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

Abstract

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+-C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ('cis') or plasma ('trans') membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (≤500 μs). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptlc vesicles. This novel mode of clustering may allow for rapid Ca2+-triggered oligomerization of the protein via the merebrane distal C2B domain.