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Gwyn W. Gould - One of the best experts on this subject based on the ideXlab platform.
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tyrosine phosphorylation of munc18c on residue 521 abrogates binding to Syntaxin 4
BMC Biochemistry, 2011Co-Authors: Nia J Bryant, Veronica Aran, Gwyn W. GouldAbstract:Background Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4.
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negative regulation of Syntaxin4 snap 23 vamp2 mediated membrane fusion by munc18c in vitro
PLOS ONE, 2008Co-Authors: Fiona M Brandie, Nia J Bryant, Veronica Aran, Avani Verma, James A Mcnew, Gwyn W. GouldAbstract:Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of Syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings: Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by Syntaxin 4/ SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the Syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance: Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by Syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.
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the vesicle and target snare proteins that mediate glut4 vesicle fusion are localized in detergent insoluble lipid rafts present on distinct intracellular membranes
Journal of Biological Chemistry, 2002Co-Authors: Luke H Chamberlain, Gwyn W. GouldAbstract:Abstract Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and Syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of Syntaxin 4 (∼35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or Syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-β-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.
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the vesicle and target snare proteins that mediate glut4 vesicle fusion are localized in detergent insoluble lipid rafts present on distinct intracellular membranes
Journal of Biological Chemistry, 2002Co-Authors: Luke H Chamberlain, Gwyn W. GouldAbstract:Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and Syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of Syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or Syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.
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the synaptic vesicle protein cysteine string protein is associated with the plasma membrane in 3t3 l1 adipocytes and interacts with Syntaxin 4
Journal of Cell Science, 2001Co-Authors: Luke H Chamberlain, Robert D Burgoyne, Valerie H. Maier, M E Graham, Susan Kane, J L Jackson, Gwyn W. GouldAbstract:Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE Syntaxin 4. As Syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, Syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with Syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.
Debbie C Thurmond - One of the best experts on this subject based on the ideXlab platform.
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Munc18c depletion selectively impairs the sustained phase of insulin release. Diabetes 2009;58:1165–1174
2016Co-Authors: Debbie C ThurmondAbstract:implicated in Syntaxin 4–mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mecha-nism(s) using the islet -cell as a model system. RESEARCH DESIGN AND METHODS—Perifusion analyses of isolated Munc18c- (/) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 -cells to determine the mecha-nistic role of Munc18c in Syntaxin 4 activation and docking/ fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization
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Syntaxin 4 overexpression ameliorates effects of aging and high fat diet on glucose control and extends lifespan
Cell Metabolism, 2015Co-Authors: Richard A Miller, Debbie C ThurmondAbstract:Indirect evidence suggests that improved insulin sensitivity may contribute to improved lifespan of mice in which aging has been slowed by mutations, drugs, or dietary means, even in stocks of mice that do not show signs of late-life diabetes. Peripheral responses to insulin can be augmented by overexpression of Syntaxin 4 (Syn4), a plasma-membrane-localized SNARE protein. We show here that Syn4 transgenic (Tg) mice with high level expression of Syn4 had a significant extension of lifespan (33% increase in median) and showed increased peripheral insulin sensitivity, even at ages where controls exhibited age-related insulin resistance. Moreover, skeletal muscle GLUT4 and islet insulin granule exocytosis processes were fully protected in Syn4 Tg mice challenged with a high-fat diet. Hence, high-level expressing Syn4 Tg mice may exert better glycemic control, which slows multiple aspects of aging and extends lifespan, even in non-diabetic mice.
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Syntaxin 4 up regulation increases efficiency of insulin release in pancreatic islets from humans with and without type 2 diabetes mellitus
PMC, 2014Co-Authors: Natalie D Stull, Raghavendra G Mirmira, Debbie C ThurmondAbstract:Context: Evidence suggests that dysfunctional β-cell insulin release precedes type 1 and type 2 diabetes (T1D and T2D, respectively) and that enhancing the efficiency of insulin release from pancreatic islet β-cells may delay/prevent these diseases. We took advantage of the rare opportunity to test this paradigm using islets from human type 2 diabetic individuals. Objectives: Insulin release capacity is limited by the abundance of fusogenic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Because enrichment of Syntaxin 4, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, enhances β-cell function in mice, we investigated its potential to restore functional insulin secretion to human diabetic islets. Design: Human islets from type 2 diabetic and healthy individuals transduced to overexpress Syntaxin 4 were examined by perifusion analysis. Streptozotocin-induced diabetic recipient mice transplanted with Syntaxin 4-enriched ...
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munc18 1 regulates first phase insulin release by promoting granule docking to multiple Syntaxin isoforms
Journal of Biological Chemistry, 2012Co-Authors: Michael A Kalwat, Min Jung Kim, Matthijs Verhage, Debbie C ThurmondAbstract:Abstract Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet beta cells is coincident with Type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1+/- and beta-cell specific Munc18-1-/- knockout mice, we establish that Munc18-1 is required for the first-phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whilst cells with elevated levels of Munc18-1 exhibited a ~20% increase in pre-docked granule number. Pre-docked Syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in beta cell lysates, yet surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of Syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, Syntaxin 4 was requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for Syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with Syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating Syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.
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exocytosis mechanisms underlying insulin release and glucose uptake conserved roles for munc18c and Syntaxin 4
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2010Co-Authors: Jenna L Jewell, Debbie C ThurmondAbstract:Type 2 diabetes has been coined “a two-hit disease,” as it involves specific defects of glucose-stimulated insulin secretion from the pancreatic beta cells in addition to defects in peripheral tiss...
Thierry Galli - One of the best experts on this subject based on the ideXlab platform.
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transport of the major myelin proteolipid protein is directed by vamp3 and vamp7
The Journal of Neuroscience, 2011Co-Authors: Anke Feldmann, Jesa Amphornrat, Christine Winterstein, Madeleine Schonherr, Lydia Danglot, Torben Ruhwedel, Thierry Galli, Wiebke Mobius, Klaus-armin Nave, Dieter BrunsAbstract:CNS myelination by oligodendrocytes requires directed transport of myelin membrane components and a timely and spatially controlled membrane expansion. In this study, we show the functional involvement of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) proteins VAMP3/cellubrevin and VAMP7/TI-VAMP in myelin membrane trafficking. VAMP3 and VAMP7 colocalize with the major myelin proteolipid protein (PLP) in recycling endosomes and late endosomes/lysosomes, respectively. Interference with VAMP3 or VAMP7 function using small interfering RNA-mediated silencing and exogenous expression of dominant-negative proteins diminished transport of PLP to the oligodendroglial cell surface. In addition, the association of PLP with myelin-like membranes produced by oligodendrocytes cocultured with cortical neurons was reduced. We furthermore identified Syntaxin-4 and Syntaxin-3 as prime acceptor Q-SNAREs of VAMP3 and VAMP7, respectively. Analysis of VAMP3-deficient mice revealed no myelination defects. Interestingly, AP-3δ-deficient mocha mice, which suffer from impaired secretion of lysosome-related organelles and missorting of VAMP7, exhibit a mild dysmyelination characterized by reduced levels of select myelin proteins, including PLP. We conclude that PLP reaches the cell surface via at least two trafficking pathways with distinct regulations: (1) VAMP3 mediates fusion of recycling endosome-derived vesicles with the oligodendroglial plasma membrane in the course of the secretory pathway; (2) VAMP7 controls exocytosis of PLP from late endosomal/lysosomal organelles as part of a transcytosis pathway. Our in vivo data suggest that exocytosis of lysosome-related organelles controlled by VAMP7 contributes to myelin biogenesis by delivering cargo to the myelin membrane.
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v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells.
Journal of Cell Biology, 2007Co-Authors: Ian C. Fields, Véronique Proux-gillardeaux, Thierry Galli, Elina Shteyn, Marc Pypaert, Richard S. Kang, Heike FölschAbstract:The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE Syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with Syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.
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identification of snares involved in synaptotagmin vii regulated lysosomal exocytosis
Journal of Biological Chemistry, 2004Co-Authors: Swathi Rao, Veronique Prouxgillardeaux, Thierry Galli, Chau Huynh, Norma W AndrewsAbstract:Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by Syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and Syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, Syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of Syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.
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vimentin filaments in fibroblasts are a reservoir for snap23 a component of the membrane fusion machinery
Molecular Biology of the Cell, 2000Co-Authors: Wolfgang Faigle, Emma Colucciguyon, Daniel Louvard, Sebastian Amigorena, Thierry GalliAbstract:Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with Syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.
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soluble nsf attachment protein receptors snares in rbl 2h3 mast cells functional role of Syntaxin 4 in exocytosis and identification of a vesicle associated membrane protein 8 containing secretory compartment
Journal of Immunology, 2000Co-Authors: Fabienne Paumet, Thierry Galli, Joelle Le Mao, Sophie Martin, B David, Ulrich Blank, Michele RoaAbstract:Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the Syntaxin family, Syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of Syntaxin 4, but not of Syntaxin 2 nor Syntaxin 3, caused inhibition of FceRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that Syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.
Hiromi Shimomura - One of the best experts on this subject based on the ideXlab platform.
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characterization of cysteine string protein in rat parotid acinar cells
Archives of Biochemistry and Biophysics, 2013Co-Authors: Hiromi Shimomura, Akane Imai, Tomoko NashidaAbstract:Abstract Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70 kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with Syntaxin 4, but not with Syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11–82, containing the J-domain only, nor GST-CSP183–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.
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roles of munc18 3 in amylase release from rat parotid acinar cells
Archives of Biochemistry and Biophysics, 2004Co-Authors: Akane Imai, Tomoko Nashida, Hiromi ShimomuraAbstract:Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, Syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and Syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with Syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.
Luke H Chamberlain - One of the best experts on this subject based on the ideXlab platform.
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the vesicle and target snare proteins that mediate glut4 vesicle fusion are localized in detergent insoluble lipid rafts present on distinct intracellular membranes
Journal of Biological Chemistry, 2002Co-Authors: Luke H Chamberlain, Gwyn W. GouldAbstract:Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and Syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of Syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or Syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.
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the vesicle and target snare proteins that mediate glut4 vesicle fusion are localized in detergent insoluble lipid rafts present on distinct intracellular membranes
Journal of Biological Chemistry, 2002Co-Authors: Luke H Chamberlain, Gwyn W. GouldAbstract:Abstract Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and Syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of Syntaxin 4 (∼35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or Syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-β-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.
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the synaptic vesicle protein cysteine string protein is associated with the plasma membrane in 3t3 l1 adipocytes and interacts with Syntaxin 4
Journal of Cell Science, 2001Co-Authors: Luke H Chamberlain, Robert D Burgoyne, Valerie H. Maier, M E Graham, Susan Kane, J L Jackson, Gwyn W. GouldAbstract:Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE Syntaxin 4. As Syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, Syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with Syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.
