Productive Hemifusion Intermediates in Fast Vesicle Fusion Driven by Neuronal SNAREs

¹ U. California-Berkeley
² U. Wisconsin-Madison

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

Submitted on March 1, 2007
Revised on May 22, 2007
Accepted on 12 September 2007

	Abstract

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 POPC in the v-SNARE vesicle with 0 or 20% DOPE 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 are 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 %DOPE. This suggests similar underlying molecular pathways for protein-free and neuronal SNARE-driven fusion. Removal of DOPS from the v-SNARE vesicle has no effect on docking or fusion.

Key Words: hemifusion, in vitro fusion assay, lipids, membrane, neuronal SNAREs, single-vesicle