title: Visualization of synaptotagmin I oligomers assembled onto lipid monolayers.
abstract: Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B.membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.
authors.affiliation: Department of Biological Sciences and Biotechnology, State-Key Laboratory of Biomembranes, Tsinghua University, Beijing 100084, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wu
authors.forename: Yuhong
authors.initials: Y
authors.lastname: He
authors.forename: Jihong
authors.initials: J
authors.lastname: Bai
authors.forename: Shang-Rong
authors.initials: SR
authors.lastname: Ji
authors.forename: Ward C
authors.initials: WC
authors.lastname: Tucker
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
authors.forename: Sen-Fang
authors.initials: SF
authors.lastname: Sui
mesh_terms: D000818:Animals; D001704:Biopolymers / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D050356:Lipid Metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D001704:Biopolymers; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium