The assembly of AP-3 adaptor complex-containing clathrin-coated vesicles on synthetic liposomes

The heterotetrameric adaptor protein complex AP-3 has been shown to functio n in the sorting of proteins to the endosomal/lysosomal system. However, th e mechanism of AP-3 recruitment onto membranes is poorly understood, and it is still uncertain whether AP-3 nucleates clathrin-coated vesicles. Using purified components, we show that AP-3 and clathrin are recruited onto prot ein-free liposomes and Golgi-enriched membranes by a process that requires ADP-ribosylation factor (ARF) and GTP but no other proteins or nucleotides. The efficiency of recruitment onto the two sources of membranes is compara ble and independent of the composition of the liposomes. Clathrin binding o ccurred in a cooperative manner as a function of the membrane concentration of AP-3. Thin-section electron microscopy of liposomes and Golgi-enriched membranes that had been incubated with AP-3, clathrin, and ARF . GTP showed the presence of clathrin-coated buds and vesicles. These results establish that AP-3-containing clathrin-coated vesicles form in vitro and are consis tent with AP-3-dependent protein transport being mediated by clathrin-coate d vesicles.