Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin - PubMed (original) (raw)

Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin

G Schiavo et al. Proc Natl Acad Sci U S A. 1996.

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Abstract

The synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium-dependent, neurotransmitter release and is likely to be the calcium sensor for exocytosis, because of its many calcium-dependent properties. Polyphosphoinositides are needed for exocytosis, but it has not been known why. We now provide a possible connection between these observations with the finding that the C2B domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate (PIns-4,5-P2), its isomer phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P3). Calcium ions switch the specificity of this binding from PIns-3,4,5-P3 (at calcium concentrations found in resting nerve terminals) to PIns-4,5-P2 (at concentration of calcium required for transmitter release). Inositol polyphosphates, known blockers of neurotransmitter release, inhibit the binding of both PIns-4,5-P2 and PIns-3,4,5-P3 to tagmin. Our findings imply that tagmin may operate as a bimodal calcium sensor, switching bound lipids during exocytosis. This connection to polyphosphoinositides, compounds whose levels are physiologically regulated, could be important for long-term memory and learning.

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Figures

Figure 2

Figure 2

Calcium dependency of PIns-4,5-P2 and PIns-3,4,5-P3 binding to tagmin. GST-tagmin beads were simultaneously incubated with two populations of PC liposomes either containing PIns-4,5-P2 or PIns-3,4,5-P3 at variable Ca2+ concentrations (○, PIns-4,5-P2; ▪, PIns-3,4,5-P3). Liposome binding to tagmin and its domains was determined as described in Fig. 1.

Figure 4

Figure 4

The binding of PIns-4,5-P2 to recombinant and native tagmin is saturable and is competed by InsP6. (A) GST-tagmin (•) or GST alone (○) was incubated at increasing concentration of radioactive PIns-4,5-P2 in detergent (OG) micelles in the presence of 2 mM EGTA. Samples were analyzed as described in Fig. 1. (A) Tagmin/PIns-4,5-P2 molar ratio as function of the micellar PIns-4,5-P2 concentration. Dashed line represents the PIns-4,5-P2 specifically associated with tagmin. Parallel experiments using Triton X-100 [0.02% (wt/vol)] gave the same results (not shown). (B) GST-tagmin was incubated in the presence of saturable amount of PIns-4,5-P2 (6 μM) with increasing amount of InsP6 in the presence of 2 mM EGTA. (C) Immunoprecipitated native tagmin (•) or antitagmin antibody alone (○) were incubated with radioactive PIns-4,5-P2 in detergent micelles in the presence of 2 mM EGTA. Dashed line represents the PIns-4,5-P2 specifically associated with tagmin. (D) SDS/PAGE profile of the immunopurified native tagmin used in C. In addition to the tagmin monomer with an apparent molecular weight of 65 kDa, an SDS-resistant tagmin dimer is also visible (37).

Figure 1

Figure 1

Tagmin binds specifically to polyphosphoinositide-containing liposomes in a calcium-dependent manner. GST-tagmin was incubated with liposomes containing pure PC or PC together with distinct polyphosphoinositides (as indicated) in the absence (shaded bars, 2 mM EGTA) or presence of calcium ions (solid bars, 100 μM free Ca2+). Lipid binding was quantified by liquid scintillation counting of the radioactive PC used as tracer and expressed as percent of total radioactivity used. Specific binding was calculated by subtracting the nonspecific lipid interaction of GST (2.4 ± 0.7%) from individual samples. Similar results were obtained both with small and large unilamellar vesicles, thus suggesting that size and curvature of the liposome do not influence the binding.

Figure 3

Figure 3

The polyphosphoinositides binding site on tagmin is localized to the C2B domain. GST fusion proteins containing tagmin or its N-terminal (C2A; aa 96–265; 10 μg) or the C-terminal (C2B; aa 248–421; 10 μg) domains were incubated with liposomes containing PC and 25% (wt/wt) PS (A), 1% (wt/wt) PIns-4,5-P2 (B), or 1% (wt/wt) PIns-3,4,5-P3 (C) (shaded bars, 2 mM EGTA; solid bars, 100 μM free Ca2+). Liposome binding to tagmin and its domains was determined as described in Fig. 1.

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