Figure 2.
The interaction between Exo70 and phospholipids in vitro. (A) GST-Exo70 purified from bacteria (0.15 μM) was incubated with liposomes (200 μM) containing 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS. After centrifugation at 150,000 × g for 30 min, the proteins in supernatant (S) and pellet (P) were subjected to SDS-PAGE and visualized by SYPRORed staining. Exo70 bound to vesicles containing 5% PI(4,5)P2 or 60% PS. It also bound weakly to vesicles containing 40% PS. GST did not bind to liposomes with any lipid composition in the cosedimentation assay. (B and C) The interaction of Exo70 with phospholipids. Exo70, 0.15 μM, was incubated with increasing concentrations of LUVs composed of (B) 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS or (C) PI(3)P, PI(4)P, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3. The percentage of bound Exo70 was plotted with the increasing liposome concentration with a single rectangular hyperbola equation (B = BmaxX/[Kd + X]) using SigmaPlot. Each point is the average of three measurements. Error bars, SD.