Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin.

Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2.

Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin.

The identification and functional characterization of proteins localized to synaptic vesicles has contributed significantly to our understanding of neurotransmission. Studies of synaptic vesicle protein interactions have both led to the identification of novel synaptic proteins and suggested hypotheses of protein function. Synaptic vesicle protein 2 (SV2), is an integral membrane glycoprotein present in all synaptic vesicles. There are two characterized isoforms, SV2A and SV2B. Despite their homology to transporter proteins, the function of the SV2s remains unknown. In an effort to determine SV2 function and identify cofactors required for SV2 activity, we examined the protein interactions of SV2 using a combination of cross-linking, immunoprecipitation, and recombinant protein affinity chromatography. We report that SV2 is part of a large protein complex that contains the synaptic vesicle protein synaptotagmin. The interaction between SV2 and synaptotagmin is direct, specific to SV2A, and inhibited by calcium with an EC50 of approximately 10 microM. Interaction is mediated by the cytoplasmic amino terminus of SV2A and the C2B domain of synaptotagmin. Our observations suggest a regulatory relationship between these two proteins.