Uncoating activities of ArfGAPs measured by light scattering. (A–D) The coat assembly–disassembly cycle was measured by light scattering at 350 nm in a spectrofluorometer. Liposomes supplemented with 2 mol% of p23 lipopeptide and extruded through 200-, 80-, or 30-nm polycarbonate filters (dynamic light-scattering assays showed a mean diameter of 160 nm, 60 nm, and 40 nm, respectively; Fig. S2, available) or salt-washed Golgi membranes were incubated with Arf1, coatomer, and EDTA. At the time points indicated, GTP or its slowly hydrolyzable analogue GTPγS was added to start the coating reaction. After 10 min, the GTP state was stabilized with MgCl2. ArfGAPs (ArfGAP1, blue lines; ArfGAP2, gray lines; ArfGAP3, black lines) were added at the indicated time points to induce coat disassembly. Scattering at time point 0 was set to 0 AU, and scattering of the coated state was normalized to 100 AU. (A) 160 nm liposomes and 2.5 nM ArfGAPs; (B) 60 nm liposomes and 2.5 nM ArfGAPs; (C) 40 nm liposomes and 2.5 nM ArfGAPs; (D) Golgi membranes and 1 and 5 nM ArfGAPs.
