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Productive Hemifusion Intermediates in Fast Vesicle Fusion Driven by Neuronal SNAREs

Tingting Liu ^*, Tingting Wang ^*, Edwin R. Chapman  and James C. Weisshaar ^*

^* Departments of Chemistry and Howard Hughes Medical Institute and Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin

Correspondence: Address reprint requests to James C. Weisshaar, E-mail: weisshaar{at}chem.wisc.edu.

An in vitro fusion assay uses fluorescence microscopy of labeled lipids to monitor single v-SNARE vesicle docking and fusion events on a planar lipid bilayer containing t-SNAREs. For vesicles and bilayer comprising phosphatidylcholine (POPC, 84–85% by mol) and phosphatidylserine (DOPS, 15% by mol), previous work demonstrated prompt, full fusion (_fus = 25 ms). Substitution of 20–60% phosphatidylethanolamine (DOPE) for phosphatidylcholine in the v-SNARE vesicle with either 0 or 20% DOPE included in the t-SNARE bilayer gives rise to hemifusion events. Labeled lipids diffuse into the planar bilayer as two temporally distinct waves, presumably hemifusion of the outer leaflet followed by inner leaflet (core) fusion. The fusion kinetics with DOPE is markedly heterogeneous. Some vesicle/docking site pairs exhibit prompt, full fusion while others exhibit hemifusion. Hemifusion events are roughly half productive (leading to subsequent core fusion within 20 s) and half dead-end. In qualitative accord with expectations from studies of protein-free vesicle-vesicle fusion, the hemifusion rate k_hemi is 15–20 times faster than the core fusion rate k_core, and the fraction of hemifusion events increases with increasing percentage of DOPE. This suggests similar underlying molecular pathways for protein-free and neuronal SNARE-driven fusion. Removal of phosphatidylserine from the v-SNARE vesicle has no effect on docking or fusion.