Figure 5.

AP-1 binding to liposomes requires cytosolic factor(s) recruited by ARF⋅GTPγS. (A) ARF-depleted Golgi membranes or liposomes prepared from soybean 20% PC material were incubated with 15 μg/ml soluble rat liver CCV coat fraction with or without ARF1 and GTPγS as noted. AP-1 binding was detected with the RY/1 antibody to μ1. The low level of AP-1 binding to the Golgi membranes in the absence of ARF1 may reflect incomplete depletion of ARF during the preincubation (see Materials and Methods). (A) Liposomes were primed with ARF⋅GTPγS in a first-stage incubation with 4 μM ARF1 and 100 μM GTPγS for 30 min at 37°C as noted. After recovery of the liposomes, either 5 mg/ml rat liver cytosol or 15 μg/ml soluble CCV coat fraction was mixed with the primed liposomes on ice for 15 min as indicated. The binding of AP-1 and ARF was determined by immunoblotting. (C) Liposomes were incubated with 4 μM ARF1 and 100 μM GTPγS, with or without 5 mg/ml AP-1-depleted bovine adrenal cytosol for 15 min at 37°C as noted. The liposomes were recovered and mixed with 15 μg/ml soluble CCV coat fraction with or without AP-1-depleted bovine adrenal cytosol on ice for 15 min. The binding of AP-1 and ARF was determined as above.