Figure 7.

Clathrin is recruited onto AP-3–containing membranes in a cooperative manner. Liposomes (200 μg/ml) prepared from soybean 20% PC material were incubated in 200-μl reactions containing increasing concentrations of AP-3 as noted and 10 μg/ml purified rat brain clathrin in the presence of 100 μM GTPγS. All reactions contained recombinant myristoylated ARF1 (50 μg/ml) and BSA (2.5 mg/ml). After recruitment reactions, membranes were recovered, separated by 12% SDS-PAGE, and transferred to nitrocellulose, and bound proteins were analyzed by immunoblotting with antibodies directed against the clathrin light chain (CLC) or AP-3 ς followed by densitometric quantitation. Clathrin binding in the absence of added AP-3 (inset, lane 1) was considered to be nonspecific and was subtracted from the other values. The amount of clathrin bound (in arbitrary units) to the liposomal membranes is plotted as a function of the amount of AP-3 bound (in arbitrary units). This experiment was done three times with similar results.