A Novel Site of Action for -SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of -SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of -SNAP in clusters on plasma membranes. Clustering is mediated by the binding of -SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of -SNAP potently inhibits Ca2 -dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an -SNAP mutant defective in NSF activation is used. We conclude that -SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for -SNAP in the SNARE cycle that drives exocytotic membrane fusion.