Figure 3.

Identification of the GAT PI4P-binding site. (A) Recombinant GGA GST-GAT and its subdomains were incubated with nitrocellulose membranes dotted with 100, 50, and 25 pmol lipids. These lipid dot blots were made in the laboratory. A Coomassie blue–stained gel of the recombinant GST-GAT and GST-C-GAT used is shown. (B) Comparing the binding of wt and mutant GGA2 GAT to liposomes. 0.02 mg/ml (0.4 μM) GGA2 GST-GAT was incubated with 0.2 mg/ml mixed lipid vesicles. The vesicles were sedimented by centrifugation and bound proteins were detected by Coomassie blue staining after SDS-PAGE. The bottom panel shows the ratios of protein cosedimentated with PI4P versus PS liposomes (PI4P-binding index), as mean ± SE from two to four experiments. (C) Comparing the effect of Arf1-GTP on GAT binding to PS liposomes. PS liposomes were preloaded with or without recombinant myr-Arf1-GTPγS. The percent of GAT sedimented was shown in the right panel (mean ± SE, n = 3). **p = 0.003, *p = 0.02, NS (not significant), p = 0.15.