Distinct Ca(2+)-dependent properties of the first and second C2-domains of synaptotagmin I.
Synaptotagmin 1 (SytI) is a synaptic vesicle protein that binds Ca2+ and is essential for fast, Ca(2+)-dependent neurotransmitter release in the hippocampus, suggesting that it serves as a Ca2+ sensor for exocytosis. Although SytI has two cytoplasmic C2-domains, only the first C2-domain was shown to exhibit Ca2+ regulation; it binds phospholipids and syntaxin in a Ca(2+)-dependent manner. By contrast, the second C2-domain is inactive in these assays and only binds putative interacting molecules in a Ca(2+)-independent manner. We have now discovered in a yeast two-hybrid screen for SytI-interacting molecules that the C2-domains of SytI interact with themselves. Using immobilized recombinant C2-domains from SytI and SytII, we found that only the second but not the first C2-domains of these synaptotagmins are capable of affinity-purifying native rat brain SytI and that this binding is Ca(2+)-dependent, suggesting that only the second C2-domain is capable of a Ca(2+)-triggered self-association. A relatively high Ca2+ concentration (> 100 microM) is required for binding in the presence of Mg2+; Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+. Our data suggest that the second C2-domain of SytI is also a Ca(2+)-regulated domain similar to the first C2-domain but with distinct binding activities.
Sugita
S
S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Hata
Y
Y
Südhof
T C
TC
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D005089:Exocytosis; D006624:Hippocampus / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013572:Synaptic Vesicles / Q000378:metabolism; D050863:Synaptotagmin I; D050861:Synaptotagmin II; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; D050861:Synaptotagmin II; C494312:Syt1 protein, rat; C494264:Syt2 protein, rat; D050857:Synaptotagmins; D002118:Calcium