Figure 6.

Gcs1p and Age2p ARFGAP activity. Recombinant Gcs1p or Age2p (as indicated at bottom) were titrated into assays containing myristoylated yeast ARF1 loaded with [α-³²P]GTP and standard vesicles composed of 600 μM PC and 400 μM phosphatidylserine (stippled bars). When DAG (solid bars) or PA (hatched bars) was incorporated into vesicles, the concentration of these lipids was 100 μM, and PS concentration was reduced to 300 μM. ARFGAP activity was measured by conversion of [α-³²P]GTP to [α-³²P]GDP in a single round of GTP hydrolysis by ARF1 (2 μM) preloaded with [α-³²P]GTP in a 2-min reaction (Kam et al., 2000). Mock controls were represented by reactions devoid of ARFGAP, and these low background values were subtracted from all other assay reactions. ARFGAP activity for each condition is expressed as ln(S₀/S) min⁻¹ μg protein⁻¹ units as described in the text and by Randazzo and Kahn (1994). Representative results obtained with liposomes generated by sonication are shown.