An activated Q‐SNARE/SM protein complex as a possible intermediate in SNARE assembly

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.     

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Munc13‐1 is crucial for neurotransmitter release and, together with Munc18‐1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin‐1, SNAP‐25, and synaptobrevin. Assembly starts with syntaxin‐1 folded into a self‐inhibited closed conformation that binds to Munc18‐1. Munc13‐1 is believed to catalyze the opening of syntaxin‐1 to facilitate SNARE complex formation. However, different types of Munc13‐1‐syntaxin‐1 interactions have been reported to underlie this activity, and the critical nature of Munc13‐1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13‐1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin‐1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13‐1 fragments, even though binding of this linker region to Munc13‐1 is barely detectable. Conversely, the syntaxin‐1 SNARE motif clearly binds to Munc13‐1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13‐1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13‐1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin‐1 via interactions with the linker.     

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.     

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.     

Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.     

神经末梢突触囊泡释放神经递质过程的调控蛋白

神经末梢突触囊泡释放神经递质是一个复杂且受到精细调控的过程,涉及多种蛋白质间的相互作用。位于突触囊泡膜上的突触囊泡蛋白／突触囊泡相关膜蛋白（synaptobrevin/VAMP),与位于突触前膜上的syntaxin和突触小体相关蛋白SNAP－25,三者聚合形成的可溶性N－甲基马来酰胺敏感因子（NSF）附着蛋白受体（SNARE）核心复合物是突触囊泡胞吐过程中的核心成分。本文主要围绕参与空触囊泡胞吐过程,以及调节SNARE核心复合物的形成,解离及其功能的蛋白质,并对突触囊泡胞吐过程的分子模型作一概述。     

Synaptobrevin N-terminally bound to syntaxin–SNAP-25 defines the primed vesicle state in regulated exocytosis

Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)–dependent exocytosis pathway at an intermediate “cocked” state, from which fusion can be triggered by Ca²⁺. It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin–SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca²⁺-triggered C-terminal assembly and membrane fusion.     

Structure and Dynamics of a Two‐Helix SNARE Complex in Live Cells

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q_a‐, Q_b‐, Q_c‐ and R‐SNARE‐motif form a complex. The core complex represents a Q_aQ_bQ_cR‐SNARE‐motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)‐studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q_a), SNAP25 (Q_b/Q_c) and synaptobrevin 2 (R)]. We find that the Q_b‐SNARE‐motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q_b‐SNARE‐motif and depends on its position within the molecule. We conclude that a zippered Q_aQ_b‐SNARE complex represents a short‐lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.     

Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca²⁺-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1–SNARE–complexin macromolecular assembly that is poised for Ca²⁺ triggering of fusion.     

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.     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

The SNARE protein vti1a functions in dense‐core vesicle biogenesis

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans‐Golgi network, partially overlapping with syntaxin‐6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca²⁺‐channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin‐2 content. In contrast, release kinetics and Ca²⁺‐sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long‐term re‐expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short‐term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca²⁺‐channel trafficking, but is dispensable for transmitter release.     

Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.     

Mechanism of arachidonic acid action on syntaxin-Munc18

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.     

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.     

S-nitrosylation of syntaxin 1 at Cys(145) is a regulatory switch controlling Munc18-1 binding

Exocytosis is regulated by NO in many cell types, including neurons. In the present study we show that syntaxin 1a is a substrate for S-nitrosylation and that NO disrupts the binding of Munc18-1 to the closed conformation of syntaxin 1a in vitro. In contrast, NO does not inhibit SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor} complex formation or binding of Munc18-1 to the SNARE complex. Cys(145) of syntaxin 1a is the target of NO, as a non-nitrosylatable C145S mutant is resistant to NO and novel nitrosomimetic Cys(145) mutants mimic the effect of NO on Munc18-1 binding in vitro. Furthermore, expression of nitrosomimetic syntaxin 1a in living cells affects Munc18-1 localization and alters exocytosis release kinetics and quantal size. Molecular dynamic simulations suggest that NO regulates the syntaxin-Munc18 interaction by local rearrangement of the syntaxin linker and H3c regions. Thus S-nitrosylation of Cys(145) may be a molecular switch to disrupt Munc18-1 binding to the closed conformation of syntaxin 1a, thereby facilitating its engagement with the membrane fusion machinery.     

Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.

According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.     

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.     

Interaction of the Complexin Accessory Helix with the C-Terminus of the SNARE Complex: Molecular-Dynamics Model of the Fusion Clamp

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx^3-69Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.