Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth

Abstract

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2(E301K)) suppressed the ubi4Δ growth defect. The rpt2(E301K) mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2(E301K) mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.

Keywords: gene transcription; proteasome; transcription promoter; ubiquitin; yeast; zinc.

FIGURE 1.

Many mutants defective for ubiquitin metabolism show growth defects in low zinc. A, analysis of growth by flow cytometry. Untagged wild-type (BY4743) cells or isogenic mutants of the indicated genotype were mixed with approximately equal numbers of GFP-expressing BY4743 cells, inoculated into zinc-replete (ZnR; LZM + 100 μm ZnCl₂) or zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) medium, and grown for 15 generations prior to analysis. Approximately 20,000 total cells per culture were assessed for GFP fluorescence by flow cytometry, and the graph shows the percentage of untagged cells in the cultures. The data are the means from six replicate cultures, and the error bars indicate ±1 S.D. The dashed line at 50% indicates the expected result if both mutant and wild-type cells grew equally well. B, the mean percentages of untagged (control or mutant) cells in the zinc-deficient cultures divided by their percentages in the zinc-replete cultures. Ratios less than 1.0 indicate a stronger growth defect in zinc-deficient medium.

FIGURE 2.

ubi4Δ mutants exhibit a severe growth defect in zinc-deficient conditions. A and B, time course of wild-type (BY4741) and isogenic ubi4::KanMX4 mutant growth in zinc-replete (ZnR; A) or zinc-deficient (ZnD; B) medium (LZM with 100 or 1 μm added zinc, respectively). Cultures were inoculated with log phase cells to a starting A₆₀₀ of 0.01, and culture densities were recorded at the indicated times. C, effect of varying zinc concentration on growth of wild-type and ubi4Δ strains. Cultures were inoculated as described for A, and cell densities were measured after 48-h incubation. D, log phase cultures of wild-type and ubi4Δ mutant cells in zinc-replete medium were used to inoculate zinc-deficient medium. Cell viability was monitored over time by plating aliquots on YPD plates and counting colony-forming units following 2 days of incubation. E, the ubi4Δ growth defect in zinc-deficient medium is rescued specifically by zinc. Aliquots of LZM + 1 μm added zinc were supplemented with 100 μm ZnCl₂, FeCl₃, CuCl₂, MnCl₂, or CoCl₂ and inoculated with wild-type or ubi4Δ cells as described for A. Control (C) indicates LZM + 1 μm ZnCl₂ with no additional metal supplement. Cell densities were measured after 48 h. F, a ubi4Δ mutant was not hypersensitive to copper or iron deficiency. Aliquots of YPD were supplemented with 100 μm copper chelator bathocuproine disulfonic acid (BCS) or iron chelator bathophenanthroline disulfonate (BPS) with or without 100 μm added CuCl₂ or FeCl₃ and inoculated with wild-type or ubi4Δ cells as described for A. Control (C) indicates YPD with no additional metal or chelator supplement. Cultures were grown for 24 h, and cell densities were recorded. For all panels, data points represent averages of three independent cultures, and error bars denote ±1 S.D.

FIGURE 3.

UBI4 is the predominant source of ubiquitin for zinc-limited cells. A, wild-type (BY4741) and ubi4::KanMX4 cells were grown in zinc-deficient (D; LZM + 1 μm ZnCl₂) or zinc-replete (R; LZM + 100 μm ZnCl₂) medium. The abundance of ubiquitin and a loading control protein (Pgk1) was assayed by immunoblotting (one representative immunoblot is shown). Levels of ubiquitin conjugates (Ub-C) and monomeric ubiquitin (Ub-M) were quantified from three replicates. B, effect of zinc status and ubi4Δ mutation on expression of ubiquitin precursor genes and the ribosomal subunit gene RPL1B. Wild-type (BY4741), ubi4::KanMX4, and ubi4Δ rpt2^E301K (CWM280) cells were grown to log phase in SD medium and then used to inoculate zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) or -replete (ZnR; LZM + 100 μm ZnCl₂) medium at low starting densities. Cultures were maintained in log phase by dilution with fresh media for 24 h. The mRNA abundance of the indicated genes was then determined by RT-qPCR. Target transcript abundance was normalized to the average abundance of three control transcripts (18S rRNA, TAF10, and ACT1). All plotted data points represent the means of three replicates, and error bars denote ±1 S.D. A.U., arbitrary units.

FIGURE 4.

Mutation of proteasome subunit genes restored ubiquitin supply and growth in zinc-deficient ubi4Δ cells. A, a recessive rpt2 allele suppressed the ubi4Δ growth defect in zinc-deficient conditions. Wild-type (BY4741), ubi4::KanMX4, and ubi4Δ rpt2^E301K (CWM280) strains were grown to saturation in SD medium and inoculated into zinc-deficient (LZM + 1 μm ZnCl₂) cultures. Growth was monitored by measuring cell densities. Each data point represents the mean of three independent cultures, and error bars show ±1 S.D. B, effect of the rpt2^E301K suppressor mutation on ubiquitin accumulation. Strains listed in A were grown in zinc-deficient conditions and assayed for ubiquitin by immunoblotting. One representative immunoblot is shown. Pgk1 was detected as a loading control. Adjacent panels show quantitation of ubiquitin conjugates (Ub-C) and monomers (Ub-M) in three replicate immunoblots including the example shown. C, hypomorphic alleles of the PRE1 and PRE6 proteasomal subunit genes suppressed the ubi4Δ growth defect in zinc-deficient conditions. Cell densities of wild-type (BY4741), ubi4Δ (CWM260), pre1^DAmP, pre6 ^DAmP, ubi4Δ rpt2^E301K (CWM280), ubi4Δ pre1 ^DAmP (CWM278), and ubi4Δ pre6 ^DAmP (CWM279) strains were compared after 48 h of growth in zinc-deficient (LZM + 1 μm ZnCl₂) medium as described for A. Means of three replicates are shown; error bars represent ± 1 S.D. D, wild-type (BY4741), ubi4Δ (CWM260), and ubi4Δ pre1^DAmP (CWM278) cells were grown in zinc-deficient conditions and assayed for ubiquitin by immunoblotting. One representative immunoblot is shown, and Pgk1 was detected as a loading control. Adjacent panels show quantitation of ubiquitin conjugates (Ub-C) and monomers (Ub-M) in three replicate immunoblots including the example shown. A.U., arbitrary units.

FIGURE 5.

Reduced proteasome activity restores ubiquitin-dependent degradation. A, mODC accumulates following proteasome inhibition (PI). BY4743 pdr5Δ cells transformed with empty vector (pRS315; V) or p415-GFP-FLAG-mODC (pmODC; expressing FLAG-tagged mouse ornithine decarboxylase) were grown to log phase in LZM + 100 μm ZnCl₂ and treated for 1 h with DMSO carrier (−) or proteasome inhibitors (50 μm MG132 and 250 μm bortezomib; +) before harvesting. Protein extracts were analyzed by immunoblotting with anti-FLAG antibodies. B, mODC accumulates in rpt2^E301K mutants independently of zinc supply. Wild-type (BY4741), ubi4::KanMX4, and ubi4Δ rpt2^E301K (CWM280) strains transformed with p415-GFP-FLAG-mODC were grown to log phase in LZM + 100 μm ZnCl₂ (ZnR) or 1 μm ZnCl₂ (ZnD) prior to immunoblotting. C, GFP-VHL accumulates following proteasome inhibition. BY4743 pdr5Δ was transformed with empty vector (V) or pESC-LEU-GAL1p-GFP-VHL (pGFP-VHL; expressing GFP-tagged von Hippel-Lindau protein), and cells were assayed for mODC as described in A. The position of the GFP-VHL band is shown, and two nonspecific bands are indicated by asterisks. D, GFP-VHL is stabilized in zinc-deficient ubi4Δ cells and degraded in ubi4Δ rpt2^E301K. GFP-VHL accumulation was assayed as described for mODC in B. For all immunoblots in A–D, one blot representative of three replicates is shown, and Pgk1 was detected as a loading control. E, quantitation of GFP-VHL bands for three replicates, including the blot shown in D. Error bars denote ±1 S.D. F, expression of an Hsf1-regulated reporter gene in wild-type and ubi4Δ cells. Strains transformed with the pHSE-lacZ plasmid were grown to log phase in zinc-replete (ZnR; LZM + 100 μm ZnCl₂) or zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) medium and assayed for β-galactosidase activity. As a positive control for reporter activity, aliquots of zinc-replete cells were also subjected to heat shock at 37 °C for 1 h before assay (HS). G and H, wild-type (BY4743), tsa1Δ, and ubi4Δ diploid strains transformed with pHSP104-GFP were grown in LZM + 1000 μm ZnCl₂ (ZnR) or 1 μm ZnCl₂ (ZnD) medium for at least four generations, maintaining cell density below an A₅₉₅ of 0.4 by dilution with fresh media. Cells were examined by fluorescence microscopy to determine the proportion of cells with detectable GFP fluorescence that also displayed foci. Data points indicate the average of three replicates, and error bars show ±1 S.D. A.U., arbitrary units; M.U., Miller units.

FIGURE 6.

Zap1 induces UBI4 expression in zinc-deficient cells via an intragenic promoter. A, UBI4 induction parallels Zap1 target gene expression in zinc-deficient conditions. Log phase cultures of wild-type and ubi4Δ mutant cells grown in zinc-replete medium (LZM + 100 μm ZnCl₂) were used to inoculate zinc-deficient medium at time 0 (LZM + 1 μm ZnCl₂). Cells were harvested at the indicated times, and expression of the indicated genes was assayed by RT-qPCR. Each data point is the mean of three replicates, and the error bars denote ±1 S.D. B, location of potential Zap1 binding sites (ZREs) and transcription start sites in UBI4. The structure of the UBI4 gene is shown with its previously mapped promoter elements for Hsf1 (HSE)- and Msn2/Msn4 (STRE)-dependent regulation and TATA box. R1–R5 represent the ubiquitin repeats, and the locations of the potential intragenic ZREs are marked (Z1–Z3). The graphs below show the transcription start sites mapped by RLM-RACE using mRNA from zinc-replete (ZnR; LZM + 100 μm ZnCl₂) and zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) cells. C and D, short 0.5-kb UBI4 transcripts are dependent on zinc supply and Zap1. Northern blotting analysis of UBI4 expressed from the chromosomal locus (C) or a UBI4 plasmid bearing the full promoter (pRS315-UBI4; D) is shown. For C, wild-type, zap1Δ, and ubi4Δ haploid strains were transformed with pGK-ZRT1 to allow growth of zap1Δ mutants in low zinc and then grown in zinc-replete (R; LZM + 100 μm ZnCl₂) or zinc-deficient (D; LZM + 1 μm ZnCl₂) conditions prior to RNA extraction. For D, zap1Δ (CWM260) and zap1Δ ubi4Δ (CWM276) strains were transformed with both pRS315-UBI4 and pGK-ZRT1 prior to growth in zinc-deficient or -replete medium and RNA extraction. For C and D, UBI4 mRNA was detected using a probe specific for the 3′-UTR, and TAF10 was also detected as a loading control. E, mutation of the UBI4 HSE blocks UBI4 induction by heat shock but only in an msn2Δ msn4Δ mutant background. Wild-type (BY4741) or msn2Δ msn4Δ (DBY9435) strains were transformed with pRS315-UBI4 (wild-type UBI4) or pRS315-UBI4^HSEmut (HSE mutant). Cells were grown to log phase at 25 °C in SD medium or subjected to heat shock (HS; 20 min at 39 °C) before harvesting. Total RNA was isolated and assayed for UBI4 expression using RT-qPCR. Each data point is the mean of three replicates, and the error bars denote ±1 S.D. F, the UBI4 ZRE is essential for production of intragenic transcripts. Yeast strains ubi4Δ (CWM260) and ubi4Δ msn2Δ msn4Δ (CWM274) were transformed with either the wild-type UBI4 plasmid (pRS315-UBI4), a plasmid in which the three intragenic ZREs were mutated (pRS315-UBI4^ZREmut), the HSE mutant plasmid (pRS315-UBI4^HSEmut), or a plasmid in which both the ZREs and the HSE were mutated (pRS315-UBI4^HSEmutZREmut). Cells were grown and analyzed by Northern blotting as described for C and D. G, the ZREs are required for UBI4 induction in zinc deficiency. Mutant ubi4Δ (CWM260) and ubi4Δ zap1Δ (CWM276) cells were transformed with pGK-ZRT1 together with either the wild-type UBI4 plasmid (pRS315-UBI4) or the ZRE mutant plasmid (pRS315-UBI4^ZREmut). Cells were grown to log phase in zinc-replete (ZnR; LZM + 100 μm ZnCl₂) or zinc-deficient (ZnD; LZM + 1 μm ZnCl₂) medium prior to RNA extraction. Quantitative RT-PCR was used to measure total UBI4 transcripts using primers specific to the fifth ubiquitin repeat that is present in both full-length and short transcripts. A Zap1 target gene (ADH4) was also detected as a positive control for loss of Zap1 function. Each data point is the mean of three replicates, and the error bars denote ±1 S.D. A.U., arbitrary units.

FIGURE 7.

Zap1-dependent regulation of UBI4 is important for adaptation to zinc deficiency. A, a short UBI4 transcript generated from the GAL1 promoter complemented ubi4Δ in low zinc. The ubi4::KanMX4 strain was co-transformed with pGEV-HIS3 and either the empty vector pRS316-GAL1 (V) or pGAL1-UBI4S (S) or pGAL1-UBI4L (L) plasmids encoding short and long UBI4 transcripts, respectively. Strains were grown in SD medium prior to inoculating cultures of zinc-deficient (ZnD; LZM + 1 μm zinc) or -replete (ZnR; LZM + 100 μm zinc) medium to low initial densities. LZM also contained 1 μm β-estradiol to induce the GAL1 promoter. Cultures were grown for 15 (replete) or 63 h (deficient) before recording cell densities. B, mutation of the ZREs in the UBI4 coding sequence caused poor growth in zinc-deficient conditions. Wild-type (CWM286), ubi4Δ (CWM281), and ubi4^ZREmut (CWM285) strains were cultured in SD medium prior to inoculating zinc-deficient (ZnD; LZM + 0.5 μm zinc) or zinc-replete (ZnR; LZM + 100 μm zinc) medium. Cell densities were determined after 16 (zinc-replete) or 65 h (zinc-deficient). Each data point is the mean of three replicates, and the error bars denote ±1 S.D.