Abstract
SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion in neuronal exocytosis and intracellular membrane trafficking. It is widely accepted that the zippering interaction between syntaxin1a, SNAP25 and synaptobrevin2 brings plasma- and vesicle membrane together and leads to their merger. Membrane fusion has been extensively studied by liposome and single vesicle-planar membrane fusion assays. However, both approaches have been criticized because of slow fusion rates incompatible with cellular rates or lack of physiological specificity, respectively. Here we present a fluorescence-basedsingle vesicle-planar bilayer fusion assay with millisecond time resolution and an improved reconstitution procedure that preserves the native topology and mobility of the SNAREs. The acceptor-SNARE complex, composed of syntaxin1a (SyxH3), SNAP25 and a short synaptobrevin2 peptide (Syb49-96), was reconstituted into planar bilayers by a combined Langmuir-Blodgett - vesicle fusion technique. Docking and fusion of single Rh-DOPE labeled Syb vesicles to the supported acceptor-SNARE membranes were observed by total internal reflection fluorescence microscopy. No docking or fusion was observed in protein-free control membranes. Vesicle SNARE docking was dependent on the acceptor-SNARE complex density in the membranes. Moreover, docking was SNAP25-dependent, and subsequent fusion did not require Ca2+ and was efficient at ambient temperature. A detailed kinetic analysis of >1000 single fusion events revealed that each fusion reaction consists of 6 to 9 activating steps with 8 steps fitting the data best. This could be interpreted by fusion sites consisting of 8 SNARE complexes that each activate in a single rate-limiting step in 8 ms. We find that different lipid compositions of the supported and the vesicle membrane have a relatively minor influence on docking, but modulate the fusion efficiency and fusion kinetics more dramatically. Supported by NIH grant GM072694
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