Figure 1.

The C2 domain binds to the cell surface in a Ca²⁺-dependent manner

(A) Rationale behind using the C2 domain (gray) to label recycling vesicles (amber) and the crystal structure of the cPLA₂ C2 domain bound to Ca²⁺ (yellow spheres) (PDB: IRLW; Perisic et al., 1998).

(B) Adherent PC12 cells were incubated with the indicated concentrations of purified C2 domain for 5 min on ice, and the amount of C2 domain bound to cells after washing was detected by immunoblotting (black trace; n = 6 dishes). To examine the lipid interactions involved in membrane binding, cells were pre-treated with either phosphatidylcholine-specific phospholipase D (red trace; n = 5 dishes) or sphingomyelinase (blue trace; n = 5 dishes) for 1 h. Cell integrity was assayed by checking for β-actin leakage under all conditions (data not shown).

(C) Divalent cation specificity of membrane binding. Cells were incubated with 1 μM C2 domain for 5 min in the indicated free Ca²⁺ and Mg²⁺ concentrations (n = 3).

(D) Reversibility of membrane binding. Cells were incubated with 1 μM C2 domain (no wash) or further incubated without the C2 domain for 5 min in normal extracellular solution (Ca²⁺ wash), in a pH 5.5 solution containing 2 mM Ca²⁺ (pH 5.5 wash), or in 1 mM EGTA solution containing 1 mM Mg²⁺ (EGTA wash) (n = 4). Error bars, SEM.