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1
Amrouche, Fethia, Bouziane Mahmah, Maiouf Belhamel y Hocine Benmoussa. "Modélisation d’une pile à combustible PEMFC alimentée directement en hydrogène-oxygène et validation expérimentale". Journal of Renewable Energies 8, n.º 2 (31 de diciembre de 2005): 109–21. http://dx.doi.org/10.54966/jreen.v8i2.856.
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La pile à combustible (PAC) est connue depuis longtemps comme un convertisseur d’hydrogène en énergie (électrique + thermique) possédant de très bons rendements, les recherches sur cette technologie se développent partout dans le monde de manière considérable. Les raisons sont bien connues: la réponse aux contraintes environnementales, aux problèmes posés par la production centralisée d’électricité, la nécessité d’avoir des alternatives énergétiques (vecteur hydrogène) et certaines exigences technologiques spécifiques telles que les applications spatiales, sous-marines, électroniques portables, alimentation électrique de sites isolés et de microsystèmes. Il est certain que nous assisterons dans les prochaines décennies à l’émergence de la filière hydrogène dans notre vie quotidienne comme vecteur énergétique. Le choix de la technologie des piles à combustible à membrane échangeuse de protons (PEMFC) est implicite vu les performances intéressantes (faible poids, robuste, électrolyte solide, démarrage rapide, large gamme de puissance de 1 W à10 MW, etc.). Il est donc important de pousser encore plus loin les efforts de recherche/développement autour de cette technologie pour pouvoir la maîtriser et étendre son application. Cet article présente les résultats de la modélisation de la cinétique électrochimique et la production électrique des piles à combustible PEMFC alimentée directement en gaz pur (hydrogène et oxygène) et la validation expérimentale grâce à une base de données établie au niveau du ‘’Laboratoire d’Hydrogène en Réseau – CDER‘’, dans le but d’exploiter et d’améliorer les modèles électrochimiques existants.
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Tian, Ye, Charles Schwieters, Stanley Opella y Francesca Marassi. "NMR-Restrained Structure Calculations of Membrane Proteins in Implicit Lipid Bilayer Membranes". Biophysical Journal 108, n.º 2 (enero de 2015): 251a. http://dx.doi.org/10.1016/j.bpj.2014.11.1389.
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Sáenz, James P., Daniel Grosser, Alexander S. Bradley, Thibaut J. Lagny, Oksana Lavrynenko, Martyna Broda y Kai Simons. "Hopanoids as functional analogues of cholesterol in bacterial membranes". Proceedings of the National Academy of Sciences 112, n.º 38 (8 de septiembre de 2015): 11971–76. http://dx.doi.org/10.1073/pnas.1515607112.
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The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negativeMethylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.
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Petrie, Emma J., Richard W. Birkinshaw, Akiko Koide, Eric Denbaum, Joanne M. Hildebrand, Sarah E. Garnish, Katherine A. Davies et al. "Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies". Proceedings of the National Academy of Sciences 117, n.º 15 (31 de marzo de 2020): 8468–75. http://dx.doi.org/10.1073/pnas.1919960117.
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The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.
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Zelhof, Andrew C., Hong Bao, Robert W. Hardy, Azam Razzaq, Bing Zhang y Chris Q. Doe. "DrosophilaAmphiphysin is implicated in protein localization and membrane morphogenesis but not in synaptic vesicle endocytosis". Development 128, n.º 24 (15 de diciembre de 2001): 5005–15. http://dx.doi.org/10.1242/dev.128.24.5005.
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Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. We show that Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribble, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and lack of phenotypes suggests that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types.
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Mc Dermott, Ray, Umit Ziylan, Danièle Spehner, Huguette Bausinger, Dan Lipsker, Mieke Mommaas, Jean-Pierre Cazenave et al. "Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates". Molecular Biology of the Cell 13, n.º 1 (enero de 2002): 317–35. http://dx.doi.org/10.1091/mbc.01-06-0300.
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Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.
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Wilson, D. W., S. W. Whiteheart, M. Wiedmann, M. Brunner y J. E. Rothman. "A multisubunit particle implicated in membrane fusion." Journal of Cell Biology 117, n.º 3 (1 de mayo de 1992): 531–38. http://dx.doi.org/10.1083/jcb.117.3.531.
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The N-ethylmaleimide sensitive fusion protein (NSF) is required for fusion of lipid bilayers at many locations within eukaryotic cells. Binding of NSF to Golgi membranes is known to require an integral membrane receptor and one or more members of a family of related soluble NSF attachment proteins (alpha-, beta-, and gamma-SNAPs). Here we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component in a detergent solubilized system. We show that NSF only binds to SNAPs in the presence of the integral receptor, resulting in the formation of a multisubunit protein complex with a sedimentation coefficient of 20S. Particle assembly reveals striking differences between members of the SNAP protein family; gamma-SNAP associates with the complex via a binding site distinct from that used by alpha- and beta-SNAPs, which are themselves equivalent, alternative subunits of the particle. Once formed, the 20S particle is subsequently able to disassemble in a process coupled to the hydrolysis of ATP. We suggest how cycles of complex assembly and disassembly could help confer specificity to the generalized NSF-dependent fusion apparatus.
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Parodi, Emily M., Crystal S. Baker, Cayla Tetzlaff, Sasha Villahermosa y Linda S. Huang. "SPO71 Mediates Prospore Membrane Size and Maturation in Saccharomyces cerevisiae". Eukaryotic Cell 11, n.º 10 (18 de mayo de 2012): 1191–200. http://dx.doi.org/10.1128/ec.00076-12.
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ABSTRACT The mechanisms that control the size and shape of membranes are not well understood, despite the importance of these structures in determining organelle and cell morphology. The prospore membrane, a double lipid bilayer that is synthesized de novo during sporulation in S. cerevisiae , grows to surround the four meiotic products. This membrane determines the shape of the newly formed spores and serves as the template for spore wall deposition. Ultimately, the inner leaflet of the prospore membrane will become the new plasma membrane of the cell upon germination. Here we show that Spo71, a pleckstrin homology domain protein whose expression is induced during sporulation, is critical for the appropriate growth of the prospore membrane. Without SPO71 , prospore membranes surround the nuclei but are abnormally small, and spore wall deposition is disrupted. Sporulating spo71 Δ cells have prospore membranes that properly localize components to their growing leading edges yet cannot properly localize septin structures. We also found that SPO71 genetically interacts with SPO1 , a gene with homology to the phospholipase B gene that has been previously implicated in determining the shape of the prospore membrane. Together, these results show that SPO71 plays a critical role in prospore membrane development.
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He, Yi, Lidia Prieto y Themis Lazaridis. "Electrostatic Interactions between Antimicrobial Peptides and Anionic Membranes: Insights from an Implicit Membrane Model". Biophysical Journal 100, n.º 3 (febrero de 2011): 497a. http://dx.doi.org/10.1016/j.bpj.2010.12.2914.
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Boyd, RB, JP Burke, J. Atkin, VW Thompson y JF Nugent. "Significance of capillary basement membrane changes in diabetes mellitus". Journal of the American Podiatric Medical Association 80, n.º 6 (1 de junio de 1990): 307–13. http://dx.doi.org/10.7547/87507315-80-6-307.
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Diabetes mellitus is a disease in which the capillary basement membranes are substantially altered. This diabetic microangiopathy is characterized by a thickening of the basement membrane and changes in its permeability characteristic due to a disturbance in the production and distribution of its functional components. Glucose metabolism and insulin imbalance have been implicated in these basement membrane modifications. The authors describe normal capillary basement membrane architecture and then discuss how pathologic changes caused by diabetes mellitus are related to diabetic foot pathology.
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GILLOOLY, David J., Anne SIMONSEN y Harald STENMARK. "Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins". Biochemical Journal 355, n.º 2 (6 de abril de 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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Yuzlenko, Olga y Themis Lazaridis. "Membrane protein native state discrimination by implicit membrane models". Journal of Computational Chemistry 34, n.º 9 (7 de diciembre de 2012): 731–38. http://dx.doi.org/10.1002/jcc.23189.
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Bean, Björn D. M., Samantha K. Dziurdzik, Kathleen L. Kolehmainen, Claire M. S. Fowler, Waldan K. Kwong, Leslie I. Grad, Michael Davey, Cayetana Schluter y Elizabeth Conibear. "Competitive organelle-specific adaptors recruit Vps13 to membrane contact sites". Journal of Cell Biology 217, n.º 10 (17 de julio de 2018): 3593–607. http://dx.doi.org/10.1083/jcb.201804111.
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The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13–Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.
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Saller, Manfred J., Zht Cheng Wu, Jeanine de Keyzer y Arnold J. M. Driessen. "The YidC/Oxa1/Alb3 protein family: common principles and distinct features". Biological Chemistry 393, n.º 11 (1 de noviembre de 2012): 1279–90. http://dx.doi.org/10.1515/hsz-2012-0199.
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Abstract The members of the YidC/Oxa1/Alb3 protein family are evolutionary conserved in all three domains of life. They facilitate the insertion of membrane proteins into bacterial, mitochondrial, and thylakoid membranes and have been implicated in membrane protein folding and complex formation. The major classes of substrates are small hydrophobic subunits of large energy-transducing complexes involved in respiration and light capturing. All YidC-like proteins share a conserved membrane region, whereas the N- and C-terminal regions are diverse and fulfill accessory functions in protein targeting.
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Chae, Gyung Joon, Sang Bae Lee, Ui Won Jung, Yong Keun Lee, Chong Kwan Kim y Seong Ho Choi. "The Effects of Antibiotics Blended Chitosan Membranes on the Calvarial Critical Size Defect in Sprague Dawley Rats". Key Engineering Materials 342-343 (julio de 2007): 857–60. http://dx.doi.org/10.4028/www.scientific.net/kem.342-343.857.
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The purpose of this study was to evaluate the osteogenesis of tetracycline blended chitosan membranes on the calvarial critical size defect in Sprague Dawley. An 8 mm surgical defect was created with a trephine bur in the area of the midsagittal suture. Forty rats were divided into four groups: negative control group, positive control group and two experimental groups. Three types of membranes were made and a comparative study was done. One type of non-woven membrane was made by chitosan for positive control. The other two types of non-woven membranes were made by immersing non-woven chitosan into either the tetracycline solution or the chitosan-tetracycline solution. Histologic analysis was done at 2 weeks and 8 weeks of healing periods. We concluded that that the use of tetracycline blended chitosan membrane on the calvarial defects in rats has a significant effect on the regeneration of bone tissue in itself. In addition it implicates that tetracycline blended chitosan membrane may be useful for guided tissue regeneration.
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Titorenko, Vladimir I. y Richard A. Rachubinski. "Peroxisomal Membrane Fusion Requires Two Aaa Family Atpases, Pex1p and Pex6p". Journal of Cell Biology 150, n.º 4 (21 de agosto de 2000): 881–86. http://dx.doi.org/10.1083/jcb.150.4.881.
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Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway. We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners. The next step, peroxisome docking, requires release of Pex1p from one partner. Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.
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Jost, M., D. Zeuschner, J. Seemann, K. Weber y V. Gerke. "Identification and characterization of a novel type of annexin-membrane interaction: Ca2+ is not required for the association of annexin II with early endosomes". Journal of Cell Science 110, n.º 2 (15 de enero de 1997): 221–28. http://dx.doi.org/10.1242/jcs.110.2.221.
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Annexin II, a member of a family of Ca2+ and membrane binding proteins, has been implicated in regulating membrane organization and membrane transport during endocytosis and Ca2+ regulated secretion. To characterize the mechanistic aspects of the annexin. II action we studied parameters which determine the endosomal association of annexin II. Immunoblot analysis of subcellular membrane fractions prepared from BHK cells in the presence of a Ca2+ chelating agent reveals that annexin II remains associated with endosomal membranes under such conditions. This annexin II behaviour is atypical for the Ca2+ regulated annexins and is corroborated by the finding that ectopically expressed annexin II mutants with inactivated Ca2+ binding sites continue to co-fractionate with endosomal membranes. The Ca(2+)-independent membrane association of annexin II is also not affected by introducing mutations interfering with the complex formation of annexin II with its intracellular protein ligand p11. However, a deletion of the unique N-terminal domain of annexin II, in particular the sequence spanning residues 15 to 24, abolishes the Ca(2+)-independent association of the protein with endosomes. These results describe a novel, Ca(2+)-independent type of annexin-membrane interaction and provide a first explanation for the observed preference of different annexins for different cellular membranes. In the case of annexin II this specificity could be mediated through specific membrane receptors interacting with a unique sequence in the annexin II molecule.
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Hitt, A. L., J. H. Hartwig y E. J. Luna. "Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium." Journal of Cell Biology 126, n.º 6 (15 de septiembre de 1994): 1433–44. http://dx.doi.org/10.1083/jcb.126.6.1433.
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Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.
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Quinones, B., K. Riento, V. M. Olkkonen, S. Hardy y M. K. Bennett. "Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations". Journal of Cell Science 112, n.º 23 (1 de diciembre de 1999): 4291–304. http://dx.doi.org/10.1242/jcs.112.23.4291.
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The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2′), 2C (2″) and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.
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Feher, J. J. y G. D. Ford. "A simple student laboratory on osmotic flow, osmotic pressure, and the reflection coefficient." Advances in Physiology Education 268, n.º 6 (junio de 1995): S10. http://dx.doi.org/10.1152/advances.1995.268.6.s10.
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Osmosis is usually taught from the point of view of the osmotic pressure developed when solutions of different concentrations of solute are separated by an ideal semipermeable membrane. The osmotic pressure is defined at equilibrium when there is no net flow, and it takes some time to reach this equilibrium. Although the osmotic pressure is certainly important, teaching only this point of view implicitly diminishes the importance of osmotic flow, which begins almost instantaneously across a membrane. A device was constructed with which students could measure the flow across a model membrane (dialysis tubing) as a function of concentration for solutes of different sizes. The device produced flows that were linearly proportional to the concentration, providing a confirmation of van't Hoff's law. Separate student groups repeated these experiments using both different solutes and different dialysis membranes. The combined results of four student groups showed that the flow across these nonideal membranes depends on the solute and membrane as well as the concentration of solute. Given a value for area times filtration coefficient (A x Lp) for the membranes (determined beforehand by their instructor), the students could calculate the reflection coefficient (sigma) for three solutes and two membranes. The results showed that large solutes had large sigma and that less porous membranes had larger sigma. A concurrent demonstration using this device and membranes showed that the osmotic flow can generate large pressures. These experiments and demonstration provide a balanced view of osmotic flow and pressure.
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Angelova, Plamena R., Minee L. Choi, Alexey V. Berezhnov, Mathew H. Horrocks, Craig D. Hughes, Suman De, Margarida Rodrigues et al. "Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation". Cell Death & Differentiation 27, n.º 10 (27 de abril de 2020): 2781–96. http://dx.doi.org/10.1038/s41418-020-0542-z.
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Abstract Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson’s disease, and highlights a new mechanism by which lipid peroxidation causes cell death.
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Krausser, Johannes, Tuomas P. J. Knowles y Anđela Šarić. "Physical mechanisms of amyloid nucleation on fluid membranes". Proceedings of the National Academy of Sciences 117, n.º 52 (16 de diciembre de 2020): 33090–98. http://dx.doi.org/10.1073/pnas.2007694117.
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Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context.
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Ríos-Medina, Yolanda, Pedro Rico-Chávez, Ivette Martínez-Vieyra, Juan C. Durán-Álvarez, Mario Rodriguez-Varela, Ruth Rincón-Heredia, César Reyes-López y Doris Cerecedo. "Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis". International Journal of Molecular Sciences 25, n.º 9 (30 de abril de 2024): 4939. http://dx.doi.org/10.3390/ijms25094939.
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Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.
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Jovanovic, Olivera A., Fraser D. Brown y Julie G. Donaldson. "An Effector Domain Mutant of Arf6 Implicates Phospholipase D in Endosomal Membrane Recycling". Molecular Biology of the Cell 17, n.º 1 (enero de 2006): 327–35. http://dx.doi.org/10.1091/mbc.e05-06-0523.
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In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.
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Schulz, Timothy A., Mal-Gi Choi, Sumana Raychaudhuri, Jason A. Mears, Rodolfo Ghirlando, Jenny E. Hinshaw y William A. Prinz. "Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues". Journal of Cell Biology 187, n.º 6 (14 de diciembre de 2009): 889–903. http://dx.doi.org/10.1083/jcb.200905007.
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Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.
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Ilangumaran, Subburaj, Stephan Arni, Gerhild van Echten-Deckert, Bettina Borisch y Daniel C. Hoessli. "Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes". Molecular Biology of the Cell 10, n.º 4 (abril de 1999): 891–905. http://dx.doi.org/10.1091/mbc.10.4.891.
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Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.
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Bandorowicz-Pikuła, J. y S. Pikuła. "Annexins and ATP in membrane traffic: a comparison with membrane fusion machinery." Acta Biochimica Polonica 45, n.º 3 (30 de septiembre de 1998): 721–33. http://dx.doi.org/10.18388/abp.1998_4211.
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Annexins, calcium- and membrane-binding multifunctional proteins, have been implicated in N-ethylmaleimide (NEM)-independent fusion of vesicular structures involved in membrane traffic. This view is based on intracellular localization of annexins, which are frequently associated with endosomes, chromaffin granules, caveolae, clathrin-coated pits, and other membrane compartments, engaged in endo- and exocytosis. Moreover, annexins were found to modulate budding and aggregation of vesicle membranes, to interact with cytoskeletal proteins, and, upon binding to membranes, to change the structure of lipid bilayer, leading to membrane fusion. In addition, some annexins are substrates for various protein kinases and, in membrane-bound form, reveal calcium channel activity. Recently, annexins were observed to interact in vitro and in vivo with nucleotides, ATP, GTP or cAMP, which are potent mediators of membrane traffic processes. In addition, annexin VII showed hydrolytic activity towards GTP, and similarities in the mechanism of action to that of small GTP-binding proteins were found. The aim of the present review is to summarize the observations implying annexins as possible effectors in endo- and exocytosis and to compare them with well known complexes of cytosolic and membrane proteins forming the true membrane fusion machinery within a cell, conserved from yeast to the neurons of humans.
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Zhang, Guangzhi y Hélène Sanfaçon. "Characterization of Membrane Association Domains within the Tomato Ringspot Nepovirus X2 Protein, an Endoplasmic Reticulum-Targeted Polytopic MembraneProtein". Journal of Virology 80, n.º 21 (23 de agosto de 2006): 10847–57. http://dx.doi.org/10.1128/jvi.00789-06.
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ABSTRACT Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.
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Beraki, Tsebaot, Xiaoyu Hu, Malgorzata Broncel, Joanna C. Young, William J. O’Shaughnessy, Dominika Borek, Moritz Treeck y Michael L. Reese. "Divergent kinase regulates membrane ultrastructure of theToxoplasmaparasitophorous vacuole". Proceedings of the National Academy of Sciences 116, n.º 13 (8 de marzo de 2019): 6361–70. http://dx.doi.org/10.1073/pnas.1816161116.
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Apicomplexan parasites replicate within a protective organelle, called the parasitophorous vacuole (PV). TheToxoplasma gondiiPV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network’s functions. Here, we identify the parasite-secreted kinase WNG1 (With-No-Gly-loop) as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, we show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, our results describe a unique family ofToxoplasmakinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.
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Hinshaw, J. E. y K. R. Miller. "Localization of light-harvesting complex II to the occluded surfaces of photosynthetic membranes." Journal of Cell Biology 109, n.º 4 (1 de octubre de 1989): 1725–31. http://dx.doi.org/10.1083/jcb.109.4.1725.
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The photosynthetic membranes of green plants are organized into stacked regions interconnected by nonstacked regions that have been shown to be biochemically and structurally distinct. Because the stacking process occludes the surfaces of appressed membranes, it has been impossible to conduct structural or biochemical studies of the outer surfaces of the photosynthetic membrane in regions of membrane stacking. Although stacking is mediated at this surface, it has not been possible to determine whether membrane components implicated in the stacking process, including a major light-harvesting complex (LHC-II), are in fact exposed at the membrane surface. We have been able to expose this surface for study in the electron microscope and directly label it with antibodies to determine protein exposure. The appearance of the newly exposed outer stacked surface highlights the extreme lateral heterogeneity of the photosynthetic membrane. The surface is smooth in contrast to the neighboring nonstacked surface that is covered with distinct particles. Although some investigators have suggested the existence of a cytochrome b6/f-rich boundary region between stacked and nonstacked membranes, our results provide no structural support for this concept. To explore the biochemical nature of the occluded membrane surface, we have used an mAb against the amino terminal region of the LHC-II. This mAb clearly labels the newly exposed outer stacked surface but does not label the inner surface or the outer nonstacked surface. These experimental results confirm the presence of the amino terminal region of this complex at the outer surface of the membrane in stacked regions, and also show that this complex is largely absent from nonstacked membranes.
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Fan, Weiliang, Jia Guo, Beichen Gao, Wenbin Zhang, Liucong Ling, Tao Xu, Chenjie Pan et al. "Flotillin-mediated endocytosis and ALIX–syntenin-1–mediated exocytosis protect the cell membrane from damage caused by necroptosis". Science Signaling 12, n.º 583 (28 de mayo de 2019): eaaw3423. http://dx.doi.org/10.1126/scisignal.aaw3423.
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Necroptosis is a form of regulated necrosis that is implicated in various human diseases including Alzheimer’s disease. Necroptosis requires the translocation of the pseudokinase MLKL from the cytosol to the plasma membrane after its phosphorylation by the kinase RIPK3. Using protein cross-linking followed by affinity purification, we detected the lipid raft–associated proteins flotillin-1 and flotillin-2 and the ESCRT-associated proteins ALIX and syntenin-1 in membrane-localized MLKL immunoprecipitates. Phosphorylated MLKL was removed from membranes through either flotillin-mediated endocytosis followed by lysosomal degradation or ALIX–syntenin-1–mediated exocytosis. Thus, cells undergoing necroptosis need to overcome these independent suppressive mechanisms before plasma membrane disruption can occur.
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Lanrezac, André, Benoist Laurent, Hubert Santuz, Nicolas Férey y Marc Baaden. "Fast and Interactive Positioning of Proteins within Membranes". Algorithms 15, n.º 11 (7 de noviembre de 2022): 415. http://dx.doi.org/10.3390/a15110415.
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(1) Background: We developed an algorithm to perform interactive molecular simulations (IMS) of protein alignment in membranes, allowing on-the-fly monitoring and manipulation of such molecular systems at various scales. (2) Methods: UnityMol, an advanced molecular visualization software; MDDriver, a socket for data communication; and BioSpring, a Spring network simulation engine, were extended to perform IMS. These components are designed to easily communicate with each other, adapt to other molecular simulation software, and provide a development framework for adding new interaction models to simulate biological phenomena such as protein alignment in the membrane at a fast enough rate for real-time experiments. (3) Results: We describe in detail the integration of an implicit membrane model for Integral Membrane Protein And Lipid Association (IMPALA) into our IMS framework. Our implementation can cover multiple levels of representation, and the degrees of freedom can be tuned to optimize the experience. We explain the validation of this model in an interactive and exhaustive search mode. (4) Conclusions: Protein positioning in model membranes can now be performed interactively in real time.
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Schulman, G., R. Hakim, R. Arias, M. Silverberg, A. P. Kaplan y L. Arbeit. "Bradykinin generation by dialysis membranes: possible role in anaphylactic reaction." Journal of the American Society of Nephrology 3, n.º 9 (marzo de 1993): 1563–69. http://dx.doi.org/10.1681/asn.v391563.
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Several recent reports have described a high incidence of anaphylactic reactions in patients being dialyzed with high-flux membranes while simultaneously using angiotensin-converting enzyme inhibitors. Many of these reports implicate polyacrylonitrile (PAN) as the membrane commonly involved in these reactions. To elucidate potential mechanisms of these anaphylactic reactions, whether dialysis membranes can activate the Hageman factor-dependent (contact) pathways as assessed by the in vitro generation of activated Hageman factor (Hfa), as well as the formation of kallikrein and subsequent bradykinin generation was examined. Both cuprophane (CUP) and PAN membranes were able to activate Hageman factor and convert prekallikrein to kallikrein as measured by an ELISA against kallikrein-C1-inactivator complexes. Subsequently, the active kallikrein was able to cleave bradykinin from its endogenous substrate, high-molecular-weight kininogen. However, it was found that the PAN membrane consistently led to an earlier and significantly higher formation of Hfa and kallikrein when compared with CUP. Importantly, there was also a pronounced but transient generation of bradykinin by the PAN membrane, in contrast to slower bradykinin formation by CUP, with both normal and uremic blood. It was proposed that the early and vigorous bradykinin generation induced by the contact of blood with PAN could explain, in part, the pathogenesis of the reported anaphylactoid reactions.
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Sharma, Mahak, Sai Srinivas Panapakkam Giridharan, Juliati Rahajeng, Naava Naslavsky y Steve Caplan. "MICAL-L1 Links EHD1 to Tubular Recycling Endosomes and Regulates Receptor Recycling". Molecular Biology of the Cell 20, n.º 24 (15 de diciembre de 2009): 5181–94. http://dx.doi.org/10.1091/mbc.e09-06-0535.
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Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.
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Klotz, K. N., K. L. Krotec, J. Gripentrog y A. J. Jesaitis. "Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils." Journal of Immunology 152, n.º 2 (15 de enero de 1994): 801–10. http://dx.doi.org/10.4049/jimmunol.152.2.801.
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Abstract The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCl, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-[125I]ASD at 15 degrees C, were solubilized, nearly all receptors were recovered in the pellet fraction. Incubation of cells with the ligand at 4 degrees C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. In these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.
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Levi, Valeria, Ana M. Villamil Giraldo, Pablo R. Castello, Juan P. F. C. Rossi y F. Luis González Flecha. "Effects of phosphatidylethanolamine glycation on lipid–protein interactions and membrane protein thermal stability". Biochemical Journal 416, n.º 1 (28 de octubre de 2008): 145–52. http://dx.doi.org/10.1042/bj20080618.
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Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca2+-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by ∼30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.
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Ahn, Anna, Don L. Gibbons y Margaret Kielian. "The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains". Journal of Virology 76, n.º 7 (1 de abril de 2002): 3267–75. http://dx.doi.org/10.1128/jvi.76.7.3267-3275.2002.
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ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl β-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.
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Wall, J., F. Ayoub y P. O'Shea. "Interactions of macromolecules with the mammalian cell surface". Journal of Cell Science 108, n.º 7 (1 de julio de 1995): 2673–82. http://dx.doi.org/10.1242/jcs.108.7.2673.
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The characterisation of fluoresceinphosphatidylethanolamine (FPE) as a real-time indicator of the electrostatic nature of the cell membrane surface is described. The conditions appropriate for the labelling of cell membranes and the implementation of FPE as a tool to monitor the interactions of various proteins and peptides with membranes are outlined. Some complications attributed to the erythrocyte glycocalyx are examined. In addition it is shown using neuraminidase as an example, that some types of enzyme-catalysed reactions on the cell surface may be monitored in real time. It is also shown that information concerning the binding of several proteins such as serum albumin and monoclonal antibodies are accessible with this technique. The albumin in particular is shown to exhibit a saturation of binding, the analysis of which indicates that the dissociation constant for erythrocytes was determined to be 8 microM and for lymphocytes to be almost 3 microM. On the basis of this comparison together with artificial membranes, the membrane protein components of the lymphocyte surface are implicated in the binding of albumin or the erythrocyte membrane proteins reduce the affinity of the cell surface for albumin.
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Liu, Hualan, W. Keith Ray, Richard F. Helm, David L. Popham y Stephen B. Melville. "Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly". Journal of Bacteriology 198, n.º 12 (11 de abril de 2016): 1773–82. http://dx.doi.org/10.1128/jb.00212-16.
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ABSTRACTHeat-resistant endospore formation plays an important role inClostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores ofC. perfringensstrain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed ofl-arginine-poly(l-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species ofClostridium, but their role has not been defined. To determine the function of cyanophycin inC. perfringens, a mutation was introduced into thecphAgene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulatingC. perfringenscells, anEscherichia colistrain expressing thecphAgene made copious amounts of cyanophycin, confirming thatcphAencodes a cyanophycin synthetase.IMPORTANCEClostridium perfringensis a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. HowC. perfringenscontrols the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role inC. perfringensthan it does in cyanobacteria.
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Pavlov, Evgeny V., Muriel Priault, Dawn Pietkiewicz, Emily H. Y. Cheng, Bruno Antonsson, Stephen Manon, Stanley J. Korsmeyer, Carmen A. Mannella y Kathleen W. Kinnally. "A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast". Journal of Cell Biology 155, n.º 5 (26 de noviembre de 2001): 725–32. http://dx.doi.org/10.1083/jcb.200107057.
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During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.
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Ngo, Mike H., Terry R. Colbourne y Neale D. Ridgway. "Functional implications of sterol transport by the oxysterol-binding protein gene family". Biochemical Journal 429, n.º 1 (14 de junio de 2010): 13–24. http://dx.doi.org/10.1042/bj20100263.
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Cholesterol and its numerous oxygenated derivatives (oxysterols) profoundly affect the biophysical properties of membranes, and positively and negatively regulate sterol homoeostasis through interaction with effector proteins. As the bulk of cellular sterols are segregated from the sensory machinery that controls homoeostatic responses, an important regulatory step involves sterol transport or signalling between membrane compartments. Evidence for rapid, energy-independent transport between organelles has implicated transport proteins, such as the eukaryotic family of OSBP (oxysterol-binding protein)/ORPs (OSBP-related proteins). Since the founding member of this family was identified more than 25 years ago, accumulated evidence has implicated OSBP/ORPs in sterol signalling and/or sterol transport functions. However, recent evidence of sterol transfer activity by OSBP/ORPs suggests that other seemingly disparate functions could be the result of alterations in membrane sterol distribution or ancillary to this primary activity.
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Nepal, Binod, John Leveritt y Themis Lazaridis. "Membrane Curvature Sensing by Amphipathic Helices: Insights from Implicit Membrane Modeling". Biophysical Journal 114, n.º 9 (mayo de 2018): 2128–41. http://dx.doi.org/10.1016/j.bpj.2018.03.030.
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Kweon, Youngseok, Anca Rothe, Elizabeth Conibear y Tom H. Stevens. "Ykt6p Is a Multifunctional Yeast R-SNARE That Is Required for Multiple Membrane Transport Pathways to the Vacuole". Molecular Biology of the Cell 14, n.º 5 (mayo de 2003): 1868–81. http://dx.doi.org/10.1091/mbc.e02-10-0687.
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Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.
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Ponmalar, Ilanila I., Ramesh Cheerla, K. Ganapathy Ayappa y Jaydeep K. Basu. "Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins". Proceedings of the National Academy of Sciences 116, n.º 26 (12 de junio de 2019): 12839–44. http://dx.doi.org/10.1073/pnas.1821897116.
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Pore-forming toxins (PFTs) are a class of proteins implicated in a wide range of virulent bacterial infections and diseases. These toxins bind to target membranes and subsequently oligomerize to form functional pores that eventually lead to cell lysis. While the protein undergoes large conformational changes on the bilayer, the connection between intermediate oligomeric states and lipid reorganization during pore formation is largely unexplored. Cholesterol-dependent cytolysins (CDCs) are a subclass of PFTs widely implicated in food poisoning and other related infections. Using a prototypical CDC, listeriolysin O (LLO), we provide a microscopic connection between pore formation, lipid dynamics, and leakage kinetics by using a combination of Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) measurements on single giant unilamellar vesicles (GUVs). Upon exposure to LLO, two distinct populations of GUVs with widely different leakage kinetics emerge. We attribute these differences to the existence of oligomeric intermediates, sampling various membrane-bound conformational states of the protein, and their intimate coupling to lipid rearrangement and dynamics. Molecular dynamics simulations capture the influence of various membrane-bound conformational states on the lipid and cholesterol dynamics, providing molecular interpretations to the FRET and FCS experiments. Our study establishes a microscopic connection between membrane binding and conformational changes and their influence on lipid reorganization during PFT-mediated cell lysis. Additionally, our study provides insights into membrane-mediated protein interactions widely implicated in cell signaling, fusion, folding, and other biomolecular processes.
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Sorek, Nadav, Limor Poraty, Hasana Sternberg, Enat Bar, Efraim Lewinsohn y Shaul Yalovsky. "Activation Status-Coupled Transient S Acylation Determines MembranePartitioning of a Plant Rho-Related GTPase". Molecular and Cellular Biology 27, n.º 6 (22 de enero de 2007): 2144–54. http://dx.doi.org/10.1128/mcb.02347-06.
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ABSTRACT ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His6-GFP-Atrop6CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His6-GFP-Atrop6CAmS156 in which cysteine156 was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.
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Bkaily, Ghassan, Yanick Simon, Ashley Jazzar, Houssein Najibeddine, Alexandre Normand y Danielle Jacques. "High Na+ Salt Diet and Remodeling of Vascular Smooth Muscle and Endothelial Cells". Biomedicines 9, n.º 8 (24 de julio de 2021): 883. http://dx.doi.org/10.3390/biomedicines9080883.
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Our knowledge on essential hypertension is vast, and its treatment is well known. Not all hypertensives are salt-sensitive. The available evidence suggests that even normotensive individuals are at high cardiovascular risk and lower survival rate, as blood pressure eventually rises later in life with a high salt diet. In addition, little is known about high sodium (Na+) salt diet-sensitive hypertension. There is no doubt that direct and indirect Na+ transporters, such as the Na/Ca exchanger and the Na/H exchanger, and the Na/K pump could be implicated in the development of high salt-induced hypertension in humans. These mechanisms could be involved following the destruction of the cell membrane glycocalyx and changes in vascular endothelial and smooth muscle cells membranes’ permeability and osmolarity. Thus, it is vital to determine the membrane and intracellular mechanisms implicated in this type of hypertension and its treatment.
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Mayer, A., I. E. Ivanov, D. Gravotta, M. Adesnik y D. D. Sabatini. "Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells". Journal of Cell Science 109, n.º 7 (1 de julio de 1996): 1667–76. http://dx.doi.org/10.1242/jcs.109.7.1667.
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An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15–30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.
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Georgatos, S. D., D. C. Weaver y V. T. Marchesi. "Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains." Journal of Cell Biology 100, n.º 6 (1 de junio de 1985): 1962–67. http://dx.doi.org/10.1083/jcb.100.6.1962.
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Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.
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Deretic, D., L. A. Huber, N. Ransom, M. Mancini, K. Simons y D. S. Papermaster. "rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis". Journal of Cell Science 108, n.º 1 (1 de enero de 1995): 215–24. http://dx.doi.org/10.1242/jcs.108.1.215.
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Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.
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Siess, Katharina M. y Thomas A. Leonard. "Lipid-dependent Akt-ivity: where, when, and how". Biochemical Society Transactions 47, n.º 3 (30 de mayo de 2019): 897–908. http://dx.doi.org/10.1042/bst20190013.
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Abstract Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3. Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2. While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.