title: Calcium-dependent self-association of synaptotagmin I.
abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1-5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1-5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1-5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.
authors.affiliation: Neuroscience Graduate Program, University of Virginia, Charlottesville 22908, USA.
authors.forename: C K
authors.initials: CK
authors.lastname: Damer
authors.forename: C E
authors.initials: CE
authors.lastname: Creutz
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm; D008027:Light; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure*; D010446:Peptide Fragments / Q000737:chemistry; D010743:Phospholipids; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D012542:Scattering, Radiation; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium