FIGURE 2. Mutations on CPX-I designed to mimic hydrophobic layers on VAMP2 stabilize the clamp.

A) Dose dependent inhibition of the cell fusion reaction using different soluble CPX-I mutants (sCPX-I). Increasing concentration of each recombinant sCPX were added at the time the two cell populations were mixed. Cells were allowed to fuse overnight and the fusion efficiency was determined as the percentage of fusion. Results are mean ± SEM of three independent experiments. B) Effect of different cell surface expressed “super-clamp” CPX-I-GPI mutants on cell fusion (blue bars), on the cell fusion recovery after addition of PI-PLC in the absence (green bars) or presence of SYT-I and Calcium (red bars). Experiments are the mean±SEM of three independent experiments. Dashed lines show the maximum cell fusion recovery in the absence (green) or presence of Ca/SYT-I (red) and the total overnight clamping (blue). C) The SYT-I requirement of CPX-I-^D27l/E34F-GPI was tested by performing a cell fusion experiment as described in Fig. 2B. In this case the cell fusion recovery was carried out at 200 μM Free Ca²⁺ and samples were fixed at the indicated time every 5 min. The level of fusion was determined as percentage of transfected v-cells that fused. Results are mean ± SEM of three independent experiments. D) Differential V_C-Peptide sensitivity of CPX-I “super-clamp” mutant constructs. Increasing concentrations of V_C-peptide were added at the time the two cell populations were mixed. Cells were allowed to fuse overnight and the fusion efficiency was determined as the percentage of fusion. Dashed lines correspond to the basal level of overnight fusion in the absence of CPX-I (green) or in the presence of CPX-I (red). Results are mean ± SEM of three independent experiments