PI-PLC–based Scramblase Assay

For the PI-PLC–based scramblase assay, a previously reported one-pot method was used to make liposomes and proteoliposomes (27 (link), 36 (link)) using a mixture of lipids, including egg PC and brain PS at a molar ratio of 9:1. Briefly, 8.2 μmol egg PC, 0.92 μmol brain PS, and a trace amount of [³H]PI (∼25,000 cpm), [³H]GlcN-PI (∼12,000 cpm), or [³H]GlcNAc-PI (∼12,000 cpm) were combined in a glass screw-cap tube, dried under a stream of N₂ gas, and solubilized in 1.9 mL of buffer A (10 mM Hepes-NaOH, pH 7.4,100 mM NaCl, 1% [wt/vol] Triton X-100). The lipid solution was vortexed at moderate speed for 20 min until the solution become completely transparent. The solubilized lipids were divided into two aliquots and mixed with or without purified CLPTM1L-FH in a final volume of 1 mL buffer A, and incubated at 4 °C for 1 h with end-over-end rotation. Proteoliposomes or protein-free liposomes were generated by two rounds of treatment with a total of 300 mg of wet SM-2 Bio-Beads (Bio-Rad) for 1 mL of sample. The samples were placed in 2-mL Eppendorf tubes, and end-over-end mixed with 100 mg of Bio-beads at room temperature for 3 h. A second portion of 200 mg of Bio-beads was added and the incubation was continued at 4 °C for 16 h. Finally, the samples were transferred to 1.5-mL Eppendorf tubes without disturbing the Bio-beads and used immediately for scramblase assays.