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J. Biol. Chem., Vol. 282, Issue 36, 26335-26343, September 7, 2007

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Complexin/Synaptotagmin Interplay Controls Acrosomal Exocytosis^*

Carlos M. Roggero¹, Gerardo A. De Blas¹, Han Dai, Claudia N. Tomes, Josep Rizo, and Luis S. Mayorga²

From the Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza 5500, Argentina and the Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca²⁺ and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca²⁺. We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca²⁺ and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain.

Received for publication, January 30, 2007 , and in revised form, July 3, 2007.

^* This work was supported in part by an International Research Scholar Award from the Howard Hughes Medical Institute (to L. M.) and grants from Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina), Agencia Nacional de Promoción Científica y Tecnológica (Argentina), and National Institutes of Health Grant NS40944. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Casilla de Correo 56, 5500 Mendoza, Argentina. Tel.: 54-261-449-4143; Fax: 54-261-449-4117; E-mail: lmayorga{at}fcm.uncu.edu.ar.

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