Biochemical and functional characterization of the interaction between the synaptic vesicle proteins SV2 and synaptotagmin.

摘要

The exocytosis of synaptic vesicles is a highly regulated form of membrane trafficking. Studying the molecules specific to the synapse can help us understand how neurotransmission is so exquisitely regulated. This dissertation examines the protein-protein interaction between two synaptic vesicle proteins, Synaptic Vesicle Protein 2 (SV2) and synaptotagmin. SV2 is a glycoprotein with twelve putative transmembrane domains and homology to transporters. There are three known isoforms of SV2, SV2A, SV2B, and SV2C, all of which are specific to secretory tissues. Synaptotagmins, a family of at least twelve isoforms, contain two PKC-homology (C2) domains which bind calcium and mediate their molecular interactions.; Through crosslinking, GST-fusion protein chromatography, and immunoprecipitation techniques, the following biochemical properties of the SV2/synaptotagmin interaction were identified: (1) There is a direct interaction between SV2 and synaptotagmin. (2) SV2A and SV2C, but not SV2B, interact with synaptotagmin. (3) Synaptotagmins 1-8 all bind SV2. (4) The amino terminus of SV2 and the lysine-rich region of the C2B domain of synaptotagmin mediate the interaction. (5) Calcium and phosphorylation regulate the SV2/synaptotagmin interaction.; The SV2/synaptotagmin interaction could be functioning in any of three ways. First, synaptotagmin could be a modulator of SV2 transport activity. Second, SV2 may be regulating synaptotagmin's participation in calcium-dependent exocytosis. Finally, this complex may be involved in ensuring proper endocytosis of SV2 after synaptic vesicle fusion with the plasma membrane through synaptotagmin's interaction with the clathrin adaptor complex. Future studies will continue to examine the role of SV2 and synaptotagmin in neurosecretion.