Figure 3.

ARF1 and ARF5 mediate recruitment of AP-3 onto liposomes and Golgi-enriched membranes. Liposomes (200 μg/ml) prepared from soybean 20% PC material (lower panel) or AP-1– and ARF-depleted Golgi-enriched membranes (50 μg/ml; upper panel) were incubated in a reaction mixture containing ARF-depleted bovine adrenal cytosol at 5 mg/ml supplemented with recombinant myristoylated ARF1 or ARF5 at a final concentration of 50 μg/ml. Nucleotides were added as indicated above each lane. Final guanine nucleotide concentrations were 1 mM GTP and 100 μM GTPγS. The ATP regeneration system consisted of 1 mM ATP, 10 mM creatine phosphate, and 5 U/ml creatine kinase. After recruitment reactions, membranes were recovered by centrifugation and subjected to SDS-PAGE as described in Figure 1. Portions of each blot were probed with antibodies specific for the AP-1 μ and AP-3 ς coat proteins.