SNARE-driven, 25-millisecond vesicle fusion in vitro

Abstract

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate kdock and the unimolecular fusion rate kfus. On low t-SNARE-density bilayers at 37°C, docking is efficient: k dOCk = 2.2 × 107 M-1 s-1, ∼40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (Dlipid = 0.6 μm2 s-1); ∼80% of the docked vesicles fuse promptly as a homogeneous subpopulation with kfus = 40 ± 15 s -1 (τfus = 25 ms). This is 103-10 4 times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains ∼40 times slower than the fastest, submillisecond presynaptic vesicle population response. © 2005 by the Biophysical Society.