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