Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I.
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.
Hui
Enfu
E
Howard Hughes Medical Institute and Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706, USA.
Bai
Jihong
J
Chapman
Edwin R
ER
D002118:Calcium / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008567:Membranes, Artificial; D010539:Permeability; D010743:Phospholipids / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D050863:Synaptotagmin I / Q000737:chemistry*
D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010743:Phospholipids; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D002118:Calcium