CYTOSOLIC AND MEMBRANE-ASSOCIATED PROTEINS INVOLVED IN THE RECRUITMENT OF AP-1 ADAPTERS 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 coa t that drives vesicle budding. The GTPase ARF1 has been shown to be re quired for AP-1 recruitment, and here we demonstrate that we can recon stitute full GTP gamma S-dependent recruitment of adaptors onto an enr iched trans-Golgi network membrane fraction by adding purified AP-1 an d recombinant myristylated ARF1, indicating that these are the only so luble proteins required for binding. To identify some of the membrane proteins involved in recruitment, we have incubated permeabilized meta bolically labeled cells with cytosol under conditions that promote ada ptor binding, then cross-linked the samples with 3,3'-dithiobis(sulfos uccinimidylproprionate), denatured by boiling in SDS, and immunoprecip itated with antibodies against the various subunits. Under these condi tions, the adaptor subunits co-precipitate not only with each other an d with clathrin, but also with three novel proteins: p75, which is spe cifically cross-linked to gamma-adaptin; p80, which is specifically cr oss-linked to beta'-adaptin; and p60, which is specifically cross-link ed to AP47. These proteins are all candidates for components of the ad aptor docking site on the trans-Golgi network membrane.