The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast - PubMed (original) (raw)
The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast
S Swaminathan et al. Mol Biol Cell. 1999 Aug.
Free PMC article
Abstract
Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.
Figures
Figure 1
Characteristics of stationary phase doa4Δ cells. (A) Analysis of ubiquitin levels in wild-type and doa4Δ cells at distinct stages in the growth cycle. Lanes 1–3, wild type (vector); lanes 4–6, wild type (YEp96); lanes 7–9, doa4Δ (vector); lanes 10–12, doa4Δ (YEp96). At the indicated time points, extracts for immunoblot analysis were made from the same yeast cultures that were used in B. Anti-ubiquitin antibody binding was detected by enhanced chemiluminescence, whereas anti-Pgk1 antibody binding was followed by [125I]protein A. The * and bracket indicate the positions of ubiquitinated species that accumulate in doa4Δ cells. The slight increase in ubiquitin (Ub) levels in wild-type cells carrying YEp96 at 6 h is most likely caused by upregulation of the CUP1 promoter in response to nutrient starvation (Tamai et al., 1994). The position of size standards (in kilodaltons) is indicated on the left. (B) Viability of wild-type and doa4Δ cells in the presence and absence of exogenously supplied ubiquitin. Rates of survival of wild-type (MHY501) and congenic doa4Δ (MHY623) cells carrying pES12 (vector) or YEp96 (CUP1UB) were measured by propidium iodide staining. Viability measurements were begun when yeast cultures were in logarithmic growth at an OD600 of ∼0.5, ∼10 h before growth levels off. The symbols denote the following strains: wild type (vector; ▪), wild type (CUP1UB; ▴), doa4Δ (vector; ●), and doa4Δ (CUP1UB; ♦).
Figure 2
The deubiquitinating activity of Doa4 is required to maintain ubiquitin levels in stationary phase. Anti-ubiquitin immunoblots (ECL detection) of doa4Δ (MHY623) cells carrying YCplac33-based plasmids expressing either the HA-tagged active site mutant, doa4C571S, or wild-type HA-Doa4. Lanes 1–3 and 4–6 show extracts from logarithmic phase cells (OD600 ∼ 1.0) and early stationary phase cells (OD600 ∼ 2.5), respectively. Positions of putative ubiquitin-peptide species in doa4Δ cells are indicated by the * and bracket; free ubiquitin and unanchored ubiquitin chain positions are also marked. The blot (middle) showing free ubiquitin alone is a shorter exposure of the top immunoblot. As a loading control, Pgk1 levels (bottom blot) were followed, with [125I]protein A as the secondary antibody. Very weak suppression of ubiquitin depletion is detected in cells expressing doa4C571S; the mechanistic basis of this suppression is unknown.
Figure 3
Suppression of the heat, canavanine, and cadmium sensitivities of doa4Δ cells by ubiquitin expression. Tenfold serial dilutions of wild-type (MHY501) or doa4Δ (MHY623) cells transformed with pES12 (vector), YEp96 (_CUP1_-UB), YEp90 (_CUP1_-UBK48R), and pTER103 (_CUP1_-UBK63R) were spotted onto selective plates containing 0.8 μg/ml canavanine sulfate or 30 μM CdCl2. No copper was added. Plates were incubated at either 30 or 38°C for 3–5 d.
Figure 4
Pulse-chase analysis of Ub-Pro-βgal (A), _Deg1_-Ura3 (B and C), and α2 (B–E) in doa4Δ cells supplemented with ubiquitin. Turnover rates of Ub-Pro-βgal and _Deg1_-Ura3 were followed in wild-type (MHY501) and doa4Δ (MHY623) cells carrying plasmids expressing Ub-Pro-βgal from the GAL1 promoter and _Deg1_-Ura3 from the α2 promoter. Endogenous α2 levels were followed in wild-type (MHY501) and doa4Δ (MHY623) cells transformed with a plasmid carrying _Deg1_-Ura3. Where indicated, 100 μM CuSO4 was added to cultures ∼3 h before harvesting. For D and E, the symbols denote the following strains: wild type (vector; ▪), wild type (CUP1UB; ▴), doa4Δ (vector; ●), and doa4Δ (CUP1UB; ♦).
Figure 5
Levels of ubiquitin in doa4Δ cells carrying the ubiquitin-encoding YEp96 multicopy plasmid. Anti-ubiquitin immunoblot analysis of logarithmic phase extracts from wild-type (MHY501) and doa4Δ (MHY623) cells cotransformed with plasmid-borne _Deg1_-Ura3 and either pES12 (vector) or YEp96 (CUP1UB). The same transformants were used for the pulse-chase analysis in Figure 4, B–D. [125I]protein A was used to detect ubiquitin and Pgk1 levels.
Figure 6
Analysis of ubiquitin synthesis rates in doa4Δ cells. (A) Northern analysis of UBI4. Total RNA was prepared from wild-type (MHY501) and doa4Δ (MHY623) cells grown in minimal media in early logarithmic phase (t = 0 h) and stationary phase (t = 8 and 14 h). UBI4 transcripts were detected using a 1.3-kb _Bst_XI-_Bcl_I fragment from UBI4. The blot was stripped and reprobed with a 560-bp _Cla_I fragment from the ACT1 gene. (B) Pulse labeling of ubiquitin. Radiolabeled ubiquitin was immunoprecipitated from wild-type (MHY501) and doa4Δ (MHY623) cells in late logarithmic phase (OD600 ∼ 1.5) with anti-ubiquitin antiserum, which does not efficiently immunoprecipitate ubiquitin–protein conjugates. Cells were pulse-labeled with 35S-TransLabel for 30 min.
Figure 7
Analysis of ubiquitin degradation in doa4Δ cells. (A) Rates of ubiquitin turnover determined in wild-type (MHY501) cells in late logarithmic phase (OD600 ∼ 1.5). Cycloheximide was added to a final concentration of 50 μg/ml, and at the indicated time points, extracts from equal aliquots of cells were processed for anti-ubiquitin immunoblotting. Ubiquitin and ubiquitin-containing species were detected using affinity-purified anti-ubiquitin antibodies (from C. Pickart) and [125I]protein A as the secondary antibody. (B) doa4Δ (MHY623) cells analyzed as described in A. (C) doa4Δ doa3-1 (MHY1063) cells analyzed as described in A. (D) doa4Δ pep4 prb1Δ (MHY1046) cells analyzed as described in A. (E) Quantitation of the rates of ubiquitin disappearance in A–D. Rates of ubiquitin turnover were derived from PhosphorImager (Molecular Dynamics) quantitation of the blots. (F) Quantitation of rates of Pgk1 disappearance in A–D. The symbols denote the following strains: wild type (▪), doa4Δ (▴), doa4Δ doa3-1 (●), doa4Δ pep4 prb1Δ (♦).
Figure 8
Suppression of doa4Δ defects by mutations in the endocytic pathway. (A) end3-1 suppresses accumulation of low molecular mass ubiquitinated species (bracket) in doa4Δ cells. Anti-ubiquitin immunoblotting (ECL detection) of extracts from cells in logarithmic growth is shown. Wild-type (MHY501), doa4Δ (MHY623), end3-1 (MHY1475), and end3-1 doa4Δ (MHY1479) cells were grown at 23°C. Cultures were shifted to 30°C for ∼2–3 h before extracts for immunoblot analysis were made. (B) Suppression of ubiquitin depletion in stationary phase doa4Δ cells by mutations in VPS24, VPS27, or END3 as assayed by anti-ubiquitin immunoblot analysis (ECL detection) is shown. Extracts were made from cells at an OD600 of ∼2.5. (C) Inhibition of the vacuolar protein–sorting pathway or of vacuolar proteases suppresses the accumulation of the low molecular mass ubiquitinated species (bracket) in doa4Δ cells. Anti-ubiquitin immunoblot analysis (ECL detection) of logarithmic phase cells from wild-type (MHY501), doa4Δ (MHY623), pep4 prb1Δ (MHY1061), doa4Δ pep4 prb1Δ (MHY1046), vps24Δ (MHY1232), vps24Δ doa4Δ (MHY1251), vps27Δ (MHY1269), and vps27Δ doa4Δ (MHY1275) cells is shown. Bottom, blots in A–C were stripped and reprobed with an anti-Pgk1 antibody, using [125I]protein A for antibody detection.
References
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