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J. Biol. Chem., Vol. 281, Issue 21, 14823-14832, May 26, 2006

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SNARE Complex Zero Layer Residues Are Not Critical for N-Ethylmaleimide-sensitive Factor-mediated Disassembly^*

Joshua M. Lauer, Seema Dalal, Karla E. Marz, Michael L. Nonet, and Phyllis I. Hanson¹

From the Departments of Cell Biology and Physiology and Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110

Membrane-anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNARE complexes are parallel four-helix bundles stabilized in part by hydrophobic interactions within their core. At the center of SNARE complexes is a distinctive zero layer that consists of one arginine and three glutamines. This zero layer is thought to play a special role in the biology of the SNARE complex. One proposal is that the polar residues of the zero layer enable N-ethylmaleimide-sensitive factor (NSF)-mediated SNARE complex disassembly. Here, we studied the effects of manipulating the zero layer of the well studied synaptic SNARE complex in vitro and in vivo. Using a fluorescence-based assay to follow SNARE complex disassembly in real time, we found that the maximal rate at which NSF disassembles complexes was unaffected by mutations in the zero layer, including single replacement of the syntaxin glutamine with arginine as well as multiple replacement of all four layer residues with non-polar amino acids. To determine whether syntaxin with arginine instead of glutamine in its zero layer can support SNARE function in vivo, we introduced it as a transgene into a Caenorhabditis elegans syntaxin-null strain. Mutant syntaxin rescued viability and locomotory defects similarly to wild-type syntaxin, demonstrating that SNARE complexes with two glutamines and two arginines in the zero layer can support neurotransmission. These findings show that residues of the zero layer do not play an essential role in NSF-mediated disassembly.

Received for publication, November 28, 2005 , and in revised form, February 28, 2006.

^* This work was supported by grants from the National Institutes of Health (to P. I. H. and M. L. N.) and by a W. M. Keck Foundation distinguished young scholar award, a Searle scholar award, and the Andrew and Virginia Craig Faculty Research Fund (to P. I. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid, Campus Box 8228, St. Louis, MO 63110. Tel.: 314-747-4233; Fax: 314-362-7463; E-mail: phanson{at}cellbiology.wustl.edu.

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