Abstract
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.
Highlights
Formation and dissociation of protein complexes in the synapse mediate and regulate the events of the synaptic vesicle cycle
Following treatment with dithiobis(succinimidyl propionate) (DSP), approximately 50% of the Synaptic vesicle protein 2 (SV2) shifted from its normal migration at 70 – 85 kDa to the top of the gel (Fig. 1), suggesting that SV2 is part of a large protein complex
Synaptotagmin bound the SV2A but not the SV2B peptide (Fig. 3A). This indicates 1) that the interaction is isoform-specific and 2) that the amino terminus of SV2 is sufficient to mediate interaction with synaptotagmin. While these results suggest that SV2 and synaptotagmin interact, it remained possible that the observed interaction is mediated by another synaptic protein present in synaptic vesicle extracts
Summary
Formation and dissociation of protein complexes in the synapse mediate and regulate the events of the synaptic vesicle cycle. Calcium Effects on SV2-Synaptotagmin Binding—Approximately 1 g of GST fusion protein bound to glutathione-agarose beads was incubated for 1 h with 200 g of Triton X-100-solubilized synaptosome protein in HEDTA buffer (50 mM Hepes, 5 mM HEDTA, 95 mM KOAc, pH 7.5) with the indicated free calcium concentrations.
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