title: Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1.
abstract: Synaptotagmin 1 (Syt1) is a Ca(2+) sensor for SNARE-mediated, Ca(2+)-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca(2+)-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1-lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca(2+)-independent interactions are mediated via a lysine rich region of the C2B domain while Ca(2+)-dependent interactions are mediated via the Ca(2+)-binding loops.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305-5432, USA.
authors.forename: Marija
authors.initials: M
authors.lastname: Vrljic
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Hill
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006031:Glycosylation; D007313:Insecta / Q000166:cytology; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D050863:Synaptotagmin I