Isoform-specific, Calcium-regulated Interaction of the Synaptic Vesicle Proteins SV2 and Synaptotagmin

doi:10.1074/jbc.271.44.27770

Isoform-specific, Calcium-regulated Interaction of the Synaptic Vesicle Proteins SV2 and Synaptotagmin*

From the ^‡ Graduate Program in Neurobiology and Behavior and
^§Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195

^¶To whom correspondence should be addressed:
Dept. of Pharmacology, D431 HSB, Box 357280, University of Washington, Seattle, WA 98195.
Tel.: 296-616-2962; Fax: 206-685-3822.

Abstract

The identification and functional characterization of proteins localized to synaptic vesicles has contributed significantly to our understanding of neurotransmission. Studies of synaptic vesicle protein interactions have both led to the identification of novel synaptic proteins and suggested hypotheses of protein function. Synaptic vesicle protein 2 (SV2), is an integral membrane glycoprotein present in all synaptic vesicles. There are two characterized isoforms, SV2A and SV2B. Despite their homology to transporter proteins, the function of the SV2s remains unknown. In an effort to determine SV2 function and identify cofactors required for SV2 activity, we examined the protein interactions of SV2 using a combination of cross-linking, immunoprecipitation, and recombinant protein affinity chromatography. We report that SV2 is part of a large protein complex that contains the synaptic vesicle protein synaptotagmin. The interaction between SV2 and synaptotagmin is direct, specific to SV2A, and inhibited by calcium with an EC₅₀ of approximately 10 µM. Interaction is mediated by the cytoplasmic amino terminus of SV2A and the C2B domain of synaptotagmin. Our observations suggest a regulatory relationship between these two proteins.

Footnotes

↵* This work was supported in part by the Training in Molecular and Cellular Biology Grant (5 T32 GM07270-21) (to A. E. S.), the Pharmaceutical Research and Manufacturers of America Foundation, and the Alfred P. Sloan Foundation (to S. M. B.). 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.
↵¹ The abbreviations used are:

SV2

synaptic vesicle protein 2

DSP

dithiobis(succinimidyl propionate)

HBS

Hepes-buffered saline

IB

incubation buffer

PAGE

polyacrylamide gel electrophoresis.
↵²S. M. Bajjalieh, unpublished results.
- Received July 1, 1996.
- Revision received August 16, 1996.