Vps10p transport from the trans-golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins betw een the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Ap12p, one large subun it of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vac uolar protein receptor, are associated with clathrin molecules. Vps10p pack aging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that b lock Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Ap12p. Interestingly, a Vps10C(t)Deltap mutant lacking i ts C-terminal cytoplasmic domain, the portion of the receptor responsible f or carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coa ts. The Vps10p C-terminal domain appears to play a principal role in retrie val of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.