Fig. 2.

Ca²⁺- and phospholipid-dependent membrane binding by E-Syt2 C₂ domains. (A) Recombinant GST-fusion proteins containing the E-Syt2 domains indicated on the right were used in phospholipid-binding assays. Proteins were incubated in the absence of divalent cations, in 1 mM Ca²⁺, or in 1 mM Mg²⁺ with liposomes composed of 25% PS/75% PC (wt/wt). Liposomes were pelleted by centrifugation, and bound proteins were analyzed by SDS/PAGE and Coomassie staining. (B) Ca²⁺ dependence of phospholipid binding. Recombinant GST-fusion proteins containing either the E-Syt juxtamembranous X domain together with the C₂Aand C₂B domains (XC₂AB), or only the C₂A or C₂C domain from E-Syt2, were incubated with liposomes composed of 25% PS/75% PC in the presence of the indicated concentrations of free Ca²⁺. Proteins bound to the liposomes were analyzed by centrifugation, followed by SDS/PAGE and Coomassie blue staining. (C) Phospholipid dependence of Ca²⁺-dependent membrane binding of the E-Syt2 C₂AB domains. Phospholipid binding of a GST fusion protein containing the E-Syt2 C₂AB domains was tested at the indicated Ca²⁺ concentrations and in the presence of 1 mM Mg²⁺ (as a negative control) in liposomes with the four different phospholipid compositions shown on the right. (PIP, phosphatidylinositol phosphate; PE, phosphatidylethanolamine).