Figure 4.

GTPγS-dependent recruitment of AP-3 and clathrin onto liposomes. Liposomes (200 μg/ml) prepared from soybean 20% PC material were incubated in a reaction mixture containing either AP-1–depleted (A) or complete (B) bovine adrenal cytosol at 5 mg/ml in the absence or presence of 100 μM GTPγS. Cytosol devoid of AP-1 was prepared by immunodepletion with the use of mAb 100/3 specific for the γ subunit of AP-1. Reactions were supplemented with soluble unassembled clathrin at the final concentration shown above each lane in nanograms. After recruitment reactions, membranes were recovered by centrifugation and bound proteins were fractionated by 12% SDS-PAGE. Portions of each blot were probed with mAb TD.1 specific for the clathrin heavy chain (CHC) or affinity-purified antibodies against the AP-1 μ and AP-3 ς coat proteins.