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.     

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca²⁺-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms.     

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。     

A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.     

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.     

Single molecule mechanical probing of the SNARE protein interactions

Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction.     

SNAP23 Regulates Endothelial Exocytosis of von Willebrand Factor

Endothelial exocytosis regulates vascular thrombosis and inflammation. The trafficking and release of endothelial vesicles is mediated by SNARE (Soluble NSF Attachment protein REceptors) molecules, but the exact identity of endothelial SNAREs has been unclear. Three SNARE molecules form a ternary complex, including isoforms of the syntaxin (STX), vesicle-associated membrane protein (VAMP), and synaptosomal-associated protein (SNAP) families. We now identify SNAP23 as the predominant endothelial SNAP isoform that mediates endothelial exocytosis of von Willebrand Factor (VWF). SNAP23 was localized to the plasma membrane. Knockdown of SNAP23 decreased endothelial exocytosis, suggesting it is important for endothelial exocytosis. SNAP23 interacted with the endothelial exocytic machinery, and formed complexes with other known endothelial SNARE molecules. Taken together, these data suggest that SNAP23 is a key component of the endothelial SNARE machinery that mediates endothelial exocytosis.     

Intrinsic Disorder of the Neuronal SNARE Protein SNAP25a in its Pre-fusion Conformation

The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population < 25%). We speculate that the N-terminal helices (A5 to Q20 and S25 to V36) which constitute the N-terminus of SN1 act as a nucleation site for initiating SNARE zippering.     

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.     

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.     

Lipid raft association of SNARE proteins regulates exocytosis in PC12 cells

SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wild-type SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.     

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.     

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Septin Dynamics Are Essential for Exocytosis

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.     

Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.     

Sequential SNARE assembly underlies priming and triggering of exocytosis

Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibited exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibited exocytosis less effectively after priming. We propose that SNAREs partially and reversibly assemble during priming, and that the syntaxin H3 domain is prevented from fully joining the complex until the arrival of the Ca2+ trigger. Furthermore, we find that mutation of hydrophobic residues of the SNAP25 C-terminal coil that contribute to SNARE core interactions affects the maximal rate of exocytosis, while mutation of charged residues on the surface of the complex affects the apparent affinity of the coil domain for the partially assembled complex.     

Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.     

Vacuolar sequential exocytosis of large dense-core vesicles in adrenal medulla

Individual exocytic events in intact adrenal medulla were visualized by two-photon extracellular polar-tracer imaging. Exocytosis of chromaffin vesicles often occurred in a sequential manner, involving first vesicles located at the cell periphery and then those present deeper within the cytoplasm. Sequential exocytosis occurred preferentially at regions of the plasma membrane facing the intercellular space. The compound vesicles swelled to more than five times their original volume and formed vacuolar exocytic lumens as a result of expansion of intravesicular gels and their confinement within the lumen by the fusion pore and the narrow intercellular space. Such luminal swelling greatly promoted sequential exocytosis. The SNARE protein SNAP25 rapidly migrated from the plasma membrane to the membrane of fused vesicles. These data indicate that vesicles present deeper within the cytoplasm can be fusion ready like those at the cell periphery, and that swelling of exocytic lumens promotes assembly of the fusion machinery. We suggest the existence of two molecular configurations for fusion-ready states in Ca2+ -dependent exocytosis.     

Mechanism of neurotransmitter release coming into focus

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca²⁺‐triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca²⁺‐dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N‐ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin‐2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N‐ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18‐1 and Munc13‐1 orchestrate SNARE complex formation in an NSF‐SNAP‐resistant manner by a mechanism whereby Munc18‐1 binds to synaptobrevin and to a self‐inhibited “closed” conformation of syntaxin‐1, thus forming a template to assemble the SNARE complex, and Munc13‐1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin‐1. Synaptotagmin‐1 functions as the major Ca²⁺ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.