The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways

Abstract

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

Figure 1

Suppression of the doa4 degradation defect by did mutations. Deg1-βgal pulse-chase analysis in doa4-1 and did doa4-1 mutants. Average Deg1-βgal half-lives from one to five independent measurements were as follows: wild type, 11 min; did1-1 doa4-1, 13 min; did2-1 doa4-1, 12 min; did4-1 doa4-1, 14 min; and did5-1 doa4-1, 22 min. Half-lives were calculated from quantitative Phosphorimager data from time points up to 30 min. During this time, degradation is close to first order; as described originally by Hochstrasser and Varshavsky (1990), Deg1-βgal degradation rates slow gradually, particularly with long chase times. The reporter protein was expressed from a chromosomally integrated copy of Deg1-lacZ. 90 kD indicates a proteolytic fragment of Deg1-βgal generated in cells in which the reporter protein is short-lived.

Figure 2

Mutations in DID genes suppress the canavanine- and temperature-sensitivity of the doa4 mutant and cause phenotypic abnormalities associated with mutants with impaired intracellular proteolysis. Growth of did single and doa4 did double mutants on canavanine-containing media (A and B) and at increased temperatures (C and D) is shown.

Figure 3

Did2 is encoded by YKR035w-A, an ORF embedded in another gene. (A) Protein expression from YKR035w-A assayed by epitope tagging and immunoprecipitation from radiolabeled yeast cells. Molecular mass standards are indicated (kDa). (B) A mutation that disrupts the predicted protein-coding sequence of YKL035w-A but not that of YKR035c on the opposite strand cannot complement a did2Δ mutation. Cells transformed with the indicated plasmids were plated on 1.0 μg/ml canavanine at 30°C.

Figure 4

The Did1–Did4 proteins are related. (A) Sequence alignment of Did1 (YLR025w), Did2 (YKR035w-A), Did3 (YKL041w), and Did4 (YKL002w). (B) Alignment of Did2 with an alternative reading frame in the human PRSM1 gene (Scott et al., 1996). (C) Similarity between yeast Did4 and the human BC-2 breast adenocarcinoma marker protein (GenBank accession number 2828147).

Figure 5

Substrate-specific suppression of doa4 degradation defects by mutations in the DID genes. Pulse-chase analysis of α2 (A), Leu-βgal (B), and Ub-Proβgal (C) in congenic wild-type, doa4Δ, and doa4Δ didΔ strains. For analysis of α2 degradation, cells expressed the transcriptional repressor from the chromosomal MAT locus. For analysis of Leu-βgal and Ub-Proβgal degradation, expression of plasmid-derived fusion proteins was induced with galactose (Bachmair et al., 1986).

Figure 6

Suppression by did mutations of the small ubiquitinated species that accumulate in doa4 cells and accumulation of ubiquitinated proteins in did single mutants. Extracts from a congenic set of strains were analyzed by anti-ubiquitin Western immunoblotting. Proteins were separated on a 16% Tricine gel; doa4 cell-specific species are marked with an asterisk. Monoubiquitinated species are detected poorly with the anti-ubiquitin mAb. The bottom panels show a section of the Coomassie blue–stained filters used for immunoblotting to indicate the relative loading of protein in each lane.

Figure 7

Vacuolar protein-sorting defect of did mutants. (A) Aberrant secretion of a vacuolar enzyme in the did mutants. Sorting of the vacuolar hydrolase CPY was determined by pulse-chase analysis. Cells spheroplasts were labeled for 10 min with ³⁵S-TransLabel, and after a 30-min chase period, CPY from intracellular (I) and extracellular (E) fractions was precipitated with anti-CPY antibodies and analyzed by SDS-PAGE. The intermediate secreted from did1 cells had a slightly slower electrophoretic mobility than did the p2 form observed in wild-type cells and the other strains. This might be due to altered glycosylation of CPY in the did1 mutant, although this has not been tested. (B) FM 4-64 staining of did1–did4 mutants. Cells were labeled with FM 4-64 for 10 min and chased for 1 h at 30°C. The cells were then viewed with epifluorescence optics.

Figure 8

Ste3 receptor turnover in doa4Δ and didΔ mutants as measured by anti-Ste3 immunoblotting after the addition of the protein synthesis inhibitor cycloheximide. Logarithmically growing cells at 30°C were sampled (250 μl) at the indicated times.

Figure 9

Mutations in genes involved in late stages of vacuolar protein sorting compensate for doa4 deficiency in yeast. (A) Pulse-chase analysis of Deg1-βgal degradation in doa4Δ vps45Δ, doa4Δ vps27Δ, and doa4Δ vps33Δ double mutants. (B) Pulse-chase analysis of α2 degradation in doa4Δ ypt1-A136D double mutant.

Figure 10

Did2 and Doa4 accumulate in the class E compartment in vps4 mutants. (A) Cellular distribution of T7-tagged Did2 and CPY analyzed by indirect immunofluorescence and confocal microscopy. Cells were grown at 24°C and then incubated for 30 min at 37°C. (B) Expression of Doa4–GFP in wild-type and vps4Δ cells measured by anti-GFP immunoblot analysis. (C) Localization by intrinsic fluorescence of Doa4–GFP in wild-type, vps4Δ, and did3Δ vps4Δ cells. (D) Immunofluorescence colocalization of Doa4–GFP and Did3–HA in wild-type and vps4Δ cells by anti-GFP and anti-HA antibodies. All samples were viewed by laser scanning confocal microscopy.