title: Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay.
abstract: In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the V(C) peptide synaptobrevin-2 (syb(57-92)) increases the docking efficiency by a factor of ∼30, but docking remains rate-limiting. In the presence of V(C) peptide, the fusion step occurs on a timescale of ∼10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca(2+), Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step.
authors.affiliation: Graduate Program in Molecular Biophysics, University of Wisconsin, Madison, Wisconsin, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Smith
authors.forename: James C
authors.initials: JC
authors.lastname: Weisshaar
mesh_terms: D001681:Biological Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D007834:Lasers; D008561:Membrane Fusion / Q000187:drug effects; D008954:Models, Biological; D010455:Peptides / Q000494:pharmacology; D050600:SNARE Proteins / Q000378:metabolism*; D013997:Time Factors; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000494:pharmacology
chemical_list: D010455:Peptides; D050600:SNARE Proteins; D050988:Vesicle-Associated Membrane Protein 2