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Vol. 19, Issue 3, 776-784, March 2008

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

Marcin Barszczewski^*, John J. Chua^*, Alexander Stein^*, Ulrike Winter^*, Rainer Heintzmann^,, Felipe E. Zilly^*,, Dirk Fasshauer^*, Thorsten Lang^*,||, and Reinhard Jahn^*

Departments of *Neurobiology and Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany

Submitted May 25, 2007; Revised October 24, 2007; Accepted December 10, 2007
Monitoring Editor: Adam Linstedt

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 -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.

Present addresses: King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London SE1 1UL, United Kingdom;

Department of Synthetic Organic Chemistry, Max-Planck-Institute for Coal Research, 45470 Mülheim an der Ruhr, Germany;

^|| LIMES-Institute, Laboratory for Membrane Biochemistry, University of Bonn, 53115 Bonn, Germany.

Address correspondence to: Reinhard Jahn (rjahn{at}gwdg.de)