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. INTRODUCTIONCommunication between neurons is mediated by neurotransmitters that are released from presynaptic nerve endings by Ca 2ϩ -dependent exocytosis of synaptic vesicles. Exocytotic fusion of the vesicle with the synaptic plasma membrane is mediated by the proteins synaptobrevin 2/VAMP2, SNAP-25, and syntaxin 1 (Jahn and Scheller, 2006;Rizo et al., 2006). These proteins are members of the SNARE protein family that are involved in all fusion events of the secretory pathway. SNAREs are characterized by stretches of 60-70 amino acids arranged in heptad repeats, termed SNARE motifs (Weimbs et al., 1997;Fasshauer et al., 1998b;Bock et al., 2001;Day et al., 2006). Syntaxin and synaptobrevin each contain a single SNARE motif that is located adjacent to a C-terminal transmembrane domain. In contrast, SNAP-25 contains two SNARE motifs connected by a palmitoylated linker region that serves as membrane anchor.Synaptobrevin resides in synaptic vesicles, whereas SNAP-25 and syntaxin 1 reside in the plasma membrane. The SNARE motifs of syntaxin, SNAP-25, and synaptobrevin readily assemble into quarternary bundles of ␣-helices (Fasshauer et al., 1997;Sutton et al., 1998). Assembly would thus lead to a tight connection between the membranes. According to this view, assembly is nucleated at the Nterminal ends of the SNARE-motifs and proceeds toward the C-terminal membrane anchor ("zippering"), resulting in a strained "trans"-complex . During membrane merger, the trans-complex would relax into a "cis"-complex in which the transmembrane domains are aligned in parallel. To regenerate the SNAREs for another round of fusion, SNARE complexes need to be disassembled by the AAAϩ-ATPase NEM-sensitive factor (NSF) in conjunction with cofactors termed soluble NSF attachment proteins (SNAPs; Sollner et al., 1993).Although the "zippering" hypothesis of SNARE function has received a lot of experimental support, it is still unclear...

Snapin, a 15-kDa protein, has been identified recently as a binding partner of SNAP-25. Moreover, snapin is regulated by phosphorylation and enhances synaptotagmin binding to SNAREs. Furthermore, snapin and Cterminal snapin fragments have been effective in changing the release properties of neurons and chromaffin cells. Here we have reinvestigated the role of snapin using both biochemical and electrophysiological approaches. Snapin is ubiquitously expressed at low levels with no detectable enrichment in the brain or in synaptic vesicles. Using non-equilibrium and equilibrium assays including pulldown experiments, co-immunoprecipitations, and CD and fluorescence anisotropy spectroscopy, we were unable to detect any specific interaction between snapin and SNAP-25. Similarly, overexpression of a C-terminal snapin fragment in hippocampal neurons failed to influence any of the analyzed parameters of neurotransmitter release. Initial biochemical characterization of recombinant snapin revealed that the protein is a stable dimer with a predominantly ␣-helical secondary structure. We conclude that the postulated role of snapin as a SNARE regulator in neurotransmitter release needs reconsideration, leaving the true function of this evolutionarily conserved protein to be discovered.Neurons release neurotransmitters by exocytosis of synaptic vesicles. Exocytotic membrane fusion is mediated by SNARE 1 proteins including the vesicle protein synaptobrevin (also referred to as VAMP) and the plasma membrane proteins SNAP-25 and syntaxin. Assembly of SNARE complexes is currently thought to pull the vesicle and plasma membrane close together and to initiate the fusion reaction (1).Because of the central role of SNAREs in fusion reactions, major efforts have been made to understand the molecular mechanisms by which these proteins work. According to the emerging picture, SNARE proteins undergo a complex assembly-disassembly cycle during the fusion reaction that is associated with major conformational changes (2). Essential for the conformational cycle are the SNARE motifs, stretches of 60 -70 amino acids that are characteristic of all SNARE proteins. They are unstructured as monomers but form extended helical bundles of extraordinary stability upon oligomerization. These complexes are disassembled by the chaperone-like ATPase NSF in conjunction with cofactors (3).More than three dozen proteins have been identified that are supposed to regulate the activity of neuronal SNAREs by virtue of binding either to individual SNAREs or to partially or fully assembled SNARE complexes. In some cases, e.g. complexin and Munc-18, the interaction between regulatory proteins and SNAREs is structurally well defined (4), leading to the development of models explaining how and at which state these proteins regulate the SNARE assembly-disassembly cycle. For most of the other reported binding proteins, however, it remains to be established to which conformational state of the SNAREs they bind and consequently how exactly they exert their presumed contro...

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.

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In our study, the publication by Fix et al. (2004. Proc. Natl. Acad. Sci. USA. 101:7311-7316) was erroneously quoted as providing evidence that a binary interaction between synaptobervin and syntaxin is sufficient for docking and subsequent fusion of artificial vesicles to planar lipid bilayers. This is incorrect. Fix et al. show rapid fusion of vesicles containing synaptobrevin/VAMP with planar membranes containing both syntaxin and SNAP-25.

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