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MBC in Press, published online ahead of print December 19, 2007
Mol. Biol. Cell 10.1091/mbc.E07-05-0498

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Submitted on May 25, 2007
Revised on October 24, 2007
Accepted on December 10, 2007

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

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

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

Monitoring Editor: Adam Linstedt

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable SNARE complex consisting of synaptobrevin-2/VAMP2, SNAP-25, and syntaxin 1. This complex is subsequently disassembled by the concerted action of {alpha}-SNAP and the AAA-ATPase 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 since cleavage of syntaxin with botulinum neurotoxin C1 or competition using antibodies against syntaxin SNARE motif abolishes clustering. Binding of {alpha}-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 {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 preventing SNARE assembly, revealing an unexpected site of action for {alpha}-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

Present addresses: {ddagger}King’s College London, Randall Division of Cell and Molecular Biophysics, New Hunt’s House, Guy’s Campus, London SE1 1UL, United Kingdom; {sect}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)






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