIL1 regulates motor neuron subtype-selective ER stress in ALS

    NARCIS (Netherlands)

    Filézac de L'Etang, Audrey; Maharjan, Niran; Cordeiro Braña, Marisa; Ruegsegger, Céline; Rehmann, Ruth; Goswami, Anand; Roos, Andreas; Troost, Dirk; Schneider, Bernard L.; Weis, Joachim; Saxena, Smita

    2015-01-01

    Mechanisms underlying motor neuron subtype-selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic

  1. Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study

    DEFF Research Database (Denmark)

    Das, Manabendra; Du, Yang; Ribeiro, Orquidea

    2017-01-01

    were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes......Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties....... Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n...

  2. Self-assembly of nanoscale particles with biosurfactants and membrane scaffold proteins.

    Science.gov (United States)

    Faas, Ramona; Pohle, Annelie; Moß, Karin; Henkel, Marius; Hausmann, Rudolf

    2017-12-01

    Nanodiscs are membrane mimetics which may be used as tools for biochemical and biophysical studies of a variety of membrane proteins. These nanoscale structures are composed of a phospholipid bilayer held together by an amphipathic membrane scaffold protein (MSP). In the past, nanodiscs were successfully assembled with membrane scaffold protein 1D1 and 1,2-dipalmitoyl- sn -glycero-3-phosphorylcholine with a homogeneous diameter of ∼10 nm. In this study, the formation of nanoscale particles from MSP1D1 and rhamnolipid biosurfactants is investigated. Different protein to lipid ratios of 1:80, 1:90 and 1:100 were used for the assembly reaction, which were consecutively separated, purified and analyzed by size-exclusion chromatography (SEC) and dynamic light scattering (DLS). Size distributions were measured to determine homogeneity and confirm size dimensions. In this study, first evidence is presented on the formation of nanoscale particles with rhamnolipid biosurfactants and membrane scaffold proteins.

  3. A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

    Directory of Open Access Journals (Sweden)

    Rhona Kayra Stuart

    2014-06-01

    Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

  4. The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

    Directory of Open Access Journals (Sweden)

    Andres Marilou A

    2007-09-01

    Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

  5. Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

    Science.gov (United States)

    Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

    2016-01-01

    The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

  6. CCAAT/Enhancer Binding Protein β in relation to ER Stress, Inflammation, and Metabolic Disturbances

    Directory of Open Access Journals (Sweden)

    Sophie E. van der Krieken

    2015-01-01

    Full Text Available The prevalence of the metabolic syndrome and underlying metabolic disturbances increase rapidly in developed countries. Various molecular targets are currently under investigation to unravel the molecular mechanisms that cause these disturbances. This is done in attempt to counter or prevent the negative health consequences of the metabolic disturbances. Here, we reviewed the current knowledge on the role of C/EBP-β in these metabolic disturbances. C/EBP-β deletion in mice resulted in downregulation of hepatic lipogenic genes and increased expression of β-oxidation genes in brown adipose tissue. Furthermore, C/EBP-β is important in the differentiation and maturation of adipocytes and is increased during ER stress and proinflammatory conditions. So far, studies were only conducted in animals and in cell systems. The results found that C/EBP-β is an important transcription factor within the metabolic disturbances of the metabolic system. Therefore, it is interesting to examine the potential role of C/EBP-β at molecular and physiological level in humans.

  7. Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

    International Nuclear Information System (INIS)

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  8. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    International Nuclear Information System (INIS)

    Smith, D.K.; Winkler, H.H.

    1980-01-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125 I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane

  9. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

    DEFF Research Database (Denmark)

    Garbarino, J.; Pan, M. H.; Chin, H. F.

    2012-01-01

    small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

  10. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    International Nuclear Information System (INIS)

    Morita, Mizuki; Katta, AVSK Mohan; Ahmad, Shandar; Mori, Takaharu; Sugita, Yuji; Mizuguchi, Kenji

    2011-01-01

    Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function

  11. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    Directory of Open Access Journals (Sweden)

    Morita Mizuki

    2011-12-01

    Full Text Available Abstract Background Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. Results To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. Conclusions We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function.

  12. Effect of ceramic membrane channel diameter on limiting retentate protein concentration during skim milk microfiltration.

    Science.gov (United States)

    Adams, Michael C; Barbano, David M

    2016-01-01

    Our objective was to determine the effect of retentate flow channel diameter (4 or 6mm) of nongraded permeability 100-nm pore size ceramic membranes operated in nonuniform transmembrane pressure mode on the limiting retentate protein concentration (LRPC) while microfiltering (MF) skim milk at a temperature of 50°C, a flux of 55 kg · m(-2) · h(-1), and an average cross-flow velocity of 7 m · s(-1). At the above conditions, the retentate true protein concentration was incrementally increased from 7 to 11.5%. When temperature, flux, and average cross-flow velocity were controlled, ceramic membrane retentate flow channel diameter did not affect the LRPC. This indicates that LRPC is not a function of the Reynolds number. Computational fluid dynamics data, which indicated that both membranes had similar radial velocity profiles within their retentate flow channels, supported this finding. Membranes with 6-mm flow channels can be operated at a lower pressure decrease from membrane inlet to membrane outlet (ΔP) or at a higher cross-flow velocity, depending on which is controlled, than membranes with 4-mm flow channels. This implies that 6-mm membranes could achieve a higher LRPC than 4-mm membranes at the same ΔP due to an increase in cross-flow velocity. In theory, the higher LRPC of the 6-mm membranes could facilitate 95% serum protein removal in 2 MF stages with diafiltration between stages if no serum protein were rejected by the membrane. At the same flux, retentate protein concentration, and average cross-flow velocity, 4-mm membranes require 21% more energy to remove a given amount of permeate than 6-mm membranes, despite the lower surface area of the 6-mm membranes. Equations to predict skim milk MF retentate viscosity as a function of protein concentration and temperature are provided. Retentate viscosity, retentate recirculation pump frequency required to maintain a given cross-flow velocity at a given retentate viscosity, and retentate protein

  13. Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

    NARCIS (Netherlands)

    Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2008-01-01

    We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a

  14. Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

    International Nuclear Information System (INIS)

    Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

    1984-01-01

    Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)

  15. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  16. Clustering and visualizing similarity networks of membrane proteins.

    Science.gov (United States)

    Hu, Geng-Ming; Mai, Te-Lun; Chen, Chi-Ming

    2015-08-01

    We proposed a fast and unsupervised clustering method, minimum span clustering (MSC), for analyzing the sequence-structure-function relationship of biological networks, and demonstrated its validity in clustering the sequence/structure similarity networks (SSN) of 682 membrane protein (MP) chains. The MSC clustering of MPs based on their sequence information was found to be consistent with their tertiary structures and functions. For the largest seven clusters predicted by MSC, the consistency in chain function within the same cluster is found to be 100%. From analyzing the edge distribution of SSN for MPs, we found a characteristic threshold distance for the boundary between clusters, over which SSN of MPs could be properly clustered by an unsupervised sparsification of the network distance matrix. The clustering results of MPs from both MSC and the unsupervised sparsification methods are consistent with each other, and have high intracluster similarity and low intercluster similarity in sequence, structure, and function. Our study showed a strong sequence-structure-function relationship of MPs. We discussed evidence of convergent evolution of MPs and suggested applications in finding structural similarities and predicting biological functions of MP chains based on their sequence information. © 2015 Wiley Periodicals, Inc.

  17. Epithelial membrane protein-1 is a biomarker of gefitinib resistance.

    Science.gov (United States)

    Jain, Anjali; Tindell, Charles A; Laux, Isett; Hunter, Jacob B; Curran, John; Galkin, Anna; Afar, Daniel E; Aronson, Nina; Shak, Steven; Natale, Ronald B; Agus, David B

    2005-08-16

    We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.

  18. Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

    Science.gov (United States)

    Sepulveda, Denisse; Rojas-Rivera, Diego; Rodríguez, Diego A; Groenendyk, Jody; Köhler, Andres; Lebeaupin, Cynthia; Ito, Shinya; Urra, Hery; Carreras-Sureda, Amado; Hazari, Younis; Vasseur-Cognet, Mireille; Ali, Maruf M U; Chevet, Eric; Campos, Gisela; Godoy, Patricio; Vaisar, Tomas; Bailly-Maitre, Béatrice; Nagata, Kazuhiro; Michalak, Marek; Sierralta, Jimena; Hetz, Claudio

    2018-01-18

    Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    Science.gov (United States)

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  20. High yield cell-free production of integral membrane proteins without refolding or detergents.

    Science.gov (United States)

    Wuu, Jessica J; Swartz, James R

    2008-05-01

    Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

  1. Living target of Ce(III) action on horseradish cells: proteins on/in cell membrane.

    Science.gov (United States)

    Yang, Guangmei; Sun, Zhaoguo; Lv, Xiaofen; Deng, Yunyun; Zhou, Qing; Huang, Xiaohua

    2012-12-01

    Positive and negative effects of rare earth elements (REEs) in life have been reported in many papers, but the cellular mechanisms have not been answered, especially the action sites of REEs on plasma membrane are unknown. Proteins on/in the plasma membrane perform main functions of the plasma membrane. Cerium (Ce) is the richest REEs in crust. Thus, the interaction between Ce(III) and the proteins on/in the plasma membrane, the morphology of protoplast, and the contents of nutrient elements in protoplast of horseradish were investigated using the optimized combination of the fluorescence microscopy, fluorescence spectroscopy, circular dichroism, scanning electron microscopy, and X-ray energy dispersive spectroscopy. It was found that Ce(III) at the low concentrations (10, 30 μM) could interact with proteins on/in the plasma membrane of horseradish, leading to the improvement in the structure of membrane proteins and the plasma membrane, which accelerated the intra-/extra-cellular substance exchange and further promoted the development of cells. When horseradish was treated with Ce(III) at the high concentrations (60, 80 μM), Ce(III) also could interact with the proteins on/in the plasma membrane of horseradish, leading to the destruction in the structure of membrane proteins and the plasma membrane. These effects decelerated the intra-/extra-cellular substance exchange and further inhibited the development of cells. Thus, the interaction between Ce(III) and proteins on/in the plasma membrane in plants was an important reason of the positive and negative effects of Ce(III) on plants. The results would provide some references for understanding the cellular effect mechanisms of REEs on plants.

  2. Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

    2013-06-01

    Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

  3. Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus

    Science.gov (United States)

    Hepler, John R.

    2017-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus. PMID:28934222

  4. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

    Directory of Open Access Journals (Sweden)

    Gábor J. Szebeni

    2017-10-01

    Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  5. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

    Science.gov (United States)

    Szebeni, Gábor J; Balázs, Árpád; Madarász, Ildikó; Pócz, Gábor; Ayaydin, Ferhan; Kanizsai, Iván; Fajka-Boja, Roberta; Alföldi, Róbert; Hackler, László; Puskás, László G

    2017-10-07

    Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G₀/G₁ cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5 , ATF4, XBP1 , and DDIT3 . The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  6. A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

    Science.gov (United States)

    Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

    2016-03-01

    Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

  7. Silver and gold nanoparticle coated membranes applied to protein dot blots

    International Nuclear Information System (INIS)

    Xie, F.; Drozdowicz-Tomsia, K.; Shtoyko, T.; Goldys, E. M.

    2011-01-01

    Detection and identification of low abundance biomarker proteins is frequently based on various types of membrane-based devices. Lowering of the protein detection limits is vital in commercial applications such as lateral flow assays and in Western blots widely used in proteomics. These currently suffer from insufficient detection sensitivity and low retention for small 2–5 kDa proteins. In this study, we report the deposition of two types of metal nanoparticles: gold colloids (50–95 nm diameter) and silver fractals onto a range of commonly used types of membranes including polyvinylidene fluoride (PVDF). Due to strong affinity of proteins to noble metals, such modified membranes have the potential to effectively capture trace proteins preventing their loss. The membranes modified by metal particles were characterized optically and by SEM. The membrane performance in protein dot blots was evaluated using the protein—fluorophore conjugates Deep Purple-bovine serum albumin and fluorescein—human serum albumin. We found that the metal nanoparticles increase light extinction by metals, which is balanced by increased fluorescence, so that the effective fluorescence signal is unchanged. This feature combined with the capture of proteins by the nanoparticles embedded in the membrane increases the detection limit of membrane assays.

  8. Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes: Implications for Lipid-Linked Disorders

    OpenAIRE

    Bogdanov, Mikhail; Mileykovskaya, Eugenia; Dowhan, William

    2008-01-01

    Lipids play important roles in cellular dysfunction leading to disease. Although a major role for phospholipids is in defining the membrane permeability barrier, phospholipids play a central role in a diverse range of cellular processes and therefore are important factors in cellular dysfunction and disease. This review is focused on the role of phospholipids in normal assembly and organization of the membrane proteins, multimeric protein complexes, and higher order supercomplexes. Since lipi...

  9. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  10. Higher-order assemblies of BAR domain proteins for shaping membranes.

    Science.gov (United States)

    Suetsugu, Shiro

    2016-06-01

    Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    Directory of Open Access Journals (Sweden)

    Ellen Wallace

    Full Text Available The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive

  12. A protein anomaly in erythrocyte membranes of patients with Duchenne muscular dystrophy

    Science.gov (United States)

    1983-01-01

    Raman spectroscopic comparisons of erythrocyte membranes from 20 patients with Duchenne muscular dystrophy and 8 age-matched controls indicate a prominent and consistent protein anomaly in the patient samples. This was apparent in the following: (a) CH-stretching signals from control membranes reveal a thermotropic transition at 15.6 degrees C, attributable to a protein/lipid phase that is lacking in dystrophic membranes. (b) CH-stretching signals from control membranes also show a protein transition at 39 degrees C [pH 7.4] that is shifted to 45 degrees in dystrophic membranes. (c) A reduction in pH to 5.7 shifts this transition from 39 degrees C to 7 degrees C in normal membranes and from 45 degrees C to 24 degrees C in dystrophic membranes. (d) The Amide I/Amide III regions indicate a significant proportion of beta- structured peptide in dystrophic but not normal membranes. (e) Analysis of tyrosine signals indicates greater polar exposure of tyrosine hydroxyl groups in dystrophic vs normal membranes. All of the differences between dystrophic and normal membranes are highly significant (P less than 0.001). PMID:6854213

  13. Interactions of Ras proteins with the plasma membrane and their roles in signaling.

    Science.gov (United States)

    Eisenberg, Sharon; Henis, Yoav I

    2008-01-01

    The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

  14. Different methods of membrane domains isolation result in similar 2-D distribution patterns of membrane domain proteins

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Petr; Hodný, Zdeněk; Švandová, I.; Svoboda, Petr

    2003-01-01

    Roč. 81, č. 6 (2003), s. 365-372 ISSN 0829-8211 R&D Projects: GA MŠk LN00A026 Grant - others:Wellcome Trust(GB) xx Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003; CEZ:AV0Z5039906 Keywords : membrane domain * G protein * two-dimensional electrophoresis * GPI-ancored proteins Subject RIV: CE - Biochemistry Impact factor: 2.456, year: 2003

  15. The plant membrane surrounding powdery mildew haustoria shares properties with the endoplasmic reticulum membrane

    DEFF Research Database (Denmark)

    Kwaaitaal, Mark Adrianus Cornelis J; Nielsen, Mads Eggert; Böhlenius, Henrik

    2017-01-01

    Many filamentous plant pathogens place specialized feeding structures, called haustoria, inside living host cells. As haustoria grow, they are believed to manipulate plant cells to generate a specialized, still enigmatic extrahaustorial membrane (EHM) around them. Here, we focused on revealing...... properties of the EHM. With the help of membranespecific dyes and transient expression of membrane-associated proteins fused to fluorescent tags, we studied the nature of the EHM generated by barley leaf epidermal cells around powdery mildew haustoria. Observations suggesting that endoplasmic reticulum (ER...... that it is not a continuum of the ER. Furthermore, GDP-locked Sar1 and a nucleotide-free RabD2a, which block ER to Golgi exit, did not hamper haustorium formation. These results indicated that the EHM shares features with the plant ER membrane, but that the EHM membrane is not dependent on conventional secretion...

  16. Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

    Science.gov (United States)

    Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

    2016-12-30

    GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

    Science.gov (United States)

    Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

    2014-09-03

    Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

  18. Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

    Science.gov (United States)

    Pešić, Ivana; Müller, Gerhard A; Baumann, Cosima; Dihazi, Gry H; Koziolek, Michael J; Eltoweissy, Marwa; Bramlage, Carsten; Asif, Abdul R; Dihazi, Hassan

    2013-04-01

    The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  20. Fouling kinetics in microfiltration of protein solutions using different membrane configurations

    DEFF Research Database (Denmark)

    Jakobsen, Sune; Jonsson, Gunnar Eigil

    1997-01-01

    Protein fouling in microfiltration has a large impact on the permeate flux and observed retention of the proteins despite the fact that the protein molecule is several times smaller than the average pore size in microfiltration membranes. This is due to adsorption and deposition of protein...... molecules and aggregates. The effect of membrane configuration upon protein fouling was investigated in crossflow filtration with asymmetric membranes either in a normal mode or in a reverse mode. It was observed by Jonsson et al. [1] that beer filtration in a reverse mode results in a smaller decrease...... in the flux compared to beer filtration in a normal mode. Similar results for protein filtration were observed by Bowen et al. [2]. One possible way to avoid fouling is the novel backshock technique (see Jonsson et al. [1]). The effect of backshock on protein filtration was investigated using a hollow fiber...

  1. Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

    Science.gov (United States)

    Zhang, Xi

    2015-01-01

    The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

  2. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Science.gov (United States)

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

  3. UV-C Adaptation of Shigella: Morphological, Outer Membrane Proteins, Secreted Proteins, and Lipopolysaccharides Effects.

    Science.gov (United States)

    Chourabi, Kalthoum; Campoy, Susana; Rodriguez, Jesus A; Kloula, Salma; Landoulsi, Ahmed; Chatti, Abdelwaheb

    2017-11-01

    Water UV disinfection remains extremely important, particularly in developing countries where drinking and reclaimed crop irrigation water may spread devastating infectious diseases. Enteric bacterial pathogens, among which Shigella, are possible contaminants of drinking and bathing water and foods. To study the effect of UV light on Shigella, four strains were exposed to different doses in a laboratory-made irradiation device, given that the ultraviolet radiation degree of inactivation is directly related to the UV dose applied to water. Our results showed that the UV-C rays are effective against all the tested Shigella strains. However, UV-C doses appeared as determinant factors for Shigella eradication. On the other hand, Shigella-survived strains changed their outer membrane protein profiles, secreted proteins, and lipopolysaccharides. Also, as shown by electron microscopy transmission, morphological alterations were manifested by an internal cytoplasm disorganized and membrane envelope breaks. Taken together, the focus of interest of our study is to know the adaptive mechanism of UV-C resistance of Shigella strains.

  4. Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

    Science.gov (United States)

    Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

    2014-01-09

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

  5. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    International Nuclear Information System (INIS)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A

    2008-01-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property

  6. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A [Technische Universitaet Muenchen, Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising (Germany)], E-mail: rudi.vogel@wzw.tum.de

    2008-07-15

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  7. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    Science.gov (United States)

    Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

    2008-07-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  8. Protein adsorption capability on polyurethane and modified-polyurethane membrane for periodontal guided tissue regeneration applications

    Energy Technology Data Exchange (ETDEWEB)

    Sheikh, Zeeshan [Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Fitzgerald Building, 150 College Street, Toronto, ON M5S 3E2 (Canada); School of Engineering and Materials Science, Queen Mary, University of London, Mile End Rd, London, E1 4NS (United Kingdom); Khan, Abdul Samad, E-mail: draskhan@ciitlahore.edu.pk [Interdisciplinary Research Centre in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000 (Pakistan); Roohpour, Nima [Oral Care R& D, GSK St., Georges Ave., Weybridge KT13 8PA (United Kingdom); Glogauer, Michael [Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Fitzgerald Building, 150 College Street, Toronto, ON M5S 3E2 (Canada); Rehman, Ihtesham u [Department of Materials Science and Engineering, The Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom)

    2016-11-01

    Periodontal disease if left untreated can result in creation of defects within the alveolar ridge. Barrier membranes are frequently used with or without bone replacement graft materials for achieving periodontal guided tissue regeneration (GTR). Surface properties of barrier membranes play a vital role in their functionality and clinical success. In this study polyetherurethane (PEU) membranes were synthesized by using 4,4′-methylene-diphenyl diisocyanate (MDI), polytetramethylene oxide (PTMO) and 1,4-butane diol (BDO) as a chain extender via solution polymerization. Hydroxyl terminated polydimethylsiloxane (PDMS) due to having inherent surface orientation towards air was used for surface modification of PEU on one side of the membranes. This resulting membranes had one surface being PEU and the other being PDMS coated PEU. The prepared membranes were treated with solutions of bovine serum albumin (BSA) in de-ionized water at 37 °C at a pH of 7.2. The surface protein adsorptive potential of PEU membranes was observed using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Raman spectroscopy and Confocal Raman spectroscopy. The contact angle measurement, tensile strength and modulus of prepared membranes were also evaluated. PEU membrane (89.86 ± 1.62°) exhibited less hydrophobic behavior than PEU-PDMS (105.87 ± 3.16°). The ultimate tensile strength and elastic modulus of PEU (27 ± 1 MPa and 14 ± 2 MPa) and PEU-PDMS (8 ± 1 MPa and 26 ± 1 MPa) membranes was in required range. The spectral analysis revealed adsorption of BSA proteins on the surface of non PDMS coated PEU surface. The PDMS modified PEU membranes demonstrated a lack of BSA adsorption. The non PDMS coated side of the membrane which adsorbs proteins could potentially be used facing towards the defect attracting growth factors for periodontal tissue regeneration. Whereas, the PDMS coated side could serve as an occlusive barrier for preventing gingival epithelial

  9. Protein adsorption capability on polyurethane and modified-polyurethane membrane for periodontal guided tissue regeneration applications

    International Nuclear Information System (INIS)

    Sheikh, Zeeshan; Khan, Abdul Samad; Roohpour, Nima; Glogauer, Michael; Rehman, Ihtesham u

    2016-01-01

    Periodontal disease if left untreated can result in creation of defects within the alveolar ridge. Barrier membranes are frequently used with or without bone replacement graft materials for achieving periodontal guided tissue regeneration (GTR). Surface properties of barrier membranes play a vital role in their functionality and clinical success. In this study polyetherurethane (PEU) membranes were synthesized by using 4,4′-methylene-diphenyl diisocyanate (MDI), polytetramethylene oxide (PTMO) and 1,4-butane diol (BDO) as a chain extender via solution polymerization. Hydroxyl terminated polydimethylsiloxane (PDMS) due to having inherent surface orientation towards air was used for surface modification of PEU on one side of the membranes. This resulting membranes had one surface being PEU and the other being PDMS coated PEU. The prepared membranes were treated with solutions of bovine serum albumin (BSA) in de-ionized water at 37 °C at a pH of 7.2. The surface protein adsorptive potential of PEU membranes was observed using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Raman spectroscopy and Confocal Raman spectroscopy. The contact angle measurement, tensile strength and modulus of prepared membranes were also evaluated. PEU membrane (89.86 ± 1.62°) exhibited less hydrophobic behavior than PEU-PDMS (105.87 ± 3.16°). The ultimate tensile strength and elastic modulus of PEU (27 ± 1 MPa and 14 ± 2 MPa) and PEU-PDMS (8 ± 1 MPa and 26 ± 1 MPa) membranes was in required range. The spectral analysis revealed adsorption of BSA proteins on the surface of non PDMS coated PEU surface. The PDMS modified PEU membranes demonstrated a lack of BSA adsorption. The non PDMS coated side of the membrane which adsorbs proteins could potentially be used facing towards the defect attracting growth factors for periodontal tissue regeneration. Whereas, the PDMS coated side could serve as an occlusive barrier for preventing gingival epithelial

  10. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

    Science.gov (United States)

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  11. Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

    Science.gov (United States)

    Varkey, Jobin; Langen, Ralf

    2017-07-01

    The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

  12. Evolved Escherichia coli Strains for Amplified, Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

    Gul, Nadia; Linares, Daniel M.; Ho, Franz Y.; Poolman, Bert

    2014-01-01

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several

  13. MEMBRANE TECHNOLOGIES — AN INNOVATIVE METHOD OF PROTEIN BIOLOGICAL VALUE INCREASING IN YOUNG CHILDREN FEEDING

    Directory of Open Access Journals (Sweden)

    I. V. Gmoshinskii

    2012-01-01

    Full Text Available A qualitatively new approach to protein production for milk formulas for infants is discussed in this article. The advantage of membrane technologies usage is that they allow preserving protein biological value and make it possible to control the levels of amino-acids in protein by optimizing their proportion and quantity.

  14. TEMPERATURE DEPENDENT PHASE BEHAVIOR AND PROTEIN PARTITIONING IN GIANT PLASMA MEMBRANE VESICLES

    OpenAIRE

    Johnson, SA; Stinson, BM; Go, M; Carmona, LM; Reminick, JI; Fang, X; Baumgart, T

    2010-01-01

    Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured...

  15. Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.

    Science.gov (United States)

    Muhammad, Noor; Dworeck, Tamara; Fioroni, Marco; Schwaneberg, Ulrich

    2011-03-17

    Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  16. Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

    Directory of Open Access Journals (Sweden)

    Fioroni Marco

    2011-03-01

    Full Text Available Abstract Background Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext. The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine. Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  17. Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

    Science.gov (United States)

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V.; Hong, Mei

    2016-01-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  18. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo [Massachusetts Institute of Technology, Department of Chemistry (United States); Sergeyev, Ivan V. [Bruker Biospin (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

    2016-03-15

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

  19. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  20. Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

    Directory of Open Access Journals (Sweden)

    Annie Frelet-Barrand

    Full Text Available BACKGROUND: Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system. CONCLUSIONS/SIGNIFICANCE: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

  1. Biomimetic triblock copolymer membrane arrays: a stable template for functional membrane proteins

    DEFF Research Database (Denmark)

    Gonzalez-Perez, A.; Jensen, Karin Bagger Stibius; Vissing, Thomas

    2009-01-01

    It is demonstrated that biomimetic stable triblock copolymer membrane arrays can be prepared using a scaffold containing 64 apertures of 300 μm diameter each. The membranes were made from a stock solution of block copolymers with decane as a solvent using a new deposition method. By using decane...

  2. Flux recovery of ceramic tubular membranes fouled with whey proteins: Some aspects of membrane cleaning

    Directory of Open Access Journals (Sweden)

    Popović Svetlana S.

    2008-01-01

    Full Text Available Efficiency of membrane processes is greatly affected by the flux reduction due to the deposits formation at the surface and/or in the pores of the membrane. Efficiency of membrane processes is affected by cleaning procedure applied to regenerate flux. In this work, flux recovery of ceramic tubular membranes with 50 and 200 nm pore size was investigated. The membranes were fouled with reconstituted whey solution for 1 hour. After that, the membranes were rinsed with clean water and then cleaned with sodium hydroxide solutions or formulated detergents (combination of P3 Ultrasil 67 and P3 Ultrasil 69. Flux recovery after the rinsing step was not satisfactory although fouling resistance reduction was significant so that chemical cleaning was necessary. In the case of 50 nm membrane total flux recovery was achieved after cleaning with 1.0% (w/w sodium hydroxide solution. In the case of 200 nm membrane total flux recovery was not achieved irrespective of the cleaning agent choice and concentration. Cleaning with commercial detergent was less efficient than cleaning with the sodium hydroxide solution.

  3. Membrane modification to avoid wettability changes due to protein adsorption in an emulsion/membrane bioreactor.

    NARCIS (Netherlands)

    Schroen, C.G.P.H.; Wijers, M.C.; Cohen Stuart, M.A.; Padt, van der A.; Riet, van 't K.

    1993-01-01

    This study addresses problems encountered with an emulsion/membrane bioreactor. In this reactor, enzyme- (lipase) catalyzed hydrolysis in an emulsion was combined with two in-line separation steps. One is carried out with a hydrophilic membrane, to separate the water phase, the other with a

  4. An albumin-fixed membrane for the removal of protein-bound toxins

    International Nuclear Information System (INIS)

    Ge Dongtao; Wu Dewang; Shi Wei; Ma Yuanyuan; Tian Xiangdong; Liang Pengfei; Zhang Qiqing

    2006-01-01

    Established methods for kidney dialysis do not work for liver failure because kidney dialysis removes only water-soluble toxins, while the liver normally removes albumin-bound toxins. In the present study, a polysulfone dialysis membrane with a -OH reactive group was prepared by hydrolyzing the chloromethylated polysulfone membrane, and the bovine serum albumin molecules were fixed into the membrane with 1,1'-carbonyldiimidazole activation. The content of albumin of the albumin-fixed membrane was 121.3 mg (g membrane) -1 . The albumin-fixed dialysis membranes were used to remove protein-bound toxins, bilirubin, from the bilirubin-albumin solution. The transfer rate of bilirubin of the albumin-fixed membrane was obviously higher compared to the normal dialysis membrane. The clearance of bilirubin with the albumin-fixed membrane was 49.8%. The albumin-fixed membrane can easily be regenerated by the bovine serum albumin and NaOH solution. Regeneration of the membrane suggested good mechanical and chemical stability, as well as good clearance of bilirubin. In addition, the effects of membrane thickness and bilirubin initial concentration on the removal of bilirubin were discussed

  5. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    Science.gov (United States)

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.

    Directory of Open Access Journals (Sweden)

    Chan-Ki Min

    2008-11-01

    Full Text Available Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip of T lymphotropic Herpesvirus saimiri (HVS is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.

  7. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    International Nuclear Information System (INIS)

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

    1990-01-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

  8. 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.

  9. Novel approaches to quantify estradiol-induced loss of ERβ1 protein in older mouse ovarian surface epithelium: new tools to assess the role of ER protein subtypes in predisposing to ovarian epithelial cancer?

    Science.gov (United States)

    Gulliver, Linda S M; Hurst, Peter R

    2011-08-01

    Loss of estrogen receptor-beta (ERβ) occurs in ovarian epithelial cancer (OEC), a cancer of mainly older women. OEC is linked epidemiologically to hormone replacement therapy, predominantly with estrogen-only formulations. This study introduces a novel, non-biased method to quantify levels of estradiol-induced loss of ERβ1 protein, and defines, for the first time, normal OSE expression patterns for ERα and ERβ1 with advancing age. Older (7-10 months) Swiss Webster mice were injected with estradiol valerate (EV) while age-matched diestrous controls received oil. Mice were culled after 48 h, and blood and one ovary were frozen for estradiol RIA. Contralateral ovaries were paraffin-embedded for immunohistochemistry. Subsets of serial sections, triple-labeled with immunofluroescent tags, were imaged with confocal microscopy to provide optimal visualization of ER protein subtype expression in OSE. Immunofluorescence emission profiles distinct to ERβ1 in OSE were standardized and quantified in control mice then compared to profiles from EV-exposed mice. Estradiol levels were significantly elevated in EV-treated mice, both in blood (p < 0.0001) and ovarian tissue (p < 0.001), resulting in 11-fold reduction in OSE expression of ERβ1 protein (p < 0.0001). In aging OSE, expression patterns of both ER subtypes varied within cells and with cell shape. ER co-localization appeared predominantly cytoplasmic and was infrequent in columnar compared to cuboidal-shaped OSE cells. Immunofluorescence emission profiling and multiple-label immunofluorescent tagging of ER using confocal microscopy, provides sharp definition of ER locus enabling concurrent qualitative and quantitative analysis of ER protein. It offers significant potential for assessing ER protein subtype status in predisposition to OEC.

  10. The Enzymology of Protein Translocation across the Escherichia coli Plasma Membrane

    NARCIS (Netherlands)

    Wickner, William; Driessen, Arnold J.M.; Hartl, Franz-Ulrich

    1991-01-01

    Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for

  11. Steric exclusion and protein conformation determine the localization of plasma membrane transporters

    NARCIS (Netherlands)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

  12. MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

    NARCIS (Netherlands)

    Bijlard, Marjolein; de Jonge, Jenny C.; Klunder, Bert; Nomden, Anita; Hoekstra, Dick; Baron, Wia

    2016-01-01

    In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known

  13. Protein shape and crowding drive domain formation and curvature in biological membranes

    NARCIS (Netherlands)

    Frese, R.N.; Pamies, Josep C.; Olsen, John D.; Bahatyrova, S.; van der Weij-de Wit, Chantal D.; Aartsma, Thijs J.; Otto, Cornelis; Hunter, C. Neil; Frenkel, Daan; van Grondelle, Rienk

    2007-01-01

    Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization

  14. Galactose oxidase labeling of membrane proteins from human brain white matter

    International Nuclear Information System (INIS)

    Hukkanen, V.; Frey, H.; Salmi, A.

    1981-01-01

    Membrane proteins of human autopsy brain white matter were subjected to a galactose oxidase/NaB 3 H 4 labeling procedure and the membranes labeled by this method or by [ 3 H]acetic anhydride techniques were studied by lectin affinity chromatography using Lens culinaris phytohemagglutinin (lentil lectin) attached to Sepharose 4B beads. (Auth.)

  15. Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

    Energy Technology Data Exchange (ETDEWEB)

    Natali, F. [OGG-INFM, Grenoble (France); Gliozzi, A.; Rolandi, R.; Relini, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Genova (Italy); Cavatorta, P.; Deriu, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Parma (Italy); Fasano, A. [Dipartimento di Biochimica e Biologia Molecolare, Universita di Bari (Italy); Riccio, P. [Dipartimento di Biologia D.B.A.F., Universita della Basilicata, Potenza (Italy)

    2002-07-01

    We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP. (orig.)

  16. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  17. On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii

    NARCIS (Netherlands)

    Bevers, E.M.; Wang, H.H.; Kamp, J.A.F. op den; Deenen, L.L.M. van

    1979-01-01

    About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A2 from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible

  18. 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...

  19. A general theory of non-equilibrium dynamics of lipid-protein fluid membranes

    DEFF Research Database (Denmark)

    Lomholt, Michael Andersen; Hansen, Per Lyngs; Miao, L.

    2005-01-01

    We present a general and systematic theory of non-equilibrium dynamics of multi-component fluid membranes, in general, and membranes containing transmembrane proteins, in particular. Developed based on a minimal number of principles of statistical physics and designed to be a meso...

  20. Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2017-06-20

    The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

  1. Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.

    Science.gov (United States)

    Lebeau, Paul; Byun, Jae Hyun; Yousof, Tamana; Austin, Richard C

    2018-04-22

    Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity. Copyright © 2018. Published by Elsevier Inc.

  2. Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

    Science.gov (United States)

    Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

    2017-07-05

    Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins

    Science.gov (United States)

    Carroll, Joe; Fearnley, Ian M.; Walker, John E.

    2006-01-01

    The covalent structure of a protein is incompletely defined by its gene sequence, and mass spectrometric analysis of the intact protein is needed to detect the presence of any posttranslational modifications. Because most membrane proteins are purified in detergents that are incompatible with mass spectrometric ionization techniques, this essential measurement has not been made on many hydrophobic proteins, and so proteomic data are incomplete. We have extracted membrane proteins from bovine mitochondria and detergent-purified NADH:ubiquinone oxidoreductase (complex I) with organic solvents, fractionated the mixtures by hydrophilic interaction chromatography, and measured the molecular masses of the intact membrane proteins, including those of six subunits of complex I that are encoded in mitochondrial DNA. These measurements resolve long-standing uncertainties about the interpretation of the mitochondrial genome, and they contribute significantly to the definition of the covalent composition of complex I. PMID:17060615

  4. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

    Science.gov (United States)

    Golan, Yonatan; Sherman, Eilon

    2017-06-01

    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  5. High-Throughput Simulations of Dimer and Trimer Assembly of Membrane Proteins. The DAFT Approach

    NARCIS (Netherlands)

    Wassenaar, Tsjerk A.; Pluhackova, Kristyna; Moussatova, Anastassiia; Sengupta, Durba; Marrink, Siewert J.; Tieleman, D. Peter; Boeckrnann, Rainer A.

    Interactions between membrane proteins are of great biological significance and are consequently an important target for pharmacological intervention. Unfortunately, it is still difficult to obtain detailed views on such interactions, both experimentally, where the environment hampers atomic

  6. Controlling the rejection of protein during membrane filtration by adding selected polyelectrolytes

    DEFF Research Database (Denmark)

    Pinelo, Manuel; Ferrer Roca, Carme; Meyer, Anne S.

    2012-01-01

    Electrostatic interactions among the charged groups on proteins and/or between proteins and other solutes significantly affect the aggregation/deposition phenomena that induce fouling and decrease permeate flux during membrane purification of proteins. Such interactions can be turned...... help enhance the performance of membrane filtration for fractionation/purification of a target protein by significantly reducing fouling and modifying rejection/selectivity.......) changing the pH, on the permeate flux and membrane transmission of bovin serum albumina (BSA) through a PVDF membrane. The addition of PS-co-AA to the feed solution resulted in significant increases of the BSA transmission at pH 7.4 as compared to the transmission of a pure BSA solution (1g...

  7. Numerical calculation on a two-step subdiffusion behavior of lateral protein movement in plasma membranes

    Science.gov (United States)

    Sumi, Tomonari; Okumoto, Atsushi; Goto, Hitoshi; Sekino, Hideo

    2017-10-01

    A two-step subdiffusion behavior of lateral movement of transmembrane proteins in plasma membranes has been observed by using single-molecule experiments. A nested double-compartment model where large compartments are divided into several smaller ones has been proposed in order to explain this observation. These compartments are considered to be delimited by membrane-skeleton "fences" and membrane-protein "pickets" bound to the fences. We perform numerical simulations of a master equation using a simple two-dimensional lattice model to investigate the heterogeneous diffusion dynamics behavior of transmembrane proteins within plasma membranes. We show that the experimentally observed two-step subdiffusion process can be described using fence and picket models combined with decreased local diffusivity of transmembrane proteins in the vicinity of the pickets. This allows us to explain the two-step subdiffusion behavior without explicitly introducing nested double compartments.

  8. RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

    Science.gov (United States)

    Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

  9. Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

    Science.gov (United States)

    Sahu, Indra D; Zhang, Rongfu; Dunagan, Megan M; Craig, Andrew F; Lorigan, Gary A

    2017-06-01

    EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (T m ) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the T m titration experiment indicated an increase in T m values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant

  10. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

    Science.gov (United States)

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

  11. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    International Nuclear Information System (INIS)

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-01-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [ 35 S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [ 3 H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125 I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [ 3 H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

  12. Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique.

    Science.gov (United States)

    Ohvo-Rekilä, Henna; Mattjus, Peter

    2011-01-01

    The glycolipid transfer protein (GLTP) is a protein capable of binding and transferring glycolipids. GLTP is cytosolic and it can interact through its FFAT-like (two phenylalanines in an acidic tract) motif with proteins localized on the surface of the endoplasmic reticulum. Previous in vitro work with GLTP has focused mainly on the complete transfer reaction of the protein, that is, binding and subsequent removal of the glycolipid from the donor membrane, transfer through the aqueous environment, and the final release of the glycolipid to an acceptor membrane. Using bilayer vesicles and surface plasmon resonance spectroscopy, we have now, for the first time, analyzed the binding and lipid removal capacity of GLTP with a completely label-free technique. This technique is focused on the initial steps in GLTP-mediated transfer and the parameters affecting these steps can be more precisely determined. We used the new approach for detailed structure-function studies of GLTP by examining the glycolipid transfer capacity of specific GLTP tryptophan mutants. Tryptophan 96 is crucial for the transfer activity of the protein and tryptophan 142 is an important part of the proteins membrane interacting domain. Further, we varied the composition of the used lipid vesicles and gained information on the effect of membrane properties on GLTP activity. GLTP prefers to interact with more tightly packed membranes, although GLTP-mediated transfer is faster from more fluid membranes. This technique is very useful for the study of membrane-protein interactions and lipid-transfer rates and it can easily be adapted to other membrane-interacting proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OF MEMBRANE-BOUND PROTEIN SIGNALS

    OpenAIRE

    Loss, Leandro

    2009-01-01

    Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims...

  14. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

    OpenAIRE

    Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H.; Michel, Jennifer Carlisle; Claxton, Derek P.; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K. Christopher; Gouaux, Eric

    2014-01-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient productio...

  15. A simple detection method for low-affinity membrane protein interactions by baculoviral display.

    Directory of Open Access Journals (Sweden)

    Toshiko Sakihama

    Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

  16. Plasma membrane protein trafficking in plant–microbe interactions: a plant cell point of view

    OpenAIRE

    Nathalie Leborgne-Castel,; Bouhidel, Karim

    2014-01-01

    In order to ensure their physiological and cellular functions, plasma membrane (PM) proteins must be properly conveyed from