Determinants of synaptobrevin regulation in membranes

Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion.     

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.     

NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin

Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin is largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex 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.     

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.     

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.     

SNARE complexes and neuroexocytosis: how many, how close?

Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a SNARE complex formed by the coil-coiling of three proteins: SNAP-25, syntaxin and VAMP/synaptobrevin. It seems that SNARE complexes assemble together in rosette-shaped super-complexes but there is controversy on the actual number (N) of copies of SNARE complexes that are necessary to mediate exocytosis. We discuss attempts to determine the value of N and suggest that N varies with the type of exocytic vesicles. In addition, we propose that the N value in neuroexocytosis can be estimated by the comparative use of different types of botulinum neurotoxins.     

A Highly Tilted Membrane Configuration for the Prefusion State of Synaptobrevin

The SNARE complex plays a vital role in vesicle fusion arising during neuronal exocytosis. Key components in the regulation of SNARE complex formation, and ultimately fusion, are the transmembrane and linker regions of the vesicle-associated protein, synaptobrevin. However, the membrane-embedded structure of synaptobrevin in its prefusion state, which determines its interaction with other SNARE proteins during fusion, is largely unknown. This study reports all-atom molecular-dynamics simulations of the prefusion configuration of synaptobrevin in a lipid bilayer, aimed at characterizing the insertion depth and the orientation of the protein in the membrane, as well as the nature of the amino acids involved in determining these properties. By characterizing the structural properties of both wild-type and mutant synaptobrevin, the effects of C-terminal additions on tilt and insertion depth of membrane-embedded synaptobrevin are determined. The simulations suggest a robust, highly tilted state for membrane-embedded synaptobrevin with a helical connection between the transmembrane and linker regions, leading to an apparently new characterization of structural elements in prefusion synaptobrevin and providing a framework for interpreting past mutation experiments.     

A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins

Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.     

Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis

Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.     

A Highly Tilted Membrane Configuration for the Prefusion State of Synaptobrevin

The SNARE complex plays a vital role in vesicle fusion arising during neuronal exocytosis. Key components in the regulation of SNARE complex formation, and ultimately fusion, are the transmembrane and linker regions of the vesicle-associated protein, synaptobrevin. However, the membrane-embedded structure of synaptobrevin in its prefusion state, which determines its interaction with other SNARE proteins during fusion, is largely unknown. This study reports all-atom molecular-dynamics simulations of the prefusion configuration of synaptobrevin in a lipid bilayer, aimed at characterizing the insertion depth and the orientation of the protein in the membrane, as well as the nature of the amino acids involved in determining these properties. By characterizing the structural properties of both wild-type and mutant synaptobrevin, the effects of C-terminal additions on tilt and insertion depth of membrane-embedded synaptobrevin are determined. The simulations suggest a robust, highly tilted state for membrane-embedded synaptobrevin with a helical connection between the transmembrane and linker regions, leading to an apparently new characterization of structural elements in prefusion synaptobrevin and providing a framework for interpreting past mutation experiments.     

Ternary SNARE complexes in parallel versus anti-parallel orientation: Examination of their disassembly using single-molecule force spectroscopy

Interactions between the proteins of the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, synaptobrevin 2 (Sb2), syntaxin 1A (Sx1A) and synaptosome-associated protein of 25kDa (SNAP25) can be readily assessed using force spectroscopy single-molecule measurements. We studied interactions during the disassembly of the ternary SNARE complex pre-formed by binding Sb2 in parallel or anti-parallel orientations to the binary Sx1A-SNAP25B acceptor complex. We determined the spontaneous dissociation lifetimes and found that the stability of the anti-parallel ternary SNARE complex is ～1/3 less than that of the parallel complex. While the free energies were very similar, within 0.5 k(B)T, for both orientations, the enthalpy changes (42.1 k(B)T and 39.8 k(B)T, for parallel and anti-parallel orientations, respectively) indicate that the parallel ternary complex is energetically advantageous by 2.3 k(B)T. Indeed, both ternary SNARE complex orientations were much more stable (by ～4-13 times) and energetically favorable (by ～9-13 k(B)T) than selected binary complexes, constituents of the ternary complex, in both orientations. We propose a model which considers the geometry for the vesicle approach to the plasma membrane with favorable energies and stability as the basis for preferential usage of the parallel ternary SNARE complex in exocytosis.     

Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca²⁺-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.     

v-SNARE actions during Ca(2+)-triggered exocytosis

Assembly of SNARE proteins between opposing membranes mediates fusion of synthetic liposomes, but it is unknown whether SNAREs act during exocytosis at the moment of Ca(2+) increase, providing the molecular force for fusion of secretory vesicles. Here, we show that execution of pre- and postfusional steps during chromaffin granule exocytosis depends crucially on a short molecular distance between the complex-forming SNARE motif and the transmembrane anchor of the vesicular SNARE protein synaptobrevin II. Extending the juxtamembrane region of synaptobrevin by insertion of flexible "linkers" reduces priming of granules, delays initiation of exocytosis upon stepwise elevation of intracellular calcium, attenuates fluctuations of early fusion pores, and slows rapid expansion of the pore in a linker-length dependent fashion. These observations provide evidence that v-SNARE proteins drive Ca(2+)-triggered membrane fusion at millisecond time scale and support a model wherein continuous molecular pulling by SNAREs guides the vesicle throughout the consecutive stages of exocytosis.     

Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

During exocytosis a four-helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C-terminal end affected fusion triggering in vivo and led to two-step unfolding of the SNARE complex in vitro, indicating that the C-terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C-terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N-terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N- to C-terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.     

Stability profile of the neuronal SNARE complex reflects its potency to drive fast membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form the SNARE complex to mediate most fusion events of the secretory pathway. The neuronal SNARE complex is featured by its high stability and half-zippered conformation required for driving robust and fast synaptic exocytosis. However, these two features seem to be thermodynamically mutually exclusive. In this study, we have employed temperature-dependent disassociation assays and single-molecule Förster resonance energy transfer (FRET) experiments to analyze the stability and conformation of the neuronal SNARE complex. We reclassified the amino acids of the SNARE motif into four sub-groups (core, core-side I and II, and non-contact). Our data showed that the core residues predominantly contribute to the complex stability to meet a basal requirement for SNARE-mediated membrane fusion, while the core-side residues exert an unbalanced effect on the N- and C-half bundle stability that determines the half-zippered conformation of the neuronal SNARE complex, which would accommodate essential regulations by complexins and synaptotagmins for fast Ca²⁺-triggered membrane fusion. Furthermore, our data confirmed a strong coupling of folding energy between the N- and C-half assembly of the neuronal SNARE complex, which rationalizes the strong potency of the half-zippered conformation to conduct robust and fast fusion. Overall, these results uncovered that the stability profile of the neuronal SNARE complex reflects its potency to drive fast and robust membrane fusion. Based on these results, we also developed a new parameter, the stability factor (F_s), to characterize the overall stability of the neuronal SNARE complex and resolved a linear correlation between the stability and inter-residue coulombic interactions of the neuronal SNARE complex, which would help rationally design artificial SNARE complexes and remold functional SNARE complexes with desirable stability.     

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.     

Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly

N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.     

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。     

High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.