title: Localization of synaptotagmin-binding domains on syntaxin.
abstract: Synaptotagmin, an abundant calcium- and phospholipid-binding protein of synaptic vesicles, has been proposed to regulate neurotransmitter release at the nerve terminal. To understand better the biochemical mechanism of neurotransmitter release, we have investigated the calcium-dependent and -independent protein-protein interactions between synaptotagmin I and syntaxin 1a, a subunit of the receptor for synaptic vesicles on the presynaptic plasma membrane. Soluble syntaxin 1a binds to synaptotagmin glutathione S-transferase (GST) fusion protein, and the binding was decreased in the presence of calcium. A synaptotagmin fragment containing the second C2 repeat (Syt3-5) had the same binding profile as the whole cytoplasmic domain; however, fragments containing the first C2 repeat (Syt1-3 and Syt2-3) showed calcium-dependent binding to syntaxin. In addition, the soluble full-length cytoplasmic domain of synaptotagmin binds to a syntaxin GST fusion protein in a calcium-dependent manner. Syntaxin domains required for calcium-dependent and -independent synaptotagmin-binding were localized using syntaxin deletion mutants. Amino acids 241-266 of the syntaxin C terminus were required for calcium-independent binding of synaptotagmin. The minimal domain required for calcium-dependent binding of synaptotagmin to syntaxin was localized to amino acids 220-266. The syntaxin domains required for synaptotagmin binding overlap with the domains for vesicle-associated membrane protein (or VAMP) and alpha-soluble N-ethyl-maleimide-sensitive fusion protein attachment protein (or alphaSNAP) interactions. The data suggest both calcium-dependent and -independent roles of synaptotagmin in regulating synaptic vesicle release and/or recycling.
authors.affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, California 94305, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Kee
authors.forename: R H
authors.initials: RH
authors.lastname: Scheller
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology; D001665:Binding Sites; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050863:Synaptotagmin I; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium