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J. Biol. Chem., Vol. 283, Issue 46, 31763-31775, November 14, 2008

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Synaptotagmin C2B Domain Regulates Ca²⁺-triggered Fusion in Vitro

CRITICAL RESIDUES REVEALED BY SCANNING ALANINE MUTAGENESIS^*

From the Department of Physiology and Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 53706

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca²⁺, and regulates neuronal exocytosis. Syt 1 harbors two Ca²⁺-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca²⁺-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca²⁺ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca²⁺/membrane-binding loops, decreases C2AB t-SNARE binding and Ca²⁺-triggered fusion in vitro without affecting Ca²⁺-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

Received for publication, May 1, 2008 , and in revised form, September 3, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM 56827 and MH 61876. This work was also supported by American Heart Association Grant 0440168N. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

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¹ To whom correspondence should be addressed: 1300 University Ave., 129 SMI, Madison, WI 53706. Tel.: 608-263-5512; Fax: 608-265-5512; E-mail: chapman{at}physiology.wisc.edu.

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