Ca2+ triggered simultaneous membrane penetration of the tandem C2 domains of synaptotagmin I
Enfu Hui 1, Jihong Bai 2 and Edwin R Chapman 3*
1 University of Wisconsin
2 Massachusetts General Hospital
3 University Of Wisconsin
* To whom correspondence should be addressed. E-mail: chapman{at}physiology.wisc.edu.
Submitted on December 22, 2005
Revised on February 8, 2006
Accepted on 1 June 2006
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Abstract |
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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 co-penetrate 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.
Key Words:
C2 domain, exocytosis, fluorescence, kinetics, membrane fusion
