A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

nSec1 binds a closed conformation of syntaxin1A

The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.     

Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a

SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.     

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.     

Amisyn,a novel syntaxin-binding protein that may regulate SNARE complex assembly

The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

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.     

VAMP3 is associated with endothelial weibel-palade bodies and participates in their Ca(2+)-dependent exocytosis

Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca(2+)-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca(2+)-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.     

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

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 FcepsilonRI-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.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Vesicle-associated membrane protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.     

VAMP3 is associated with endothelial Weibel-Palade bodies and participates in their Ca-dependent exocytosis

Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca²⁺-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca²⁺-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Pancreatic acinar cells express vesicle-associated membrane protein 2- and 8-specific populations of zymogen granules with distinct and overlapping roles in secretion

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca(2+)-regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca(2+)-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca(2+)-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca(2+)-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca(2+)-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca(2+)-regulated secretory pathways.     

Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.     

A Highlights from MBoC Selection: Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.