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Originally published In Press as doi:10.1074/jbc.M906729199 on June 5, 2000

J. Biol. Chem., Vol. 275, Issue 33, 25427-25435, August 18, 2000
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Membrane-embedded Synaptotagmin Penetrates cis or trans Target Membranes and Clusters via a Novel Mechanism*

Jihong Bai, Cynthia A. Earles, Jessica L. Lewis, and Edwin R. ChapmanDagger

From the Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+·C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (<= 500 µs). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca2+-triggered oligomerization of the protein via the membrane distal C2B domain.

* This study was supported by Grant GM 56827-01 from the National Institutes of Health, by Grant 9750326N from the American Heart Association, and by the Milwaukee Foundation and The Pew Charitable Trusts.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed: Dept. of Physiology, SMI 129, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Fax: 608-265-5512; E-mail: chapman@physiology.wisc.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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