Abstract
The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).
Highlights
Neurotransmitter release is achieved by a fusion of synaptic vesicles to presynaptic plasma membranes in response to a rapid increase in Ca2ϩ ions entering through voltage-gated Ca2ϩ channels [1, 2]
Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca2؉
The molecular weight shift induced by Ca2ϩ probably reflects direct clustering of the Syt VII C2B domain itself, and it is most unlikely to be mediated by certain adapter proteins (i.e. Ca2ϩdependent binding partner of Syts, such as syntaxin I) [40], because after Coomassie Brilliant Blue R-250 staining of the gel we were unable to observe any bands other than Syt molecules and the anti-T7-tag antibody used for immunoprecipitation, and syntaxin I is not expressed in COS-7 cells
Summary
Syt(s), synaptotagmin(s); cPLA2, cytosolic phospholipase A2; HRP, horseradish peroxidase; PLC␦1, phospholipase C␦1. This paper is available on line at http://www.jbc.org gomerization activity. We showed that the single C2 domain is sufficient for Ca2ϩ-dependent multimerization and identified Asn-328 in the C2B domain as a crucial residue for Ca2ϩ-dependent multimerization by site-directed mutagenesis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
