Fig. 4.

Ca²⁺/calmodulin binding to cis liposomes mediates transfer of the lipid-binding domain of VAMP. (A) Wild-type VAMP liposomes (25% DOPS/75% POPC) immobilized on beads were incubated with 15 μM calmodulin and trans ³H-liposomes (25% DOPS/75% POPC) in the presence of a Ca²⁺/EGTA buffer to yield the indicated free Ca²⁺ concentrations. ³H-liposome binding was measured as in Fig. 3. Results are the means ± SD of two independent experiments, each in triplicate. (B) Wild-type VAMP liposomes (as in A) were incubated with 15 μM calmodulin in the presence or absence of 150 μMCa²⁺ and trans liposomes (100% POPC or 25% DOPS/75% POPC) as indicated. After washing, calmodulin binding and VAMP content were monitored by Western blotting. Each experimental condition was studied in duplicate, and the products were run on adjacent gel lanes. Results are representative of two independent experiments.