Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: Crucial role of Doa4p ubiquitin isopeptidase

The Fur4p uracil permease, like most yeast plasma membrane proteins, underg oes ubiquitin-dependent endocytosis and is then targeted to the vacuole (eq uivalent to the mammalian lysosome) for degradation. The cell surface ubiqu itination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase, Ubi quitination of Fur4p occurs on two target lysines, which receive two ubiqui tin moieties Linked through ubiquitin Lys63, a type of Linkage (termed UbK6 3) different from that involved in proteasome recognition. We report that p ep4 cells deficient for vacuolar protease activities accumulate vacuolar un ubiquitinated Fur4p, In contrast, pep4 cells lacking the Doa4p ubiquitin is opeptidase accumulate ubiquitin-conjugated Fur4p, These data suggest that F ur;lp undergoes Doa4p-dependent deubiquitination prior to vacuolar degradat ion. Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane- bound ubiquitin conjugates, This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacu olar protein sorting mutants, Some of the small ubiquitin-linked peptides t hat are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation product s of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 l inkages. Doa4p thus appears to be involved in the deubiquitination of endoc ytosed plasma membrane proteins, some of them carrying UbK63 linkages.