Figure 8.

Direct, Ca²⁺-dependent C₂B interactions with t-SNAREs are intact in the syt^B-IE mutation. A, The syt^B-IE mutation does not inhibit Ca²⁺-dependent interactions with solubilized t-SNAREs. Left, A representative GST–syt pull-down assay; right, histogram of three experiments. The Ca²⁺-dependent increase in t-SNARE interactions (black bars relative to white bars) is not disrupted by the syt^B-IE mutation. Ca²⁺-independent t-SNARE interactions are decreased (white bars, C₂AB vs C₂AB I420E and C₂B vs C₂B I420E). B, The syt^B-IE mutation abolishes Ca²⁺-dependent C₂B interactions with membrane-embedded t-SNAREs. Left, A representative co-floatation assay; right, histogram of four experiments. The syt^B-IE mutation does not alter the amount of C₂B domain that binds to PS-free, t-SNARE vesicles (t-SNARE) in the absence of Ca²⁺ (−, left; EGTA, right). However, the syt^B-IE mutation abolishes the increase in t-SNARE vesicles binding induced by 1 mm Ca²⁺ (+, left; Ca²⁺, right; p < 0.05). C, The syt^B-IE mutation impairs C₂AB interactions with membrane-embedded t-SNAREs. Again, the syt^B-IE mutation does not alter the amount of C₂AB domain that binds to PS-free, t-SNARE vesicles in the absence of Ca²⁺ (EGTA, white bars). In the presence of 1 mm Ca²⁺ (Ca²⁺, black bars), the t-SNARE vesicles bind 70% of wild-type levels of C₂AB domain when the syt^B-IE mutation is present (p < 0.001; n = 6).