Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

Abstract

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimidesensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.Synaptobrevin (also referred to as VAMP), SNAP-25, and syntaxin are crucial components of the exocytotic apparatus in neurons (1-4). Synaptobrevin is exclusively localized to synaptic vesicles whereas syntaxin and SNAP-25 are mainly localized to the neuronal plasma membrane. Any interference with the function of these proteins, e.g., proteolysis by clostridial neurotoxins (5, 6) or genetic deletion (7), inhibits exocytotic neurotransmitter release. Relatives of these proteins participate in many intracellular membrane traffic steps in all eukaryotic cells (3), suggesting that membrane fusion is mediated by a common and conserved mechanism.Despite compelling evidence linking syntaxin, SNAP-25, and synaptobrevin to exocytosis, it is not understood how these proteins operate in the sequence of events that leads to vesicle docking and membrane fusion. In detergent extracts, the three proteins form a stable complex that binds the soluble proteins ␣͞␤͞␥-SNAP and N-ethylmaleimide-sensitive factor (NSF), leading to their designation as v-SNAREs (synaptobrevin, vesicular SNAp-REceptors) and t-SNAREs (syntaxin and SNAP-25, target membrane SNAp REceptor) (8, 9). ATPhydrolysis by NSF causes disassembly of the complex. A complex with very similar properties also can be formed from recombinant proteins lacking their transmembrane domains (10-12). Based on these, observations it has been suggested that v-SNARE-t-SNARE interactions are responsible for docking of vesicles at their target membrane and that the conformational change caused by NSF contributes to membrane fusion (1, 9). The specificity of v-SNARE-t-SNARE pairing would ensure that trafficking vesicles only dock at an appropriate target membrane. These proposals are referred to as the SNARE hypothesis (1) and have gained wide acceptance in the field (1-3).Recently, we have observed that, unlike other residents of the presynaptic plasma membrane, significant amounts of syntaxin and SNAP-25 are also localized to synaptic vesicles (13). Using two independent and complementary approaches, we now report that a ternary complex containing syntaxin, synaptobrevin, and SNAP-25 can form in the membrane of synaptic vesicles in the absence of plasm...

Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

“…Recent studies from our laboratory have shown that a ternary complex from native proteins can assemble with all three proteins residing as neighbors in a single membrane (29). However, the bulk of syntaxin and SNAP-25 is localized to the plasma membrane and synaptobrevin is almost exclusively localized to the membrane of synaptic vesicles (2).…”

Reduced aspartate release from rat hippocampal synaptosomes loaded with Clostridial toxin light chain by electroporation: Evidence for an exocytotic mechanism

“…None completely inhibited aspartate or glutamate release. Incomplete inhibition by the toxin light chains was expected, however, because SNARE proteins already complexed are inaccessible to them [14,21]. The quantitatively different effects of toxin light chains that hydrolyze the same SNARE protein can be explained by differences in affinity and kinetics.…”

N-Ethylmaleimide-sensitive Fusion Protein Contains High and Low Affinity ATP-binding Sites That Are Functionally Distinct

“…In this manner, NSF could be thought to act as a molecular chaperone (32), using ATP hydrolysis to modulate the structure of the SNARE proteins, much like HSP-90 appears to act on steroid receptors (33). Recently it has also been shown that 20 S complexes are present on vesicle membranes, suggesting that the v-and tSNAREs can bind each other in the same membrane (34,35). From this data, it seems possible that the NSF-mediated disassembly may in fact be needed to disengage these complexes formed in the same membrane so that they may bind to the SNAREs on opposing membranes.…”