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Ca²⁺-Triggered Simultaneous Membrane Penetration of the Tandem C2-Domains of Synaptotagmin I

Howard Hughes Medical Institute and Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706

Correspondence: Address reprint requests to Edwin R. Chapman, HHMI and Dept. of Physiology, SMI 129, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-1762; Fax: 608-265-5512; E-mail: chapman{at}physiology.wisc.edu.

Synaptotagmin I (syt), a transmembrane protein localized to secretory vesicles, functions as a Ca²⁺ sensor that facilitates SNARE-mediated membrane fusion. The cytoplasmic domain of syt harbors two C2-domains designated C2A and C2B. Upon binding Ca²⁺, C2A and C2B partially penetrate into membranes that contain anionic phospholipids. However, it is unknown whether these tandem C2-domains engage membranes at the same time, in a sequential manner, or in a mutually exclusive manner. We have used site-directed fluorescent probes to monitor the penetration of syt's C2-domains into phosphatidylserine-harboring lipid bilayers. We report that, in response to Ca²⁺, C2A and C2B copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more efficient when synthetic rather than natural phospholipids were used to prepare bilayers. The membrane penetration activity of the intact cytoplasmic domain of syt (C2A-C2B) exhibits significant resistance to changes in ionic strength. In contrast, the ability of isolated C2B to bind membranes in response to Ca²⁺ can be disrupted by subtle changes in ionic strength. Tethering C2B to a mutant version of C2A that does not bind Ca²⁺ or membranes significantly increases the stability of Ca²⁺·C2B·membrane complexes, confirming that C2A affects the membrane-binding properties of the adjacent C2B domain.