Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro*
- Claudio G. Giraudo‡,1,
- Alejandro Garcia-Diaz‡,
- William S. Eng‡,
- Ai Yamamoto§,
- Thomas J. Melia‡ and
- James E. Rothman‡,2
- ‡Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, New York, New York 10032 and the §Department of Neurology, Columbia University, New York, New York 10032
- ↵1 To whom correspondence may be addressed: Dept. of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Ave., Russ Berrie Bldg., Rm. 520, New York, New York 10032. E-mail: cgg2103{at}columbia.edu.
- ↵2 To whom correspondence may be addressed: Dept. of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 1150 St. Nicholas Ave., Russ Berrie Bldg., Rm. 520, New York, New York 10032. E-mail: jr2269{at}columbia.edu.
Abstract
In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca2+) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.
Footnotes
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↵* This work was supported, in whole or in part, by National Institutes of Health Grant RO1-GM071458 (to J. E. R.). 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.
- Received May 7, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
