Figure 4. Analysis of the time dependence of coatomer-dependent Arf GAP2 activity A. Time dependence of GTP hydrolysis and effect of p25 cargo peptide with small LUVs.

The reaction mixture contained LUVs, with PI4P and extruded through membranes with 0.03 μm diameter pores, 0.6 μM Arf1•[α³²P]GTP, 8.6 nM [1–521] Arf GAP2 His_6, 62 nM coatomer and, where indicated, 25 μM p25. B. Time dependence of GTP hydrolysis and effect of p25 cargo peptide with large LUVs. The reactions contained LUVs with PI4P and extruded through membranes with 1.0 μm pores, 0.6 μM Arf1•[α³²P]GTP, 34.2 nM [1–521] Arf GAP2 His₆, 62 nM coatomer and, where indicated, 25 μM p25. C. Effect of Arf1•GTP and curvature on coatomer binding to LUVs. Sucrose filled LUVs extruded through either 0.03 or 1 μm pore membranes were incubated with a gel filtered soluble fraction from rat liver containing coatomer but excluding Arf and either no Arf1•GTP or 0.5 μM Arf1•GTPγS for 5 min at 30°C. The LUVs were separated from bulk solution by centrifugation and coatomer associated with the LUVs measured by immunoblotting. The data are the means and standard deviations from 7 determinations. *, different than binding to 0.03 μm LUVs, p<0.05. D. Time dependence and effect of p25 cargo peptide with small LUVs and 6.7 μM Arf1•GTP. Experiment similar to that described in A except Arf1•GTP concentration was in 100 fold excess of coatomer.