Reconstituting Basic Steps of Synaptic Vesicle Fusion

Abstract

Neurotransmitter release depends critically on: the SNAREs syntaxin-1, synaptobrevin and SNAP-25, which form SNARE complexes that bridge the vesicle and plasma membranes; NSF/SNAPs, which disassemble SNARE complexes; Munc18-1, which binds to syntaxin-1 and, together with Munc13, orchestrates SNARE-complex assembly; and the Ca2+ sensor synaptotagmin-1. Previous attempts to reconstitute neurotransmitter release revealed efficient fusion of syntaxin-1/SNAP-25-liposomes with synaptobrevin-liposomes in the presence synaptotagmin-1/Ca2+, in stark contrast with physiological data showing that Munc18-1 and Munc13 are essential for neurotransmitter release. We now solve this paradox, showing that Munc18-1 displaces SNAP-25 from syntaxin-1 and that syntaxin-1/Munc18-1-liposomes fuse efficiently with synaptobrevin-liposomes in a manner that requires SNAP-25, Munc13-1 and synaptotagmin-1/Ca2+. Moreover, when starting with syntaxin-1/SNAP-25-liposomes, NSF/a-SNAP disassemble the syntaxin-1/SNAP-25 heterodimers, thus inhibiting fusion, and fusion then requires Munc18-1 and Munc13-1. These results suggest that, for the first time, our experiments reconstitute synaptic vesicle fusion with the eight major components of the release machinery. We propose a model whereby the pathway to synaptic vesicle fusion does not proceed through syntaxin-1/SNAP-25 heterodimers and starts at the syntaxin-1/Munc18-1 complex; Munc18-1 and Munc13 then orchestrate membrane fusion together with the SNAREs, synaptotagmin-1 and Ca2+ in a manner that is not inhibited by NSF/SNAPs.