Isoform-specific, Calcium-regulated Interaction of the Synaptic Vesicle Proteins SV2 and Synaptotagmin

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 EC 50 of approximately 10 M. 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.Formation and dissociation of protein complexes in the synapse mediate and regulate the events of the synaptic vesicle cycle. The identification of synaptic protein interactions has both suggested hypotheses for the role each protein plays in neurotransmission and has provided evidence for mechanistic models of synaptic vesicle docking, priming, and fusion (1).Synaptic vesicle protein 2 (SV2) 1 is a membrane glycoprotein present in all synaptic vesicles and regulated secretory vesicles of endocrine cells (2). Two isoforms of SV2, encoded by separate genes, have been characterized; . The SV2 cDNAs predict 12 transmembrane domain proteins that have significant sequence homology to the major facilitator family of transporters (3,4,7). This family of small molecule transporters includes the vesicular transporters of amines and acetylcholine (7). Based on this homology, the SV2s were initially hypothesized to be vesicular neurotransmitter transporters, with each isoform transporting a specific neurotransmitter. However, the expression of SV2A and SV2B does not correlate with neurotransmitter phenotype (8), therefore it is not likely that they serve this function.A current hypothesis of SV2 function is that it transports a constituent of synaptic vesicles other than neurotransmitters.However, attempts to demonstrate transport activity in SV2-expressing fibroblasts have been inconclusive, 2 perhaps due to the absence of a cofactor required for SV2 function. Alternatively, the structural similarity of transporters and channels (9, 10) suggests that SV2 is either a vesicular ion channel or a component of the proposed proteinaceous fusion pore which mediates neurotransmitter release.To distinguish between these hypotheses of SV2 function, and to identify potential cofactors or regulators of SV2 activit...

A Conserved Mechanism of Synaptogyrin Localization

“…Biochemical studies have revealed many protein-protein interactions between different SV proteins (Bennett et al, 1992;Calakos and Scheller, 1994;Galli et al, 1996;Schivell et al, 1996). To assess whether localization of SNG-1 is mediated by protein-protein interactions with other SV protein(s), we examined SNG-1::GFP or native SNG-1 localization in mutants lacking distinct SV-associated proteins, including RAB-3 and RBF-1 (rabphilin), SNB-1, and SNT-1 (Table 1; Nonet et al, 1993Nonet et al, , 1997Nonet et al, , 1998; Staunton, Ganetzky, and Nonet, unpublished observations).…”

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

“…This question was prompted by the finding that mutations within the C2B domain of Drosophila synaptotagmin impair excitation-secretion coupling (DiAntonio and Schwarz 1994; Littleton et al 1993, Littleton et al 1994). A number of C2B–effector interactions have been identified including the following: AP-2 (Zhang et al 1994; Jorgensen et al 1995), SV2 (Schivell et al 1996), β-SNAP (Schiavo et al 1995), Ca 2+ channels (Kim and Catterall 1997; Sheng et al 1997), inositol polyphosphates (Fukuda et al 1995) and, finally, homo-oligomerization (Chapman et al 1996; Sugita et al 1996). Of these interactions, only oligomerization was promoted by Ca 2+ .…”

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

“…This use of Ponceau-S has been described previously (Klein et al, 1995;Moore and Viselli, 2000) and actual examples occur throughout the published literature (Schivell et al, 1996;Bakiri et al, 2000). Western blotting was performed under standard conditions using 5% milk in PBS with 0.3% Tween 20 as a blocking agent.…”