title: 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 Ca(2+) 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 Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-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 (< or =500 micros). 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 synaptic vesicles. This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison 53706, USA.
authors.forename: J
authors.initials: J
authors.lastname: Bai
authors.forename: C A
authors.initials: CA
authors.lastname: Earles
authors.forename: J L
authors.initials: JL
authors.lastname: Lewis
authors.forename: E R
authors.initials: ER
authors.lastname: Chapman
mesh_terms: D020106:Acrylamide / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D013050:Spectrometry, Fluorescence; D050863:Synaptotagmin I; D050857:Synaptotagmins; D013997:Time Factors; D033921:Vesicular Transport Proteins
chemical_list: D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D020106:Acrylamide; D005982:Glutathione Transferase; D002118:Calcium