SNARE-driven, 25-millisecond vesicle fusion in vitro

¹ Dept. of Chemistry, U. Wisconsin-Madison
² Department of Physiology, U. Wisconsin-Madison
³ Dept. of Physiology, U. Wisconsin-Madison

^* To whom correspondence should be addressed. E-mail: weisshaar{at}chem.wisc.edu.

Submitted on March 9, 2005
Revised on April 22, 2005
Accepted on 14 June 2005

	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 SNAP-25) was observed in real time by widefield fluorescence microscopy. This enabled separate measurement of the docking rate kdock and the unimolecular fusion rate kfus for the first time. On low t-SNARE-density bilayers at 37 °C, docking is efficient: kdock = 2.2 x 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). About 80% of the docked vesicles fuse promptly as a homogeneous sub-population with kfus = 40 ± 15 s-1 (fus = 25 ms). This is 103-104 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 SNAP-25. At present in vitro fusion driven by SNARE complexes alone remains ~40 times slower than the fastest, sub-ms pre-synaptic vesicle population response.

Key Words: SNARE complex, in vitro fusion, vesicle docking, vesicle fusion

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