Fig. 8.

The AD3 mutation blocks Ca²⁺-driven conformational changes within the C2B domain of synaptotagmin and disrupts Ca²⁺-triggered oligomerization activity.A, Crystal structure of the C2B domain of synaptotagmin III. This image was modified from Sutton et al. (1999); the structure of the C2B domain of synaptotagmin I has not been reported, however, all known C2B domains share similar structures. The tyrosine that is mutated to an asparagine in the AD3 mutant allele of Drosophila synaptotagmin is indicated, as are five putative Ca²⁺ ligands and a single bound Mg²⁺ ion. B, The C2B domain of WT (GST-C2B_WT) and AD3 (GST-C2B_AD3) rat synaptotagmin Ib were immobilized as a GST fusion proteins (20 μg/data point) and subjected to limited proteolysis in the presence of 2 mm EGTA (−) or 1 mmCa²⁺ (+) at the indicated [chymotrypsin] for 60 min at rt. Samples were boiled in SDS sample buffer, analyzed by SDS PAGE, and stained with Coomassie blue. C,Ca²⁺-triggered synaptotagmin oligomerization is impaired by the AD3 mutation. Eight micrograms of GST or GST fused to the cytoplasmic domain of wild-type (syt_WT) or AD3 mutant (syt_AD3) Drosophilasynaptotagmin was immobilized on beads. Beads were incubated with 1.5 μm soluble WT or AD3 mutant Drosophilasynaptotagmin for 1.5 hr in 150 μl of TBS plus 0.5% Triton X-100 and either 2 mm EGTA (−), 1 mmCa²⁺ (+), or the indicated concentration of Ca²⁺. Beads were washed three times with binding buffer and boiled in SDS sample buffer. Three percent of the soluble synaptotagmin from the binding assay (left two lanes) and 25% of the bound material (remaining lanes) were subjected to SDS-PAGE and visualized by staining with Coomassie blue.Syt, Cytoplasmic domain of synaptotagmin I. Note: theasterisk indicates a proteolytic fragment present in preparations of GST-fused AD3 mutant synaptotagmin. D,Data from two oligomerization assays (as described in C) were quantified by densitometry, normalized to the pixel intensity in the “total” lanes, and plotted versus the free [Ca²⁺]. Closed circles, Wild-type synaptotagmin; open circles, AD3 synaptotagmin.E, Soluble Drosophila synaptotagmin I (5 μm) was incubated with 30 μg of either wild-type synaptotagmin IV, AD3 synaptotagmin IV, or synaptotagmin IV containing a KK to AA substitution (Chapman et al., 1998) at amino acids 385 and 386. Binding of synaptotagmin I was visualized by Western analysis with anti-synaptotagmin I DSYT2 antisera (Littleton et al., 1993). ● denotes binding reactions lacking soluble synaptotagmin.