Liposome binding assays

Preparation of liposomes: Lipids DOPA, DOPE, DOPC, Brain PI(4,5)P and Brain PI(4)P were obtained commercially (Avanti) as chloroform dissolved lipids. Volumes of lipids were separately pipetted into glass vials. Chloroform was removed by evaporation under a nitrogen stream and by desiccation overnight. Lipids were re-suspended in lipid buffer 1 (25 mM Hepes pH 7.5, 100 mM NaCl, 5% glycerol, filtered 0.22 µm) and vortexed at room temperature for 5 min to generate a cloudy solution. The solution was bath-sonicated for 5 min and freeze-thawed 5 times. Lipids were extruded by passing them 11 times through a 0.1 µm filter.

Liposomes of the following composition were prepared (weight %)

10%: 90% - DOPE: DOPC

30%: 10%: 60% - DOPA: DOPE: DOPC

30%: 10%: 60% - PI(4)P: DOPE: DOPC

25%: 10%: 65% - PI(4,5)P₂: DOPE: DOPC

Binding assays: Recombinant TgRASP2con2 and TgRASP2con2^MUT were centrifuged for 30 min at 100,000 × g at 4 °C to remove any potential precipitation. The soluble protein was transferred to a fresh tube and diluted to 40 µg/ml in protein buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT). Lipids where diluted to 1 mg/ml using lipid buffer 2 (50 mM Hepes pH 7.4, 100 mM NaCl, 1% glycerol) and diluted 2-folds in water. Lipid binding reaction was initiated by mixing 40 µl of lipids (=40 µg) with 40 µl of protein (=1.6 µg) on ice for 1 h. The mixture was then centrifuged at 100,000 × g for 30 min at 4 °C and the supernatant and pellet were separated and resuspended in Laemmli buffer before being processed for SDS–PAGE. Blots were developed with anti-His antibody and bands were analysed with Image Lab software (BioRad).