Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain.
The synaptic vesicle protein synaptotagmin has been implicated in the docking and subsequent calcium-regulated exocytosis of synaptic vesicles. We demonstrate that synaptotagmin is a major constituent of synaptic vesicle membranes, comprising 7-8% of the total vesicle protein. A proteolytic fragment of synaptotagmin, containing two repeats homologous to the C2-domain of protein kinase C, bound to a variety of natural membranes in a calcium-dependent manner (EC50 approximately 30 microM calcium). Binding was insensitive to proteolysis of the acceptor membranes suggesting an interaction with the lipid constituents. This interaction was confirmed using a recombinant fusion protein, containing both C2-like domains of synaptotagmin, that bound to artificial liposomes in a calcium-dependent manner. Phospholipid binding properties were preserved in a 114-amino acid domain corresponding to the first C2-like repeat of the protein and represents the shortest functional cassette yet reported. Furthermore, deletion of a highly conserved 9-amino acid motif, within this region, was sufficient to abolish the calcium-dependent phospholipid binding properties of this domain. This mutation may provide a means to selectively disrupt individual C2-domains in order to assess their relative contributions to function.
Chapman
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ER
Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Jahn
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D050857:Synaptotagmins; D002118:Calcium