Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis, Synaptotagmin spans the vesicle membran e once and possesses a cytoplasmic domain largely comprised of two C2 domai ns designated C2A and C2B. We have determined how deep the Ca2+-binding loo ps of Ca2+ C2A penetrate into the lipid bilayer and report mutations in syn aptotagmin that can uncouple membrane penetration from Ca2+-triggered inter actions with the SNARE complex. To determine whether C2A penetrates into th e vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-T MR) into proteoliposomes. Kinetics experiments revealed that cis interactio ns are rapid (less than or equal to 500 mu s). Binding in the trans mode wa s distinguished by the slow diffusion of trans target vesicles, Both modes of binding were observed, indicating that the linker between the membrane a nchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that sy naptotagmin may form clusters on the surface of synaptic vesicles. This nov el mode of clustering may allow for rapid Ca2+- triggered oligomerization o f the protein via the membrane distal C2B domain.