Repression of Sulfate Assimilation Is an Adaptive Response of Yeast to the Oxidative Stress of Zinc Deficiency

Heme oxygenase-1 (HO-1) degrades heme and protects cells from oxidative challenge. This antioxidant activity is thought to result from the HO-1 enzymatic activity, manifested by a decrease in the concentration of the pro-oxidant substrate heme, and an increase in the antioxidant product bilirubin. Using a global transcriptional approach, and yeast as a model, we show that HO-1 affords cellular protection via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense, rather than via its oxygenase activity. Like mammalian cells, yeast responds to oxidative stress by expressing its HO-1 homolog and, compared with the wild type, heme oxygenase-null mutant cells have increased sensitivity toward oxidants that is rescued by overexpression of human HO-1 or its yeast homolog. Increased oxidant sensitivity of heme oxygenase-null mutant cells is explained by a decrease in the expression of the genes encoding ␥-glutamylcysteine synthetase, glutathione peroxidase, catalase, and methionine sulfoxide reductase, because overexpression of any of these genes affords partial, and overexpression of all four genes provides complete, protection to the null mutant. Genes encoding antioxidant enzymes represent only a small portion of the 480 differentially expressed transcripts in heme oxygenase-null mutants. Transcriptional regulation may be explained by the nuclear localization of heme oxygenase observed in oxidantchallenged cells. Our results challenge the notion that HO-1 functions simply as a catabolic and antioxidant enzyme. They indicate much broader functions for HO-1, the unraveling of which may help explain the multiple biological responses reported in animals as a result of altered HO-1 expression.

Section: Discussionmentioning

confidence: 99%

The Yeast Homolog of Heme Oxygenase-1 Affords Cellular Antioxidant Protection via the Transcriptional Regulation of Known Antioxidant Genes

Collinson

Wimmer-Kleikamp

Gerega³

et al. 2011

“…Adaptive zinc-regulated genes include those involved in fermentation (ADH1 and ADH4) (16), phospholipid synthesis (PIS1, CKI1, and EKI1) (17,18), sulfur metabolism (MET30) (19), resistance to oxidative stress (CTT1), and NADPH production (TKL2).…”

Section: Fet4mentioning

confidence: 99%

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

MacDiarmid

Taggart

Jeong

et al. 2016

Self Cite

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 ubi4⌬ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4⌬ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2 E301K ) suppressed the ubi4⌬ 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 ubi4⌬ 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 ϳ5-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 ubi4⌬ 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.Zinc is an essential element with diverse roles in biology. Unlike transition metals, such as iron and copper, zinc ions (Zn 2ϩ ) are not redox-active under physiological conditions and do not play a direct role in redox reactions. Zn 2ϩ strongly interacts with ligands, such as cysteine, histidine, and acidic amino acids in proteins (1). When bound by three or fewer ligands, Zn 2ϩ can act as a Lewis acid to facilitate catalysis by diverse classes of enzymes, including oxidoreductases, transferases, and hydrolases (2). In contrast, binding of Zn 2ϩ by four ligands produces a relatively inert, structurally rigid tetrahedral complex, which provides stability to many classes of protein domains (1-3). Because of its catalytic and structural roles, Zn 2ϩ has been estimated to be required for the folding and function of ϳ10% of proteins encoded by eukaryotic genomes and ϳ5% of proteins in prokaryotes (4, 5). One abundant example is the enzyme alcohol dehydrogenase, which contains two Zn 2ϩ atoms per subunit, one serving in catalysis and the other playing a structural role (1, 6, 7). Consistent with the importance of zinc to Adh 2 folding, mutants of Adh lacking structural zinc site ligands are unstable and quickly degraded in ...

“…Other deletion alleles were made using overlap PCR and inserting these fragments via homologous recombination into BstXI-linearized pYef2L-Zap1-6x-myc. Reporters pDg2 (ZRE-lacZ) (13), ZRT1-lacZ, DPP1-lacZ, ZPS1-lacZ, ZRC1-lacZ (12), TSA1-lacZ (25), MET30-lacZ (26), and ZRT3-lacZ (27) were all constructed as described previously. pGEV-HIS3 (23) was used to control expression of Zap1 from the GAL1 promoter by the addition of ␤-estradiol.…”

Section: ⌬182-502mentioning

confidence: 99%

Roles of Two Activation Domains in Zap1 in the Response to Zinc Deficiency in Saccharomyces cerevisiae

Frey

Eide

2011

Self Cite

Previous studies suggested that the zinc-responsive Zap1 transcriptional activator directly regulates the expression of over 80 genes in Saccharomyces cerevisiae. Many of these genes play key roles to enhance the ability of yeast cells to grow under zinc-limiting conditions. Zap1 is unusual among transcriptional activators in that it contains two activation domains, designated AD1 and AD2, which are regulated independently by zinc. These two domains are evolutionarily conserved among Zap1 orthologs suggesting that they are both important for Zap1 function. In this study, we have examined the roles of AD1 and AD2 in low zinc growth and the regulation of Zap1 target gene expression. Using alleles that are specifically disrupted for either AD1 or AD2 function, we found that these domains are not redundant, and both are important for normal growth in low zinc. AD1 plays the primary role in zinc-responsive gene regulation, whereas AD2 is required for maximal expression of only a few target promoters. AD1 alone is capable of driving full expression of most Zap1 target genes and dictates the kinetics of Zap1 gene induction in response to zinc withdrawal. Surprisingly, we found that AD1 is less active in zinc-limited cells under heat stress and AD2 plays a more important role under those conditions. These results suggest that AD2 may contribute more to Zap1 function when zinc deficiency is combined with other environmental stresses. In the course of these studies, we also found that the heat shock response is induced under conditions of severe zinc deficiency.