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David E. James - One of the best experts on this subject based on the ideXlab platform.
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syntaxin 7 complexes with mouse vps10p tail interactor 1b syntaxin 6 vesicle associated Membrane Protein vamp 8 and vamp7 in b16 melanoma cells
2001Co-Authors: Nicholas M Wade, Nia J Bryant, Lisa M Connolly, Richard J Simpson, J P Luzio, Robert C Piper, David E. JamesAbstract:Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment Protein receptor (SNARE) involved in Membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify Proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated Protein (SNAP), Vesicle-Associated Membrane Protein (VAMP)8, VAMP7, and the Protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.
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Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes
1999Co-Authors: Caroline A Millar, Annette M Shewan, David E. James, Gilles R.x. Hickson, Gwyn W. GouldAbstract:Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma Membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (~30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPgamma S response was significantly attenuated (~85%) under the same conditions. Introduction of a GST fusion Protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPgamma S-stimulated GLUT4 translocation by ~40% but had no effect on the insulin response. Conversely, a fusion Protein encompassing the cytosolic tail of Vesicle-Associated Membrane Protein-2 had no significant effect on GTPgamma S-stimulated GLUT4 translocation but inhibited the insulin response by ~40%. GTPgamma S- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (~50%) but not by GST-Vesicle-Associated Membrane Protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPgamma S caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab Proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.
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vesicle associated Membrane Protein 2 plays a specific role in the insulin dependent trafficking of the facilitative glucose transporter glut4 in 3t3 l1 adipocytes
1998Co-Authors: Laura B Martin, Annette M Shewan, Caroline A Millar, Gwyn W. Gould, David E. JamesAbstract:Abstract Vesicle-Associated Membrane Protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625–635), suggesting that it may play a role distinct from endosomal v-SNAREs (solubleN-ethylmaleimide-sensitive factor attachment Protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathioneS-transferase (GST) fusion Proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by ∼35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5′-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.
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The glucose transporter (GLUT-4) and Vesicle-Associated Membrane Protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.
1996Co-Authors: Sally Martin, Gwyn W. Gould, Judy Tellam, C Livingstone, Jan W. Slot, David E. JamesAbstract:Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/Vesicle-Associated Membrane Protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular Membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.
Gwyn W. Gould - One of the best experts on this subject based on the ideXlab platform.
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v- and t-SNARE Protein expression in models of insulin resistance: normalization of glycemia by rosiglitazone treatment corrects overexpression of cellubrevin, Vesicle-Associated Membrane Protein-2, and syntaxin 4 in skeletal muscle of Zucker diabeti
2000Co-Authors: Valerie H. Maier, Gwyn W. Gould, Derek R. Melvin, Carolyn A. Lister, Helen Chapman, Gregory J. MurphyAbstract:Insulin stimulation of adipose and muscle cells results in the translocation of GLUT4 from an intracellular location to the plasma Membrane; this translocation is defective in insulin resistance. Studies have suggested an important role for synaptobrevin and syntaxin homologues in this event, particularly the v-soluble N-ethylmaleimide attachment Protein receptors (SNAREs) cellubrevin and Vesicle-Associated Membrane Protein-2 (VAMP-2) and the t-SNARE syntaxin 4, but the expression of these Proteins has not been studied in insulin-resistant tissues. Therefore, we examined SNARE Protein content in skeletal muscle from Zucker diabetic fatty (ZDF) rats compared with lean controls and determined the effect of the thiazolidinedione insulin sensitizer rosiglitazone on these Proteins. GLUT4 levels in skeletal muscle from ZDF rats were similar to those in lean control animals. In contrast, cellubrevin, VAMP-2, and syntaxin 4 Protein levels were elevated (2.8-fold, P = 0.02; 3.7-fold, P = 0.01; and 2.2-fold, P < 0.05, respectively) in skeletal muscle from ZDF rats compared with lean controls. Restoration of normoglycemia and normoinsulinemia in ZDF rats with rosiglitazone (30 micromol/kg) normalized cellubrevin, VAMP-2, and syntaxin 4 Protein to levels approaching those observed in lean control animals. These data show that elevated v- and t-SNARE Protein levels are associated with insulin resistance in skeletal muscle and that these increases may be reversed by rosiglitazone treatment concomitant with a restoration of glycemic control. Such increases in SNARE Protein levels were not observed in streptozotocin-induced diabetic rats, which suggests that hyperinsulinemia rather than hyperglycemia may be more important in modulating SNARE Protein expression in rodent models of insulin resistance. Consistent with this hypothesis, elevated levels of SNARE Proteins were also observed in 3T3-L1 adipocytes chronically treated with insulin (500 nmol/l for 24 h). These data argue that SNARE Protein levels may be altered in insulin-resistant states and that the levels of these Proteins are modulated by agents that increase insulin sensitivity. Moreover, these data demonstrate for the first time altered expression of Proteins known to regulate GLUT4 translocation in a model of diabetes.
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Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes
1999Co-Authors: Caroline A Millar, Annette M Shewan, David E. James, Gilles R.x. Hickson, Gwyn W. GouldAbstract:Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma Membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (~30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPgamma S response was significantly attenuated (~85%) under the same conditions. Introduction of a GST fusion Protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPgamma S-stimulated GLUT4 translocation by ~40% but had no effect on the insulin response. Conversely, a fusion Protein encompassing the cytosolic tail of Vesicle-Associated Membrane Protein-2 had no significant effect on GTPgamma S-stimulated GLUT4 translocation but inhibited the insulin response by ~40%. GTPgamma S- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (~50%) but not by GST-Vesicle-Associated Membrane Protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPgamma S caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab Proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.
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vesicle associated Membrane Protein 2 plays a specific role in the insulin dependent trafficking of the facilitative glucose transporter glut4 in 3t3 l1 adipocytes
1998Co-Authors: Laura B Martin, Annette M Shewan, Caroline A Millar, Gwyn W. Gould, David E. JamesAbstract:Abstract Vesicle-Associated Membrane Protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625–635), suggesting that it may play a role distinct from endosomal v-SNAREs (solubleN-ethylmaleimide-sensitive factor attachment Protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathioneS-transferase (GST) fusion Proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by ∼35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5′-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.
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The glucose transporter (GLUT-4) and Vesicle-Associated Membrane Protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.
1996Co-Authors: Sally Martin, Gwyn W. Gould, Judy Tellam, C Livingstone, Jan W. Slot, David E. JamesAbstract:Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/Vesicle-Associated Membrane Protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular Membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.
Thierry Galli - One of the best experts on this subject based on the ideXlab platform.
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Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice [version 2; referees: 2 approved]
2019Co-Authors: Agathe Verraes, Thierry Galli, Beatrice Cholley, Sébastien NolaAbstract:VAMP7 (Vesicle-Associated Membrane Protein) belongs to the intracellular Membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment Protein receptors) Protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and use visual scoring for immunocytochemistry staining to determine the extent of the antibodies’ specificity. Thus, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts
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role of tetanus neurotoxin insensitive vesicle associated Membrane Protein in Membrane domains transport and homeostasis
2015Co-Authors: Diana Molino, Sébastien Nola, Veronique Prouxgillardeaux, Agathe Verraes, Lydia Danglot, Gaelle Boncompain, Franck Perez, Markus R Wenk, Guanghou Shui, Thierry GalliAbstract:Biological Membranes in eukaryotes contain a large variety of Proteins and lipids often distributed in domains in plasma Membrane and endoMembranes. Molecular mechanisms responsible for the transport and the organization of these Membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in Membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored Proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored Proteins to cell surface and t...
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ti vamp vamp7 is required for optimal phagocytosis of opsonised particles in macrophages
2004Co-Authors: Virginie Braun, Thierry Galli, Philippe Chavrier, Vincent Fraisier, Graça Raposo, Ilse Hurbain, Jean-baptiste Sibarita, Florence NiedergangAbstract:Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of Membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive Vesicle-Associated Membrane Protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of Membrane delivery required for optimal FcR-mediated phagocytosis.
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Tetanus neurotoxin-insensitive Vesicle-Associated Membrane Protein localizes to a presynaptic Membrane compartment in selected terminal subsets of the rat brain.
2003Co-Authors: Aude Muzerelle, Philipp Alberts, S Martinez-arca, O Jeannequin, Jean-claude Mazie, Pierre Lafaye, Thierry Galli, P. GasparAbstract:Tetanus neurotoxin-insensitive Vesicle-Associated Membrane Protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion Protein attachment Protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in Membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.
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role of tetanus neurotoxin insensitive vesicle associated Membrane Protein ti vamp in vesicular transport mediating neurite outgrowth
2000Co-Authors: Sonia Martinezarca, Philipp Alberts, Thierry Galli, Daniel Louvard, Ahmed ZahraouiAbstract:How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma Membrane of neurons. Tetanus neurotoxin-insensitive Vesicle-Associated Membrane Protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion Protein [NSF] attachment Protein [SNAP] receptor), involved in transport to the apical plasma Membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated Protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process.
Richard H. Scheller - One of the best experts on this subject based on the ideXlab platform.
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vesicle associated Membrane Protein 4 is implicated in trans golgi network vesicle trafficking
1999Co-Authors: Martin Steegmaier, Judith Klumperman, Davide L Foletti, Richard H. SchellerAbstract:The trans-Golgi network (TGN) plays a pivotal role in directing Proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the Vesicle-Associated Membrane Protein (VAMP) family of trafficking Proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular Membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking Protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.
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snare interactions are not selective implications for Membrane fusion specificity
1999Co-Authors: Bin Yang, Martin Steegmaier, Lino C Gonzalez, Rytis Prekeris, Raj J Advani, Richard H. SchellerAbstract:The SNARE hypothesis proposes that Membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle Membrane)- and t (target Membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five Proteins of the Vesicle-Associated Membrane Protein (VAMP) family, three members of the synaptosome-associated Protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 degreesC. These results suggest that the specificity of Membrane fusion is not encoded by the interactions between SNAREs.
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three novel Proteins of the syntaxin snap 25 family
1998Co-Authors: Martin Steegmaier, Bin Yang, Jin-san Yoo, Betty Huang, Mary Shen, Ying Luo, Richard H. SchellerAbstract:Intracellular Membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment Protein receptors (SNAREs) through the formation of complexes between these Proteins present on vesicle and target Membranes. All known SNARE-mediated fusion events involve members of the syntaxin and Vesicle-Associated Membrane Protein families. The diversity of mammalian Membrane compartments predicts the existence of a large number of different syntaxin and Vesicle-Associated Membrane Protein genes. To further investigate the spectrum of SNAREs and their roles in Membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated Protein of 25 kDa) subfamilies. The Proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct Membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple Membranes. SNAP-29 does not contain a predicted Membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.
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vesicle associated Membrane Protein and synaptophysin are associated on the synaptic vesicle
1994Co-Authors: Nicole Calakos, Richard H. SchellerAbstract:Abstract The synaptic vesicle Membrane Protein VAMP (Vesicle-Associated Membrane Protein or synaptobrevin) has been implicated in synaptic vesicle docking and fusion. Synaptophsin (p38), also a synaptic vesicle Membrane Protein, has four transMembrane domains and may function as a gap junction-like pore or channel. Here we report evidence for a direct interaction between VAMP and synaptophysin using chemical cross-linking followed by the identification of immunoreactive Protein complexes. A prominent complex of 56 kDa was found to consist of VAMP and synaptophysin. Furthermore, we demonstrate that this VAMP-synaptophysin complex is enriched in the synaptic vesicle fraction of rat brain, is independent of detergent solubilization, and is present in PC12 cells subjected to in vivo cross-linking.
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a Protein assembly disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking activation and fusion
1993Co-Authors: Thomas H Sollner, Mark K Bennett, Sidney W. Whiteheart, Richard H. Scheller, James E RothmanAbstract:The SNARE hypothesis holds that a transport vesicle chooses its target for fusion when a soluble NSF attachment Protein (SNAP) receptor on the vesicle (v-SNARE) pairs with its cognate t-SNARE at the target Membrane. Three synaptosomal Membrane Proteins have previously been identified: syntaxin, SNAP-25 (t-SNAREs), and Vesicle-Associated Membrane Protein (VAMP) (v-SNARE); all assemble with SNAPs and NSF into 20S fusion particles. We now report that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin. Synaptotagmin is displaced by alpha-SNAP, suggesting that these two Proteins share binding sites on the SNARE complex and implying that synaptotagmin operates as a "clamp" to prevent fusion from proceeding in the absence of a signal. The alpha-SNAP-SNARE complex can bind NSF, and NSF-dependent hydrolysis of ATP dissociates the complex, separating syntaxin, SNAP-25, and VAMP. ATP hydrolysis by NSF may provide motion to initiate bilayer fusion.
Cesare Montecucco - One of the best experts on this subject based on the ideXlab platform.
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vamp synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes
2003Co-Authors: Paola Caccin, Giampietro Schiavo, Ornella Rossetto, Eric A Johnson, Michela Rigoni, Cesare MontecuccoAbstract:Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE Proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated Membrane Protein (VAMP), a Protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid Membranes, with electrostatic interactions playing an important role.
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structural determinants of the specificity for synaptic vesicle associated Membrane Protein synaptobrevin of tetanus and botulinum type b and g neurotoxins
1996Co-Authors: Rossella Pellizzari, Ornella Rossetto, Luisa Lozzi, Silvia Giovedi, Eric A Johnson, Clifford C Shone, Cesare MontecuccoAbstract:Abstract Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic Vesicle-Associated Membrane Protein (VAMP)/synaptobrevin, an essential Protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated Protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by site-directed mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.
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vamp synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues
1996Co-Authors: Ornella Rossetto, Giampietro Schiavo, Luisa Gorza, N Schiavo, R H Scheller, Cesare MontecuccoAbstract:VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (Vesicle-Associated Membrane Protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP Proteins in the physiology of all cells.
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vesicle associated Membrane Protein 2 synaptobrevin 2 forms a complex with synaptophysin
1995Co-Authors: Philip Washbourne, Giampietro Schiavo, Cesare MontecuccoAbstract:Vesicle-Associated Membrane Protein (VAMP) (or synaptobrevin), a type II Membrane Protein of small synaptic vesicles, is essential for neuroexocytosis because its proteolysis by tetanus and botulinum neurotoxins types B, D, F and G blocks neurotransmitter release. The addition of cross-linking reagents to isolated small synaptic vesicles induces the formation of 30 and 50 kDa complexes containing the isoform 2 of VAMP (VAMP-2). Whereas the 30 kDa band is a VAMP-2 homodimer, the 50 kDa species results from the cross-linking of VAMP-2 with synaptophysin. This heterodimer also forms in detergent-solubilized vesicles and involves the N-terminal part of VAMP-2. The implications of the existence of a synaptophysin-VAMP-2 complex in the processes of vesicle docking and fusion with the presynaptic Membrane are discussed.
