Journal
BIOPHYSICAL JOURNAL
Volume 91, Issue 5, Pages 1767-1777Publisher
BIOPHYSICAL SOCIETY
DOI: 10.1529/biophysj.105.080325
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Funding
- NIGMS NIH HHS [GM 56827, R01 GM056827] Funding Source: Medline
- NIMH NIH HHS [MH61876, R01 MH061876] Funding Source: Medline
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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 copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more effcient 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.
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