Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

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.Membrane fusion requires three proteins, syntaxin, SNAP-25, and synaptobrevin, known as SNAREs 1 (1-3). All three SNAREs are molecular targets for clostridial proteolytic toxins that potently block release of neurotransmitters (4, 5). Syntaxin and SNAP-25 reside in the plasma membrane, whereas synaptobrevin is present on the synaptic vesicle membrane. Whereas SNAP-25 associates with the plasma membrane via its palmitate chains (6), syntaxin and synaptobrevin each have one transmembrane region (TMR) (7,8). Formation of SNARE complex, composed of the cytoplasmic parts of all three SNAREs, is followed by the interaction of the complementary TMRs of syntaxin and synaptobrevin, finally mixing the opposing membranes and causing them to fuse (3, 9). SNARE-mediated synaptic vesicle exocytosis is a highly regulated event that is under the control of a number of cytosolic proteins (10 -12). Among these, complexin is unique in that it is exclusively present in neuronal cells (13)(14)(15). Gene knockout studies in mice and peptide injection studies in squid nerve terminals demonstrated that complexin positively regulates a late step in calcium-triggered exocytosis (16, 17) which, according to current understanding, is zippering of the four-helix bundle made of syntaxin, SNAP-25, and synaptobrevin (9). Interestingly, a number of human neurological diseases have also been linked to deficiency in complexin (18,19).Complexin is a small highly charged cytosolic protein that interacts selectively with the ternary SNARE complex but not with monomeric SNARE proteins (20,21). X-ray structures demonstrated that complexin binds to ...

Three evolutionarily conserved proteins known as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) mediate exocytosis from single cell eukaryotes to neurons. Among neuronal SNAREs, syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) reside on the plasma membrane, whereas synaptobrevin resides on synaptic vesicles prior to fusion. The SNARE motifs of the three proteins form a helical bundle which probably drives membrane fusion. Since studies in vivo suggested an importance for multiple SNARE complexes in the fusion process, and models appeared in the literature with large numbers of SNARE bundles executing the fusion process, we analysed the quaternary structure of the full-length native SNARE complexes in detail. By employing a preparative immunoaffinity procedure we isolated all of the SNARE complexes from brain, and have shown by size-exclusion chromatography and negative stain electron microscopy that they exist as approx. 30 nm particles containing, most frequently, 3 or 4 bundles emanating from their centre. Using highly purified, individual, full-length SNAREs we demonstrated that the oligomerization of SNAREs into star-shaped particles with 3 to 4 bundles is an intrinsic property of these proteins and is not dependent on other proteins, as previously hypothesized. The average number of the SNARE bundles in the isolated fusion particles corresponds well with the co-operativity observed in calcium-triggered neuronal exocytosis.

The SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin-synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.