Disease relevance of DOA4

• Mutations in DOA4 cause loss of the free ubiquitin pool in cells under heat stress conditions, and extra copies of UB14 restore this pool without restoring coordination of replication [1].

High impact information on DOA4

• Doa4 appears to function late in the proteolytic pathway by cleaving ubiquitin from substrate remnants still bound to protease [2].
• Ubiquitin-specific processing protease 5 (Ubp5), the closest paralogue of Doa4, has no functional overlap [3].
• We show that an N-terminal segment of Doa4 is sufficient for endosome association [3].
• Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes [4].
• Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain [5].

Biological context of DOA4

• Ume3p destruction in response to oxidative stress, but not to ethanol treatment, requires DOA4 and UMP1, two factors required for 26S proteasome activity [6].
• The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage [7].
• 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 vacuolar protein sorting mutants [8].
• Both ubiquitination and down-regulation of the permease can be restored in npi2 cells by over-expression of ubiquitin [9].
• We conclude that a ubiquitin-mediated signaling event directly involving the ubiquitin hydrolase encoded by DOA4 is needed in S. cerevisiae to prevent uncoordinated DNA replication [1].

Anatomical context of DOA4

• Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase [8].
• Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant [10].

Associations of DOA4 with chemical compounds

• Deletion of DOA4, which causes decreased levels of available ubiquitin, severely decreases the rate of glucose-induced proteolysis, and this is suppressed by the overproduction of ubiquitin [11].
• To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation [12].

Regulatory relationships of DOA4

• Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown [13].
• Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant [12].
• In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4 [14].

Other interactions of DOA4

• Taken together, these findings support a model in which Plc1p mediates an oxidative-stress signal from the plasma membrane that triggers Ume3p destruction through a Doa4p-dependent mechanism [6].
• Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered [15].
• In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p [8].
• These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation [8].
• Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons [15].

References