Cytosolic and membrane-associated proteins involved in the recruitment of AP-1 adaptors onto the trans-Golgi network

The AP-1 adaptor complex is recruited from the cytosol onto the trans-Golgi network membrane, where it co-assembles with clathrin into a coat that drives vesicle budding. The GTPase ARF1 has been shown to be required for AP-1 recruitment, and here we demonstrate that we can reconstitute full GTP gamma S-dependent recruitment of adaptors onto an enriched trans-Golgi network membrane fraction by adding purified AP-1 and recombinant myristylated ARF1, indicating that these are the only soluble proteins required for binding. To identify some of the membrane proteins involved in recruitment, we have incubated permeabilized metabolically labeled cells with cytosol under conditions that promote adaptor binding, then cross-linked the samples with 3,3'-dithiobis(sulfosuccinimidylproprionate), denatured by boiling in SDS, and immunoprecipitated with antibodies against the various subunits. Under these conditions, the adaptor subunits co-precipitate not only with each other and with clathrin, but also with three novel proteins: p75, which is specifically cross-linked to gamma-adaptin; p80, which is specifically cross-linked to beta'-adaptin; and p60, which is specifically cross-linked to AP47. These proteins are all candidates for components of the adaptor docking site on the trans-Golgi network membrane.