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...
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.Neurotransmitter secretion occurs via a tightly regulated membrane trafficking cycle localized to the presynaptic terminal. Many stages of this cycle, such as the targeting and docking of vesicles at active zone membranes, vesicle fusion, and endocytosis, are mediated by the formation of protein complexes (reviewed in Refs. 1 and 2). Protein phosphorylation, a ubiquitous mechanism of cellular regulation, plays an important role in the modulation of the synaptic vesicle cycle. Multiple examples of phosphorylation-mediated regulation of protein binding in the synapse have been reported. One well characterized example is the phosphorylation-dependent association of the actin-binding protein synapsin with synaptic vesicles, an interaction implicated in the maintenance of a reserve pool of vesicles (3, 4). Likewise, proteins involved in the docking and fusion of vesicles, termed SNAREs, 1 are substrates of several protein kinases, and phosphorylation has been reported to alter their binding to other proteins and to each other (5-8).Synaptic vesicle protein 2 (SV2), a protein common to all neurons (9, 10), has been implicated in the regulation of synaptic vesicle exocytosis (11,12). Three separately encoded isoforms, termed SV2A, SV2B, and SV2C, have been identified (13-18). Loss of the most widely expressed isoform, SV2A, results in aberrant neurotransmission and death, indicating that SV2 is an essential protein (11, 12). We previously reported that SV2A interacts with the synaptic vesicle protein synaptotagmin, a calcium-binding protein that is necessary for normal calcium-stimulated neurotransmission (19). The SV2-synaptotagmin interaction is modulated by calcium, suggesting that it plays a role in the regulation of exocytosis.The amino terminus of SV2, which mediates its interaction with synaptotagmin, contains substrate consensus sites of several protein kinases. In addition, SV2 was previously reported to be phosphorylated in vitro under conditions permissive to casein kinase I activity (20). In order to determine ...
Stochastic analysis was applied to observations of spontaneous behavior in the carnivorous mollusc Melibe leonina. Six behaviors were denned that could be easily recognized on inspection and it was found that transitions between each of these behaviors could be fully described by a first-order random process without memory of past behavioral choices. The behaviors are organized by frequency of transition into two modes, a feeding mode and a resting mode. Transitions within modes are more likely than transitions between modes, and the feeding and resting modes are linked by a preferred pair of behavioral transitions. The amount of time spent in the feeding mode is positively correlated with body size, but the average length of a feeding episode is independent of size. This suggests that body size regulates the probability of entry into feeding behavior but does not influence the basic pattern of feeding. In the presence of food the animals express nearly continuous feeding behavior, suggesting that food reduces the probability of exiting the feeding mode. This model of spontaneous behavior in Melibe is used to form hypotheses amenable to further exploration through neurophysiological experiments.
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