Article: article from journal or magazin.
A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion.
Molecular Biology of the Cell
Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive 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 alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-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 alpha-SNAP potently inhibits Ca(2+)-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 alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
Amino Acid Motifs, Animals, Binding Sites, Calcium/pharmacology, Cell Membrane/drug effects, Cell Membrane/metabolism, Cell-Free System, Exocytosis/drug effects, Humans, Membrane Fusion/drug effects, Models, Biological, N-Ethylmaleimide-Sensitive Proteins/metabolism, PC12 Cells, Protein Conformation/drug effects, Protein Transport/drug effects, Rats, SNARE Proteins/chemistry, SNARE Proteins/metabolism, Secretory Vesicles/drug effects, Secretory Vesicles/metabolism, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism, Syntaxin 1/chemistry, Syntaxin 1/metabolism
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