Journal articles on the topic 'Membrance domains'
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1
Dale, B., E. Tosti, and M. Iaccarino. "Is the plasma membrane of the human oocyte reorganised following fertilisation and early cleavage?" Zygote 3, no. 1 (February 1995): 31–36. http://dx.doi.org/10.1017/s0967199400002355.
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SummaryThe purpose of the present study was to determine whether the plasma membrance of the human oocyte is reorganised following fertlisation and during early cleavage. In order to characterise and localise the major sugar moieties on surface glycoporteins, oocytes and embroys were labelled with a range of flourescent lectins. Regional organisation of plasma membrane microvilli in oocytes and embryos was also studied using scanning electron microscopy (SEM). The plasma membrance of human oocytes, zygotes and early blastomeres stained strongly and homogeneously with concanavalin A and Triticum vulgaris lectin (WGA), indicationg the presence of plasma membrance glycoconjugates with α-D-mannosyl residues, sialic acid and β-NAc-glucosaminyl groups. We did not observe regional domains in oocytes and zygotes, suggesting that the plasma membrane is not topographically reorganised following fertilisation. SEM shows the surface of the human zygote to be organised into short microvilli 0.2–3.0 μm in length and at a density of 5–20/μm2. In early cleavage stages the microvilli are shorter and less frequent (0.2–1.0 μm; 1–5/μm2); however, there is no evidence of polarisation at this level of organisation, at either stage of development. The surface of cell fragments, common in the human embryo in vitro, differs in having few microvilli and numerous cytoplasmic blebs. In conclusion, there are no obvious morphological signs of regionalisation in the plasma membrane of the human embryo before the 8-cell stage.
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Snead, Wilton T., Wade F. Zeno, Grace Kago, Ryan W. Perkins, J. Blair Richter, Chi Zhao, Eileen M. Lafer, and Jeanne C. Stachowiak. "BAR scaffolds drive membrane fission by crowding disordered domains." Journal of Cell Biology 218, no. 2 (November 30, 2018): 664–82. http://dx.doi.org/10.1083/jcb.201807119.
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Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain–containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain–containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain–containing proteins.
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Gallop, Jennifer L., and Harvey T. McMahon. "BAR domains and membrane curvature: bringing your curves to the BAR." Biochemical Society Symposia 72 (January 1, 2005): 223–31. http://dx.doi.org/10.1042/bss0720223.
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BAR (bin, amphiphysin and Rvs161/167) domains are a unique class of dimerization domains, whose dimerization interface is edged by a membrane-binding surface. In its dimeric form, the membrane-binding interface is concave, and this gives the ability to bind better to curved membranes, i.e. to sense membrane curvature. When present at higher concentrations, the domain can stabilize membrane curvature, generating lipid tubules. This domain is found in many contexts in a wide variety of proteins, where the dimerization and membrane-binding function of this domain is likely to have a profound effect on protein activity. If these proteins function as predicted, then there will be membrane subdomains based on curvature, and thus there is an additional layer of compartmentalization on membranes. These and other possible functions of the BAR domain are discussed.
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Golantsova, Nina E., Elena E. Gorbunova, and Erich R. Mackow. "Discrete Domains within the Rotavirus VP5* Direct Peripheral Membrane Association and Membrane Permeability." Journal of Virology 78, no. 4 (February 15, 2004): 2037–44. http://dx.doi.org/10.1128/jvi.78.4.2037-2044.2004.
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ABSTRACT Cleavage of the rotavirus spike protein, VP4, is required for rotavirus-induced membrane permeability and viral entry into cells. The VP5* cleavage product selectively permeabilizes membranes and liposomes and contains an internal hydrophobic domain that is required for membrane permeability. Here we investigate VP5* domains (residues 248 to 474) that direct membrane binding. We determined that expressed VP5 fragments containing residues 248 to 474 or 265 to 474, including the internal hydrophobic domain, bind to cellular membranes but are not present in Triton X-100-resistant membrane rafts. Expressed VP5 partitions into aqueous but not detergent phases of Triton X-114, suggesting that VP5 is not integrally inserted into membranes. Since high-salt or alkaline conditions eluted VP5 from membranes, our findings demonstrate that VP5 is peripherally associated with membranes. Interestingly, mutagenesis of residue 394 (W→R) within the VP5 hydrophobic domain, which abolishes VP5-directed permeability, had no effect on VP5's peripheral membrane association. In contrast, deletion of N-terminal VP5 residues (residues 265 to 279) abolished VP5 binding to membranes. Alanine mutagenesis of two positively charged residues within this domain (residues 274R and 276K) dramatically reduced (>95%) binding of VP5 to membranes and suggested their potential interaction with polar head groups of the lipid bilayer. Mutations in either the VP5 hydrophobic or basic domain blocked VP5-directed permeability of cells. These findings indicate that there are at least two discrete domains within VP5* required for pore formation: an N-terminal basic domain that permits VP5* to peripherally associate with membranes and an internal hydrophobic domain that is essential for altering membrane permeability. These results provide a fundamental understanding of interactions between VP5* and the membrane, which are required for rotavirus entry.
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Chowdary, Tirumala Kumar, and Ekaterina E. Heldwein. "Syncytial Phenotype of C-Terminally Truncated Herpes Simplex Virus Type 1 gB Is Associated with Diminished Membrane Interactions." Journal of Virology 84, no. 10 (March 3, 2010): 4923–35. http://dx.doi.org/10.1128/jvi.00206-10.
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ABSTRACT The cytoplasmic domain of glycoprotein B (gB) from herpes simplex virus type 1 (HSV-1) is an important regulator of membrane fusion. C-terminal truncations of the cytoplasmic domain lead to either hyperfusion or fusion-null phenotypes. Currently, neither the structure of the cytoplasmic domain nor its mechanism of fusion regulation is known. Here we show, for the first time, that the full-length cytoplasmic domain of HSV-1 gB associates stably with lipid membranes, preferentially binding to membranes containing anionic head groups. This interaction involves a large increase in helical content. However, the truncated cytoplasmic domains associated with the hyperfusion phenotype show a small increase in helical structure and a diminished association with lipid membranes, whereas the one associated with the fusion-null phenotype shows no increase in helical structure and only a minimal association with lipid membranes. We hypothesize that stable binding to lipid membranes is an important part of the mechanism by which the cytoplasmic domain negatively regulates membrane fusion. Moreover, our experiments with truncated cytoplasmic domains point to two specific regions that are critical for membrane interactions. Taken together, our work provides several important new insights into the architecture of the cytoplasmic domain of HSV-1 gB and its interaction with lipid membranes.
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Yamamoto, Eiji, Jan Domański, Fiona B. Naughton, Robert B. Best, Antreas C. Kalli, Phillip J. Stansfeld, and Mark S. P. Sansom. "Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes." Science Advances 6, no. 8 (February 2020): eaay5736. http://dx.doi.org/10.1126/sciadv.aay5736.
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Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.
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Karotki, Lena, Juha T. Huiskonen, Christopher J. Stefan, Natasza E. Ziółkowska, Robyn Roth, Michal A. Surma, Nevan J. Krogan, et al. "Eisosome proteins assemble into a membrane scaffold." Journal of Cell Biology 195, no. 5 (November 28, 2011): 889–902. http://dx.doi.org/10.1083/jcb.201104040.
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Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin–Amphiphysin–Rvs domain–containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.
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Pennington, Edward Ross, E. Madison Sullivan, Amy Fix, Sahil Dadoo, Tonya N. Zeczycki, Anita DeSantis, Uwe Schlattner, et al. "Proteolipid domains form in biomimetic and cardiac mitochondrial vesicles and are regulated by cardiolipin concentration but not monolyso-cardiolipin." Journal of Biological Chemistry 293, no. 41 (August 29, 2018): 15933–46. http://dx.doi.org/10.1074/jbc.ra118.004948.
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Cardiolipin (CL) is an anionic phospholipid mainly located in the inner mitochondrial membrane, where it helps regulate bioenergetics, membrane structure, and apoptosis. Localized, phase-segregated domains of CL are hypothesized to control mitochondrial inner membrane organization. However, the existence and underlying mechanisms regulating these mitochondrial domains are unclear. Here, we first isolated detergent-resistant cardiac mitochondrial membranes that have been reported to be CL-enriched domains. Experiments with different detergents yielded only nonspecific solubilization of mitochondrial phospholipids, suggesting that CL domains are not recoverable with detergents. Next, domain formation was investigated in biomimetic giant unilamellar vesicles (GUVs) and newly synthesized giant mitochondrial vesicles (GMVs) from mouse hearts. Confocal fluorescent imaging revealed that introduction of cytochrome c into membranes promotes macroscopic proteolipid domain formation associated with membrane morphological changes in both GUVs and GMVs. Domain organization was also investigated after lowering tetralinoleoyl-CL concentration and substitution with monolyso-CL, two common modifications observed in cardiac pathologies. Loss of tetralinoleoyl-CL decreased proteolipid domain formation in GUVs, because of a favorable Gibbs-free energy of lipid mixing, whereas addition of monolyso-CL had no effect on lipid mixing. Moreover, murine GMVs generated from cardiac acyl-CoA synthetase-1 knockouts, which have remodeled CL acyl chains, did not perturb proteolipid domains. Finally, lowering the tetralinoleoyl-CL content had a stronger influence on the oxidation status of cytochrome c than did incorporation of monolyso-CL. These results indicate that proteolipid domain formation in the cardiac mitochondrial inner membrane depends on tetralinoleoyl-CL concentration, driven by underlying lipid-mixing properties, but not the presence of monolyso-CL.
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Ma, Alice D., Lawrence F. Brass, and Charles S. Abrams. "Pleckstrin Associates with Plasma Membranes and Induces the Formation of Membrane Projections: Requirements for Phosphorylation and the NH2-terminal PH Domain." Journal of Cell Biology 136, no. 5 (March 10, 1997): 1071–79. http://dx.doi.org/10.1083/jcb.136.5.1071.
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Pleckstrin homology (PH) domains are sequences of ∼100 amino acids that form “modules” that have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end of the molecule, flanking an intervening sequence of 147 residues. Evidence is accumulating to support the hypothesis that PH domains are structural motifs that target molecules to membranes, perhaps through interactions with Gβγ or phosphatidylinositol 4,5-bisphosphate (PIP2), two putative PH domain ligands. In the present studies, we show that pleckstrin associates with membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on phosphorylation of pleckstrin and requires the presence of its NH2-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH2terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually promotes the formation of membrane projections from the dorsal surface of transfected cells, and that this morphologic change is similarly PH domain dependent. Since we have shown previously that pleckstrin-mediated inhibition of PIP2 metabolism by phospholipase C or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH2-terminal PH domain, these results suggest that: (a) pleckstrin's NH2terminal PH domain may regulate pleckstrin's activity by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP2 and/or other membrane-bound ligands.
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GILLOOLY, David J., Anne SIMONSEN, and Harald STENMARK. "Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins." Biochemical Journal 355, no. 2 (April 6, 2001): 249–58. http://dx.doi.org/10.1042/bj3550249.
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PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling.
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Voleti, Rashmi, Diana R. Tomchick, Thomas C. Südhof, and Josep Rizo. "Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A domain in asynchronous neurotransmitter release." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): E8518—E8527. http://dx.doi.org/10.1073/pnas.1710708114.
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Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.
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Beaulieu, Nadine, Bari Zahedi, Rebecca E. Goulding, Ghazaleh Tazmini, Kira V. Anthony, Stephanie L. Omeis, Danielle R. de Jong, and Robert J. Kay. "Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane." Molecular Biology of the Cell 18, no. 8 (August 2007): 3156–68. http://dx.doi.org/10.1091/mbc.e06-10-0932.
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RasGRP1 is a Ras-activating exchange factor that is positively regulated by translocation to membranes. RasGRP1 contains a diacylglycerol-binding C1 domain, and it has been assumed that this domain is entirely responsible for RasGRP1 translocation. We found that the C1 domain can contribute to plasma membrane-targeted translocation of RasGRP1 induced by ligation of the B cell antigen receptor (BCR). However, this reflects cooperativity of the C1 domain with the previously unrecognized Plasma membrane Targeter (PT) domain, which is sufficient and essential for plasma membrane targeting of RasGRP1. The adjacent suppressor of PT (SuPT) domain attenuates the plasma membrane-targeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase Cγ2, the C1 domain counteracts the SuPT domain and enables efficient RasGRP1 translocation to the plasma membrane. In fibroblasts, the PT domain is inactive as a plasma membrane targeter, and the C1 domain specifies constitutive targeting of RasGRP1 to internal membranes where it can be activated and trigger oncogenic transformation. Selective use of the C1, PT, and SuPT domains may contribute to the differential targeting of RasGRP1 to the plasma membrane versus internal membranes, which has been observed in lymphocytes and other cell types.
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Lü, Junhong, Steven W. Pipe, Hongzhi Miao, Marc Jacquemin, and Gary E. Gilbert. "A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function." Blood 117, no. 11 (March 17, 2011): 3181–89. http://dx.doi.org/10.1182/blood-2010-08-301663.
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Abstract Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the Vmax for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the Vmax of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.
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Grados-Torrez, Ricardo Enrique, Carmen López-Iglesias, Joan Carles Ferrer, and Narciso Campos. "Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase." International Journal of Molecular Sciences 22, no. 17 (August 24, 2021): 9132. http://dx.doi.org/10.3390/ijms22179132.
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The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell.
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Youn, Ji-Young, Helena Friesen, Takuma Kishimoto, William M. Henne, Christoph F. Kurat, Wei Ye, Derek F. Ceccarelli, et al. "Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis." Molecular Biology of the Cell 21, no. 17 (September 2010): 3054–69. http://dx.doi.org/10.1091/mbc.e10-03-0181.
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BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161–Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.
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García-Sáez, Ana J., Salvatore Chiantia, and Petra Schwille. "Effect of Line Tension on the Lateral Organization of Lipid Membranes." Journal of Biological Chemistry 282, no. 46 (September 11, 2007): 33537–44. http://dx.doi.org/10.1074/jbc.m706162200.
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The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes.
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Lin, Sasa, Hussein Y. Naim, A. Chapin Rodriguez, and Michael G. Roth. "Mutations in the Middle of the Transmembrane Domain Reverse the Polarity of Transport of the Influenza Virus Hemagglutinin in MDCK Epithelial Cells." Journal of Cell Biology 142, no. 1 (July 13, 1998): 51–57. http://dx.doi.org/10.1083/jcb.142.1.51.
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The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting.
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Whitley, Paul, and Ismael Mingarro. "Stitching proteins into membranes, not sew simple." Biological Chemistry 395, no. 12 (December 1, 2014): 1417–24. http://dx.doi.org/10.1515/hsz-2014-0205.
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Abstract Most integral membrane proteins located within the endomembrane system of eukaryotic cells are first assembled co-translationally into the endoplasmic reticulum (ER) before being sorted and trafficked to other organelles. The assembly of membrane proteins is mediated by the ER translocon, which allows passage of lumenal domains through and lateral integration of transmembrane (TM) domains into the ER membrane. It may be convenient to imagine multi-TM domain containing membrane proteins being assembled by inserting their first TM domain in the correct orientation, with subsequent TM domains inserting with alternating orientations. However a simple threading model of assembly, with sequential insertion of one TM domain into the membrane after another, does not universally stand up to scrutiny. In this article we review some of the literature illustrating the complexities of membrane protein assembly. We also present our own thoughts on aspects that we feel are poorly understood. In short we hope to convince the readers that threading of membrane proteins into membranes is ‘not sew simple’ and a topic that requires further investigation.
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Johnson, Joanne E., Rebecca E. Goulding, Ziwei Ding, Amir Partovi, Kira V. Anthony, Nadine Beaulieu, Ghazaleh Tazmini, Rosemary B. Cornell, and Robert J. Kay. "Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs." Biochemical Journal 406, no. 2 (August 13, 2007): 223–36. http://dx.doi.org/10.1042/bj20070294.
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RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1–DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4α and RasGRP4β. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4α co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4β do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4α recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4β C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.
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Scott, Jordan L., Cary T. Frick, Kristen A. Johnson, Haining Liu, Sylvia S. Yong, Allyson G. Varney, Olaf Wiest, and Robert V. Stahelin. "Molecular Analysis of Membrane Targeting by the C2 Domain of the E3 Ubiquitin Ligase Smurf1." Biomolecules 10, no. 2 (February 4, 2020): 229. http://dx.doi.org/10.3390/biom10020229.
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SMAD ubiquitination regulatory factor 1 (Smurf1) is a Nedd4 family E3 ubiquitin ligase that regulates cell motility, polarity and TGFβ signaling. Smurf1 contains an N-terminal protein kinase C conserved 2 (C2) domain that targets cell membranes and is required for interactions with membrane-localized substrates such as RhoA. Here, we investigated the lipid-binding mechanism of Smurf1 C2, revealing a general affinity for anionic membranes in addition to a selective affinity for phosphoinositides (PIPs). We found that Smurf1 C2 localizes not only to the plasma membrane but also to negatively charged intracellular sites, acting as an anionic charge sensor and selective PIP-binding domain. Site-directed mutagenesis combined with docking/molecular dynamics simulations revealed that the Smurf1 C2 domain loop region primarily interacts with PIPs and cell membranes, as opposed to the β-surface cationic patch employed by other C2 domains. By depleting PIPs from the inner leaflet of the plasma membrane, we found that PIP binding is necessary for plasma membrane localization. Finally, we used a Smurf1 cellular ubiquitination assay to show that the amount of ubiquitin at the plasma membrane interface depends on the lipid-binding properties of Smurf1. This study shows the mechanism by which Smurf1 C2 targets membrane-based substrates and reveals a novel interaction for non-calcium-dependent C2 domains and membrane lipids.
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Moreau, A., M. Maurice, and G. Feldmann. "Analysis of hepatocyte plasma membrane domains during rat development using monoclonal antibodies." Journal of Histochemistry & Cytochemistry 36, no. 1 (January 1988): 87–94. http://dx.doi.org/10.1177/36.1.3275714.
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The plasma membrane of adult rat hepatocyte consists of three domains, which have been identified by the monoclonal antibodies A39 and A59 as markers of the sinusoidal domain, B1 of the lateral, and B10 of the canalicular domains (Eur J Cell Biol 39:122, 1985). These monoclonal antibodies were used to study, by indirect immunocytochemistry, formation of the hepatocyte plasma membrane domains during development, from day 15 of gestation to day 35 post partum. The antigens defined by A39, B1, and B10 were detected, from day 15, over the major part of the hepatocyte plasma membrane except for the membranes of newly formed bile canaliculi, which were not labeled by B1 and only poorly labeled, if at all, by A39 and B10. As soon as fetuses were 16 days old, B1 labeled predominantly the lateral domain, as in the adult. Labeling with B10 progressively intensified on the membranes of bile canaliculi, but localization was not exclusively canalicular until day 21 post partum. A39 intensely labeled the canalicular membranes at 19-21 days of gestation, while at 35 days post partum it exhibited the predominantly sinusoidal labeling observed in adult hepatocytes. The antigen defined by A59 was not detected before birth and was found exclusively on the sinusoidal domain, as in the adult. These results show that the patterns of antigen distribution on different plasma membrane domains establish themselves at different rates. The marked differences observed between fetal or neonatal and adult hepatocytes might be responsible for immaturity of liver functions in the neonate.
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Alsop, Richard J., Sebastian Himbert, Alexander Dhaliwal, Karin Schmalzl, and Maikel C. Rheinstädter. "Aspirin locally disrupts the liquid-ordered phase." Royal Society Open Science 5, no. 2 (February 2018): 171710. http://dx.doi.org/10.1098/rsos.171710.
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Local structure and dynamics of lipid membranes play an important role in membrane function. The diffusion of small molecules, the curvature of lipids around a protein and the existence of cholesterol-rich lipid domains (rafts) are examples for the membrane to serve as a functional interface. The collective fluctuations of lipid tails, in particular, are relevant for diffusion of membrane constituents and small molecules in and across membranes, and for structure and formation of membrane domains. We studied the effect of aspirin (acetylsalicylic acid, ASA) on local structure and dynamics of membranes composed of dimyristoylphosphocholine (DMPC) and cholesterol. Aspirin is a common analgesic, but is also used in the treatment of cholesterol. Using coherent inelastic neutron scattering experiments and molecular dynamics (MD) simulations, we present evidence that ASA binds to liquid-ordered, raft-like domains and disturbs domain organization and dampens collective fluctuations. By hydrogen-bonding to lipid molecules, ASA forms ‘superfluid’ complexes with lipid molecules that can organize laterally in superlattices and suppress cholesterol’s ordering effect.
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Brône, Bert, and Jan Eggermont. "PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes." American Journal of Physiology-Cell Physiology 288, no. 1 (January 2005): C20—C29. http://dx.doi.org/10.1152/ajpcell.00368.2004.
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PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J Physiol Cell Physiol 288: C20–C29, 2005; doi:10.1152/ajpcell.00368.2004.—The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane domains. In addition, the transport properties of an epithelial cell will be maintained only if these membrane transporters are retained and properly regulated in their specific membrane compartments. Recent reports have indicated that PDZ domain-containing proteins play a dual role in these processes and, in addition, that different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane domain, the PDZ network ensures that the membrane proteins are efficiently retained at the cell surface. Second, the close spatial positioning of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex (transducisome) allows fast and efficient control of membrane transport processes.
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Corbalán-García, S., M. Guerrero-Valero, C. Marín-Vicente, and J. C. Gómez-Fernández. "The C2 domains of classical/conventional PKCs are specific PtdIns(4,5)P2-sensing domains." Biochemical Society Transactions 35, no. 5 (October 25, 2007): 1046–48. http://dx.doi.org/10.1042/bst0351046.
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The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5)P2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5)P2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5)P2 in model membranes decreases the Ca2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5)P2 on the inner leaflet of the plasma membrane are sufficient to drive Ca2+-activated membrane docking during a physiological Ca2+ signal.
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Frisz, Jessica F., Haley A. Klitzing, Kaiyan Lou, Ian D. Hutcheon, Peter K. Weber, Joshua Zimmerberg, and Mary L. Kraft. "Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol." Journal of Biological Chemistry 288, no. 23 (April 22, 2013): 16855–61. http://dx.doi.org/10.1074/jbc.m113.473207.
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The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.
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STÖGBAUER, T., M. HENNIG, and J. O. RÄDLER. "ALIGNMENT AND DEFORMATION OF LIPID BILAYER DOMAINS IN VESICLES ADHERING TO MICROSTRUCTURED SUBSTRATES." Biophysical Reviews and Letters 05, no. 03 (September 2010): 153–61. http://dx.doi.org/10.1142/s1793048010001160.
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In heterogeneous lipid membranes, the lateral organization is coupled to local curvature as the membrane bending energies depend on composition. We investigate phase-separated vesicles of ternary lipid composition in contact with structured surfaces, where the distinct elastic properties of the Lo and Ld phases come into play. We show that fused silica substrates with microstructured grooves induce sorting, alignment and deformation of Lo domains in adhering vesicles. The same phenomenon is observed on flat, chemically modified substrates with alternating stripes of rough and smooth regions. In both cases it is the Lo phase which accumulates over the smooth substrate and membrane-spanned groove regions respectively. Deformation of Lo domains occurs when domain diameters grow beyond the width of the microstructured stripes. Domain alignment was also observed in binary membranes featuring gel and fluid phase coexistence showing the generic character of domain sorting on microstructured surfaces.
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Scott, Angela M., Corina E. Antal, and Alexandra C. Newton. "Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα." Journal of Biological Chemistry 288, no. 23 (April 15, 2013): 16905–15. http://dx.doi.org/10.1074/jbc.m113.467456.
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The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca2+. Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca2+-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca2+-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.
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Kaykas, Ajamete, Kathleen Worringer, and Bill Sugden. "LMP-1's Transmembrane Domains Encode Multiple Functions Required for LMP-1's Efficient Signaling." Journal of Virology 76, no. 22 (November 15, 2002): 11551–60. http://dx.doi.org/10.1128/jvi.76.22.11551-11560.2002.
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ABSTRACT The latent membrane protein-1 (LMP-1) of Epstein-Barr virus (EBV) contributes to the proliferation of infected B lymphocytes by signaling through its binding to cellular signaling molecules. It apparently mimics members of the tumor necrosis factor receptor family, in particular, CD40, by binding a similar set of cellular molecules as does CD40. LMP-1 differs dramatically in its structure from CD40. LMP-1 has six membrane-spanning domains as opposed to CD40's one. LMP-1 also differs from CD40 in its apparent independence of a ligand for its signaling. We have examined the role of LMP-1's membrane-spanning domains in its signaling. Their substitution with six membrane-spanning domains from the LMP-2A protein of EBV yields a derivative which neither coimmunoprecipitates with LMP-1 nor signals to increase the activity of NF-κB as does wild-type LMP-1. These observations indicate that LMP-1 has specific sequences in its membrane-spanning domains required for these activities. LMP-1's first and sixth membrane-spanning domains have multiple leucine residues potentially similar to leucine-heptad motifs that can mediate protein-protein interactions in membranes (Gurezka et al., J. Biol. Chem. 274:9265-9270, 1999). Substitution of seven leucines in LMP-1's sixth membrane-spanning domain has no effect on its function, whereas similar substitutions in its first membrane-spanning domain yielded a derivative which aggregates as does wild-type LMP-1 but has only 3% of wild-type's ability to signal through NF-κB. Importantly, this derivative complements a mutant of LMP-1 with wild-type membrane-spanning domains but no carboxy-terminal signaling domain. These findings together indicate that the membrane-spanning domains of LMP-1 contribute multiple functions to its signaling.
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London, Erwin. "Ordered Domain (Raft) Formation in Asymmetric Vesicles and Its Induction upon Loss of Lipid Asymmetry in Artificial and Natural Membranes." Membranes 12, no. 9 (September 9, 2022): 870. http://dx.doi.org/10.3390/membranes12090870.
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Lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets) of a membrane, is an important feature of eukaryotic plasma membranes. Investigation of the biophysical consequences of lipid asymmetry has been aided by advances in the ability to prepare artificial asymmetric membranes, especially by use of cyclodextrin-catalyzed lipid exchange. This review summarizes recent studies with artificial asymmetric membranes which have identified conditions in which asymmetry can induce or suppress the ability of membranes to form ordered domains (rafts). A consequence of the latter effect is that, under some conditions, a loss of asymmetry can induce ordered domain formation. An analogous study in plasma membrane vesicles has demonstrated that asymmetry can also suppress domain formation in natural membranes. Thus, it is possible that a loss of asymmetry can induce domain formation in vivo.
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Oancea, Elena, Mary N. Teruel, Andrew F. G. Quest, and Tobias Meyer. "Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells." Journal of Cell Biology 140, no. 3 (February 9, 1998): 485–98. http://dx.doi.org/10.1083/jcb.140.3.485.
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Cysteine-rich domains (Cys-domains) are ∼50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.
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O’Neil, Patrick K., Lynn G. L. Richardson, Yamuna D. Paila, Grzegorz Piszczek, Srinivas Chakravarthy, Nicholas Noinaj, and Danny Schnell. "The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts." Proceedings of the National Academy of Sciences 114, no. 24 (May 30, 2017): E4868—E4876. http://dx.doi.org/10.1073/pnas.1621179114.
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Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a β-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded β-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.
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Kumar, Shekhar, Steven Stayrook, James A. Huntington, Rodney M. Camire, and Sriram Krishnaswamy. "New Structural Insights into High Affinity Membrane Binding By Coagulation Factor V/Va." Blood 124, no. 21 (December 6, 2014): 4216. http://dx.doi.org/10.1182/blood.v124.21.4216.4216.
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Abstract Factor Va binds to membranes exposing phosphatidylserine (PS) with nanomolar affinity. This high affinity interaction plays a vital role in the assembly of membrane-bound prothrombinase thereby supporting robust thrombin formation at the site of vascular damage. Although the importance of the C1 and C2 domains of FVa in membrane binding has long been recognized, mechanistic details are incompletely understood. Unlike human prothrombinase, Pseudonaja textilis (common brown snake, P. tex) has evolved a membrane-independent form of prothrombinase composed of a FVa-like protein (VPtex) tightly bound to a FXa-like protein in solution. Our structural advances with FVPtex provide new tools to address major unresolved questions related to membrane binding by FVa. The high resolution (1.95Å) structure of VPtex resembles previously published structures of inactivated bovine FVa and human FVIII of lower resolution with all the structural features considered critical for membrane binding by FVa. However, VPtex bound to PS-containing membranes with very poor affinity (Kd > 1 µM). Substitution of 9 residues in the C1 and C2 domains of VPtex with residues present in the hemostatic form of FV present in the plasma of the snake yielded a derivative (VPtexC1C2) that bound to PS-containing membranes with nanomolar affinity equivalent to hFV. Interestingly, the newly acquired function of membrane binding in VPtexC1C2 does not affect its ability to function in solution, suggesting that membrane binding and solution-phase function are controlled independently. Variants containing substitutions in the individual C domains (VPtexC1 and VPtexC2) exhibited intermediate affinities (Kd=100 nM and Kd=50 nM) for binding to PS-containing membranes. However, the binding energy contributions from the individual C-domains did not additively explain the affinity of VPtexC1C2 for membranes. The large connection energy (-8.7 kcal/mole) implies substantial energetic expenditure, possibly through a conformational rearrangement, upon membrane binding. This correlates well with higher thermal factors observed in the C1 and C2 domains of structures of VPtexC1C2 and VPtexC2 as compared to VPtex. It is also supported by rapid kinetic studies illustrating equivalence in the bimolecular association rate constants for human Va and VPtex variants regardless of their membrane affinity. Thus, high affinity membrane binding results from large decreases in the dissociation rate constant expected from a conformational change that allows the protein to adopt a new stable membrane-bound configuration. A second explanation for the lack of an obvious correlation between x-ray structures of VPtexC1C2, VPtexC2 and VPtex and their affinity for membranes lies in the possibility that their solution-phase conformations differ. We explored this using small angle x-ray scattering (SAXS) of VPtex, VPtexC2 and VPtexC1C2 in solution. The low resolution SAXS envelope for VPtex could be accounted for by minor shifts in the individual domains, particularly in C1 and C2, as seen in the crystal structure. However, the SAXS envelopes for membrane binding variants (VPtexC2 and VPtexC1C2) showed major shape changes in the C-domains. Rigid body modeling revealed an increasingly extended end-on arrangement, rather than a side-by-side configuration, of the C1 and C2 domains seen in the x-ray structure and in the SAXS envelope for FVPtex. The C2-domain was found to extend away from the base of the C1 domain along the long axis of the molecule correlating major structural differences in these VPtex variants with increasing affinity for membranes. Accordingly, the spatial disposition of the C1 and C2 domains in VPtexC2 appears intermediate to their arrangement in VPtex and VPtexC1C2.These findings contrast to the arrangement seen in the crystal structures of all factor V forms, where the C-domains are arranged side-by-side, probably due to limitations imposed by crystal packing. Our SAXS studies provide clear evidence of an unforeseen framework of C-domains associated with the ability of factor V forms to bind to membranes with high affinity. The findings reveal new mechanistic insights into the structural correlates of the membrane binding function of factor V. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding.
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Puchulu-Campanella, Estela, Francesco M. Turrini, Yen-Hsing Li, and Philip S. Low. "Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3." Proceedings of the National Academy of Sciences 113, no. 48 (November 15, 2016): 13732–37. http://dx.doi.org/10.1073/pnas.1611904113.
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Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein–protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO4) withKd= 14 nM; (iii) binding of cdb3-PO4to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO4; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO4(but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO4to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3–ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties.
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McKiernan, C. J., P. F. Stabila, and I. G. Macara. "Role of the Rab3A-binding domain in targeting of rabphilin-3A to vesicle membranes of PC12 cells." Molecular and Cellular Biology 16, no. 9 (September 1996): 4985–95. http://dx.doi.org/10.1128/mcb.16.9.4985.
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Rab3A is a small GTPase implicated in the docking of secretory vesicles in neuroendocrine cells. A putative downstream target for Rab3A, rabphilin-3A, is located exclusively on secretory vesicle membranes. It contains near its C terminus two C2 domains that bind Ca2+ in a phospholipid-dependent manner and an N-terminal, Rab3A-binding domain that includes a Cys-rich region. We have determined that the Cys-rich domain binds two Zn2+ ions and is necessary but not sufficient for efficient binding of rabphilin to Rab3A. A minimal Rab3A-binding domain consists of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green fluorescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphifin in vitro colocalized with the GFP-rabphilin fusion, indicating that Rab3A targeting is independent of its interaction with rabphilin. Deletion of the C2 domains of rabphilin reduced membrane association of GFP-rabphilin but did not cause mistargeting of the membrane-associated fraction. However, disruption of the zinc fingers, which drastically reduced Rab3A binding, did not reduce membrane association. These results suggest that the C2 domains are required for efficient membrane attachment of rabphilin in PC12 cells and that Rab3A binding may act to target the protein to the correct membrane.
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Pinigin, Konstantin V., Timur R. Galimzyanov, and Sergey A. Akimov. "Amphipathic Peptides Impede Lipid Domain Fusion in Phase-Separated Membranes." Membranes 11, no. 11 (October 20, 2021): 797. http://dx.doi.org/10.3390/membranes11110797.
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Cell membranes are heterogeneous in lipid composition which leads to the phase separation with the formation of nanoscopic liquid-ordered domains, also called rafts. There are multiple cell processes whereby the clustering of these domains into a larger one might be involved, which is responsible for such important processes as signal transduction, polarized sorting, or immune response. Currently, antimicrobial amphipathic peptides are considered promising antimicrobial, antiviral, and anticancer therapeutic agents. Here, within the framework of the classical theory of elasticity adapted for lipid membranes, we investigate how the presence of the peptides in a phase-separated membrane influences the fusion of the domains. We show that the peptides tend to occupy the boundaries of liquid-ordered domains and significantly increase the energy barrier of the domain-domain fusion, which might lead to misregulation of raft clustering and adverse consequences for normal cell processes.
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Ueyama, Takehiko, Toshihiko Tatsuno, Takumi Kawasaki, Satoshi Tsujibe, Yasuhito Shirai, Hideki Sumimoto, Thomas L. Leto, and Naoaki Saito. "A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox." Molecular Biology of the Cell 18, no. 2 (February 2007): 441–54. http://dx.doi.org/10.1091/mbc.e06-08-0731.
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In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47phox, p67phox, p40phox, and Rac) translocate and associate with the membrane-spanning flavocytochrome b558, leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40phoxthat regulate its translocation to membranes upon stimulation. GFP-p40phoxtranslocates to early endosomes, whereas GFP-p47phoxtranslocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67phoxdoes not translocate to membranes when expressed alone, but it is dependent on p40phoxand p47phoxfor its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40phoxor GFP-p47phoxto their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67phoxtranslocation to either membrane is abolished by mutations that disrupt its interaction with p40phoxor p47phox. Finally, we detected a head-to-tail (PX–Phox and Bem1 [PB1] domain) intramolecular interaction within p40phoxin its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40phoxand p47phoxfunction as diverse p67phox“carrier proteins” regulated by the unmasking of membrane-targeting domains in distinct mechanisms.
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Nagel, Wolfgang, Pierre Schilcher, Lutz Zeitlmann, and Waldemar Kolanus. "The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function." Molecular Biology of the Cell 9, no. 8 (August 1998): 1981–94. http://dx.doi.org/10.1091/mbc.9.8.1981.
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Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.
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Gill, David J., Hsiangling Teo, Ji Sun, Olga Perisic, Dmitry B. Veprintsev, Yvonne Vallis, Scott D. Emr, and Roger L. Williams. "Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies." Biochemical Society Symposia 74 (January 12, 2007): 47–57. http://dx.doi.org/10.1042/bss2007c05.
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Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.
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Stanishneva-Konovalova, T. B., N. I. Derkacheva, S. V. Polevova, and O. S. Sokolova. "The Role of BAR Domain Proteins in the Regulation of Membrane Dynamics." Acta Naturae 8, no. 4 (December 15, 2016): 60–69. http://dx.doi.org/10.32607/20758251-2016-8-4-60-69.
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Many cellular processes are associated with membrane remodeling. The BAR domain protein family plays a key role in the formation and detection of local membrane curvatures and in attracting other proteins, including the regulators of actin dynamics. Based on their structural and phylogenetic properties, BAR domains are divided into several groups which affect membrane in various ways and perform different functions in cells. However, recent studies have uncovered evidence of functional differences even within the same group. This review discusses the principles underlying the interactions of different groups of BAR domains, and their individual representatives, with membranes.
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Hokanson, David E., Joseph M. Laakso, Tianming Lin, David Sept, and E. Michael Ostap. "Myo1c Binds Phosphoinositides through a Putative Pleckstrin Homology Domain." Molecular Biology of the Cell 17, no. 11 (November 2006): 4856–65. http://dx.doi.org/10.1091/mbc.e06-05-0449.
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Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP2), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.
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Ping, Holly A., Lauren M. Kraft, WeiTing Chen, Amy E. Nilles, and Laura L. Lackner. "Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities." Journal of Cell Biology 213, no. 5 (May 30, 2016): 513–24. http://dx.doi.org/10.1083/jcb.201511021.
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The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.
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Davis-Harrison, Rebecca L., Narjes Tavoosi, Mary Clay, John M. Boettcher, Chad M. Rienstra, and James H. Morrissey. "Structural Insights Into How Clotting Proteins with GLA Domains Bind to Membrane Surfaces." Blood 116, no. 21 (November 19, 2010): 1141. http://dx.doi.org/10.1182/blood.v116.21.1141.1141.
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Abstract Abstract 1141 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. In spite of their importance, a clear picture of how GLA domains bind to the membrane interface has yet to emerge. A further intriguing aspect of the membrane's role in blood coagulation is that certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. The mechanisms of this synergy, and of PE's contribution to GLA domain binding, are poorly understood – although a number of hypotheses have been put forward. We now propose a new hypothesis to explain GLA domain binding to membranes, which we term the ABC (Anything But Choline) hypothesis; it invokes two main types of protein-phospholipid interactions: a single L-serine-specific binding site in each GLA domain; and multiple “phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. We have utilized liposomes and nanoscale phospholipid bilayers (Nanodiscs) in studies employing a series of techniques including solid-state NMR (SSNMR) and surface plasmon resonance (SPR) to address the mechanism of GLA domain-membrane interactions. We provide direct evidence in favor of the ABC hypothesis for GLA domain binding to membrane surfaces. Using SSNMR, we demonstrate that two distinct PS headgroup conformations are induced by binding of calcium ions, and that a third, novel PS headgroup conformation is induced when the prothrombin GLA domain engages the membrane. SPR studies have allowed for the determination of thermodynamic profiles of GLA domains interacting with phospholipid bilayers containing PS and/or PE, providing further insights to the mechanisms of GLA domain-membrane interactions. Disclosures: No relevant conflicts of interest to declare.
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Becalska, Agata N., Charlotte F. Kelley, Cristina Berciu, Tatiana B. Stanishneva-Konovalova, Xiaofeng Fu, ShiYu Wang, Olga S. Sokolova, Daniela Nicastro, and Avital A. Rodal. "Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck." Molecular Biology of the Cell 24, no. 15 (August 2013): 2406–18. http://dx.doi.org/10.1091/mbc.e13-05-0271.
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Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.
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Moczko, M., U. Bömer, M. Kübrich, N. Zufall, A. Hönlinger, and N. Pfanner. "The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences." Molecular and Cellular Biology 17, no. 11 (November 1997): 6574–84. http://dx.doi.org/10.1128/mcb.17.11.6574.
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Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes. For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side. Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site. We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences. The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi. After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22. We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.
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Molotkovsky, Galimzyanov, Batishchev, and Akimov. "The Effect of Transmembrane Protein Shape on Surrounding Lipid Domain Formation by Wetting." Biomolecules 9, no. 11 (November 12, 2019): 729. http://dx.doi.org/10.3390/biom9110729.
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Signal transduction through cellular membranes requires the highly specific and coordinated work of specialized proteins. Proper functioning of these proteins is provided by an interplay between them and the lipid environment. Liquid-ordered lipid domains are believed to be important players here, however, it is still unclear whether conditions for a phase separation required for lipid domain formation exist in cellular membranes. Moreover, membrane leaflets are compositionally asymmetric, that could be an obstacle for the formation of symmetric domains spanning the lipid bilayer. We theoretically show that the presence of protein in the membrane leads to the formation of a stable liquid-ordered lipid phase around it by the mechanism of protein wetting by lipids, even in the absence of conditions necessary for the global phase separation in the membrane. Moreover, we show that protein shape plays a crucial role in this process, and protein conformational rearrangement can lead to changes in the size and characteristics of surrounding lipid domains.
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de Almeida, J. B., E. J. Holtzman, P. Peters, L. Ercolani, D. A. Ausiello, and J. L. Stow. "Targeting of chimeric G alpha i proteins to specific membrane domains." Journal of Cell Science 107, no. 3 (March 1, 1994): 507–15. http://dx.doi.org/10.1242/jcs.107.3.507.
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Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.
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Lipowsky, Reinhard. "Remodeling of membrane compartments: some consequences of membrane fluidity." Biological Chemistry 395, no. 3 (March 1, 2014): 253–74. http://dx.doi.org/10.1515/hsz-2013-0244.
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Abstract Biological membranes consist of fluid bilayers with many lipid and protein components. This fluidity implies a high flexibility that allows the membranes to attain a large variety of different shapes. One important shape parameter is the spontaneous curvature, which describes the asymmetry between the two leaflets of a bilayer and can be changed by adsorption of ‘particles’ such as ions or proteins from the aqueous phases. Membrane fluidity also implies that the membranes can change their local composition via lateral diffusion and form intramembrane compartments. Two mechanisms for the formation of such compartments can be distinguished: membrane segmentation arising from structured environments and domain formation as a result of phase separation within the membranes. The interplay between these two mechanisms provides a simple and generic explanation for the difficulty to observe phase domains in vivo. Intramembrane domains can form new membrane compartments via budding and tubulation processes. Which of these two processes actually occurs depends on the fluid-elastic properties of the domains, on the adsorption kinetics, and on external constraints arising, e.g., from the osmotic conditions. Vesicles are predicted to unbind from adhesive surfaces via tubulation when the spontaneous curvature of their membranes exceeds a certain threshold value.
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Li, Guangtao, Qing Wang, Shinako Kakuda, and Erwin London. "Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids." Journal of Lipid Research 61, no. 5 (January 21, 2020): 758–66. http://dx.doi.org/10.1194/jlr.ra119000565.
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The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.
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Edidin, M., and I. Stroynowski. "Differences between the lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale membrane domains." Journal of Cell Biology 112, no. 6 (March 15, 1991): 1143–50. http://dx.doi.org/10.1083/jcb.112.6.1143.
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Plasma membranes of many cells appear to be divided into domains, areas whose composition and function differ from the average for an entire membrane. We have previously used fluorescence photo-bleaching and recovery to demonstrate one type of membrane domain, with dimensions of micrometers (Yechiel, E., and M. Edidin. 1987, J. Cell Biol. 105: 755-760). The presence of membrane domains is inferred from the dependence of the apparent mobile fraction of labeled molecules on the size of the membrane area probed. We now find that by this definition classical class I MHC molecules, H-2Db, are concentrated in domains in the membranes of K78-2 hepatoma cells, while the nonclassical class I-related molecules, Qa-2, are free to pass the boundaries of these domains. The two proteins are highly homologous but differ in their mode of anchorage to the membrane lipid bilayer. H-2Db is anchored by a transmembrane peptide, while Qa-2 is anchored by a glycosylphosphatidylinositol (GPI) anchor. A mutant class I protein with its external portion derived from Qa-2 but with transmembrane and cytoplasmic sequences from a classical class I molecule shows a dependence of its mobile fraction on the area of membrane probed, while a mutant whose external portions are a mixture of classical and nonclassical class I sequences, GPI-linked to the bilayer, does not show this dependence and hence by our definition is not restricted to membrane domains.
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Liao, Maofu, Claudia Sánchez-San Martín, Aihua Zheng, and Margaret Kielian. "In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein." Journal of Virology 84, no. 11 (March 24, 2010): 5730–40. http://dx.doi.org/10.1128/jvi.00170-10.
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ABSTRACT The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.