A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion.
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.
Barszczewski
Marcin
M
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Chua
John J
JJ
Stein
Alexander
A
Winter
Ulrike
U
Heintzmann
Rainer
R
Zilly
Felipe E
FE
Fasshauer
Dirk
D
Lang
Thorsten
T
Jahn
Reinhard
R
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System; D005089:Exocytosis / Q000187:drug effects; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D008954:Models, Biological; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism; D016716:PC12 Cells; D011487:Protein Conformation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism*; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism*; D050827:Syntaxin 1 / Q000737:chemistry / Q000378:metabolism
D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D052066:N-Ethylmaleimide-Sensitive Proteins; D002118:Calcium