Calcium-dependent Oligomerization of Synaptotagmins I and II

Abstract

Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC 50 ‫؍‬ 6 ؎ 4 M). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.Neuronal communication depends upon the transduction of an electric nerve impulse for the release of neurotransmitters from their storage compartment, the small synaptic vesicles (SSV).1 This tightly regulated process is triggered by the rapid increase of the intracellular calcium due to the opening of voltage-gated calcium channels at the active zones of the synaptic plasma membrane, which causes the activation of the SSV fusion machinery (1, 2). Of the proteins involved in the physiological cycle of the SSV, the members of the synaptotagmin family represent the best candidates for the role of calcium sensors in neurotransmitter release and, more generally, in regulated exocytosis (3-5). This protein family comprises more than a dozen members having the same overall structure and a variable degree of homology (4, 5). Synaptotagmins are integral membrane proteins with broad distributions in neuronal and non-neuronal tissues that vary between isoforms (4, 5). In the central nervous system and neuroendocrine cells, synaptotagmins are localized on SSV and secretory granules (6). These proteins are characterized by a single membrane-spanning domain and by a large cytoplasmic portion containing two internal repeats that have homology with the C2 domain of protein kinase C (7,8). This domain is known to regulate the calciumdependent translocation of protein kinase C to membranes and is present in many proteins with different functions (5, 8). In synaptotagmins, the two C2 domains are re...

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

“…As noted above, the ability of native or transfected syt fragments to oligomerize in response to Ca 2ϩ is well established (22,26,39). Our studies using recombinant C2A-C2B from syt I and II, and C2A domains from syt VII and III, indicate that oligomerization requires the presence of another factor.…”

“…It was previously shown that native syt I and II heterooligomerize in response to Ca 2ϩ ; this property was recapitulated by using recombinant C2A-C2B versions of these proteins (39). However, as shown in Fig.…”

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

“…This value is similar to the [Ca 2+ ] 1/2 for the interaction of the C2A domain of synaptotagmin I with anionic phospholipids (Davis et al 1999). We note that previous measurements of the Ca 2+ dependence for synaptotagmin clustering are highly dependent on the methods and conditions in which oligomerization was measured (Chapman et al 1996; Sugita et al 1996; Osborne et al 1999). While the limited proteolysis technique employed here may only report a subset of conformational changes, this assay system has the advantage that it circumvents the need to carry out the wash steps employed in C2B-synaptotagmin pull-down assays and, therefore, accurately reports the Ca 2+ requirement for a conformational change within C2B.…”

Direct Interaction of the Rab3 Effector RIM with Ca2+Channels, SNAP-25, and Synaptotagmin

“…Inositol-1,2,3,4,5,6-hexakisphosphate (IP6) was from Calbiochem. The antibody directed against Syt-II has been described previously (35).…”

“…The beads were resuspended in 20 mM HEPES, pH 7.5, 150 mM KCl, 1 mM dithiothreitol, 5% glycerol, 0.05% Tween 20, and 1 mg/ml bovine serum albumin. They were then incubated with 35 S-labeled fragments of ␣1 subunits or with the cytoplasmic domain of syntaxin-1A produced by in vitro translation (Promega). The proteins that remained associated with the affinity columns after extensive washing were analyzed by SDS-PAGE and revealed by autoradiography or, for quantification experiments, by Molecular Imager FX (Bio-Rad).…”