The C Terminus of SNAP25 Is Essential for Ca2+-dependent Binding of Synaptotagmin to SNARE Complexes

doi:10.1074/jbc.275.9.6328

The C Terminus of SNAP25 Is Essential for Ca²⁺-dependent Binding of Synaptotagmin to SNARE Complexes*

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Abstract

The plasma membrane solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca²⁺-dependent step in regulated exocytosis, but their precise roles and regulation by Ca²⁺ are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg¹⁸⁰-Ile¹⁸¹, completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln¹⁹⁷-Arg¹⁹⁸, is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca²⁺ concentrations. BoNT A treatment essentially increased the Ca²⁺ concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca²⁺ regulation of exocytosis. Synaptotagmin, a proposed Ca²⁺ sensor for exocytosis, was found to bind SNAP25 in a Ca²⁺-stimulated manner. Ca²⁺-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca²⁺concentration required for binding. The C terminus of SNAP25 was also essential for Ca²⁺-dependent synaptotagmin binding to SNAP25·syntaxin and SNAP25·syntaxin·VAMP SNARE complexes. These results clarify classical observations on the Ca²⁺ reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca²⁺-dependent interactions between synaptotagmin and SNARE protein complexes.

Footnotes

↵* This work was supported in part by National Institutes of Health Research Grants DK25861 and DK40428 (to T. F. J. M.).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.
↵‡ Supported by National Institutes of Health Predoctoral Training Grant GM07215.
↵§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-263-2427; Fax: 608-262-3453; E-mail: tfmartin@facstaff.wisc.edu.
↵2 E. C. Larsen, unpublished data.
Abbreviations:

VAMP

vesicle-associated membrane protein

SNARE

solubleN-ethylmaleimide-sensitive factor attachment protein receptor

SNAP

synaptosome-associated protein

BoNT

botulinum neurotoxin

GST

glutathione S-transferase

CAPS

3-(cyclohexylamino)propanesulfonic acid
- Received July 2, 1999.
- Revision received November 4, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.