Gcn4 is required for the response to peroxide stress in the yeast Saccharomyces cerevisiae

Abstract

An oxidative stress occurs when reactive oxygen species overwhelm the cellular antioxidant defenses. We have examined the regulation of protein synthesis in Saccharomyces cerevisiae in response to oxidative stress induced by exposure to hydroperoxides (hydrogen peroxide, and cumene hydroperoxide), a thiol oxidant (diamide), and a heavy metal (cadmium). Examination of translational activity indicates that these oxidants inhibit translation at the initiation and postinitiation phases. Inhibition of translation initiation in response to hydroperoxides is entirely dependent on phosphorylation of the alpha subunit of eukaryotic initiation factor (eIF)2 by the Gcn2 kinase. Activation of Gcn2 is mediated by uncharged tRNA because mutation of its HisRS domain abolishes regulation in response to hydroperoxides. Furthermore, Gcn4 is translationally up-regulated in response to H(2)O(2), and it is required for hydroperoxide resistance. We used transcriptional profiling to identify a wide range of genes that mediate this response as part of the Gcn4-dependent H(2)O(2)-regulon. In contrast to hydroperoxides, regulation of translation initiation in response to cadmium and diamide depends on both Gcn2 and the eIF4E binding protein Eap1. Thus, the response to oxidative stress is mediated by oxidant-specific regulation of translation initiation, and we suggest that this is an important mechanism underlying the ability of cells to adapt to different oxidants.

Figure 1.

Inhibition of protein synthesis is a common response to oxidative stress. (A) Wild-type cells were grown to exponential phase in minimal SD media, and protein synthesis was measured by pulse labeling with [³⁵S]cysteine/methionine for 5 min. Data are shown for untreated cultures (100%) and after treatments with CHP, diamide, or cadmium for 15 min. (B) Oxidative stress inhibits translation initiation. Polyribosome traces are shown for the wild-type strain treated with the indicated concentrations of oxidants for 15 min. The peaks that contain the small ribosomal subunit (40S), the large ribosomal subunit (60S), and both subunits (80S) are indicated by arrows. The polysome peaks generated by 2, 3, 4, 5, etc., 80S ribosomes on a single mRNA are marked with a line. Numbers in brackets are the p:m ratio determined by the ratio between the area under the monosome to the polysome peaks. Representative data are shown from repeat experiments.

Figure 2.

Phosphorylation of eIF2α in response to oxidative stress. (A) Western blot analysis of eIF2α and eIF2α-P. The wild-type strain was grown to exponential phase in minimal SD media and treated with 0.5 mM H₂O₂, 0.1 mM CHP, 0.2 mM cadmium, or 4.0 mM diamide for 15 min. (B) Phosphorylation is dependent on the presence of GCN2. A gcn2 mutant was exposed to the same oxidant treatments as described above. Representative data are shown from repeat experiments.

Figure 3.

Gcn2 mediates an inhibition of translation initiation in response to different oxidants. (A) Polysome traces are shown for the wild-type and gcn2 mutant strain after treatments with oxidants as described for Figure 2A. Numbers in brackets are the p:m ratio. (B) Wild-type and gcn2 mutant cells were grown to exponential phase, treated with oxidants as described for Figure 2A, and protein synthesis measured as described for Figure 1A. Representative data are shown from repeat experiments.

Figure 4.

Oxidant-mediated inhibition of translation initiation requires the HisRS domain of Gcn2. Polysome traces are shown for a prototrophic gcn2 mutant strain (SCY51) containing wild-type, gcn2-S577A, or gcn2-m2 alleles of GCN2. Strains were treated with 0.5 mM H₂O₂, 0.1 mM CHP, 1.0 mM cadmium, or 10.0 mM diamide for 15 min. Numbers in brackets are the p:m ratio. Representative data are shown from repeat experiments.

Figure 5.

Phosphorylation of eIF2α in gcn2 mutants in response to oxidative stress. Western blot analysis of eIF2α and eIF2α-P for the prototrophic gcn2 mutant strain (SCY51) containing an empty vector, wild-type, gcn2-S577A, or gcn2-m2 alleles of GCN2. Strains was grown to exponential phase in minimal SD media and treated with H₂O₂ (A) or cadmium and diamide (B) as described for Figure 4. Note that the panel on the right of B has been overexposed relative to the panel on the left to confirm that no eIF2α is detected in a gcn2-m2 mutant. Representative data are shown from repeat experiments.

Figure 6.

Eap1 is required for the inhibition translation initiation in response to cadmium or diamide. (A) Polysome traces are shown for wild-type, gcn2, eap1, and gcn2 eap1 mutant strains treated with cadmium or diamide. Translation initiation is less inhibited in the absence of EAP1 or GCN2, and no inhibition is observed in the gcn2 eap1 double mutant. (B) Polysome analysis of the EAP1^m3 binding mutant shows that cadmium induced inhibition of translation initiation is dependent on the eIF4E binding site. Numbers in brackets are the p:m ratio. Representative data are shown from repeat experiments.

Figure 7.

Gcn4 is required for resistance to hydrogen peroxide. (A) Sensitivity to oxidative stress was determined by spotting strains onto YEPD plates containing various concentrations of H₂O₂, cadmium or diamide. Cultures of wild-type, gcn2, and gcn4 mutant strains were grown to stationary phase, and the A₆₀₀ was adjusted to 1, 0.1, 0.01, or 0.001 before spotting onto plates containing various concentrations of oxidants. Growth was monitored after 3-d incubation at 30°C. Results are shown for plates containing no oxidant (YEPD), 4.0 mM H₂O₂, 0.1 mM CHP, 10 μM cadmium, and 2.5 mM diamide. No growth of any of the strains was observed at higher concentrations of cadmium or diamide. Western blots are shown probed for Gcn4-myc in a wild-type and gcn2 mutant in response to H₂O₂ (B) and for the wild-type strain in response to cadmium and diamide (C). Induction of Gcn4 in response to an amino acid starvation is shown as a control (−AA). An amino starvation was induced by shifting cells to minimal media lacking any amino acids for 2 h. The same blots were probed with an antibody against Tef1 (eEF1α) as a loading control. Representative data are shown from repeat experiments.

Figure 8.

Identification of the Gcn4-dependent H₂O₂ regulon. Genes were identified which displayed significantly different responses to hydrogen peroxide in the gcn2 or gcn4 mutants and assigned to one of eight clusters by using a k-means clustering algorithm (left). The data for each cluster are represented as a profile of the z-transformed (for each probe set, the mean set to 0 and SD to 1 using maxdView), log 2 values for the mean of each condition. Error bars indicate the maximum and minimum values within each group. The number of genes in each cluster is specified. Displayed on the right is the same z-transformed data shown as an Eisen color plot. Red and green indicate positive and negative change from zero, respectively, with color intensity indicating the degree of deviation.

Figure 9.

Validation of microarray data. The microarray data were confirmed by RT-PCR analysis for representative genes. The numbers shown are–fold induction in response to H₂O₂ in WT, gcn4, and gcn2 mutant cells from the RT-PCR and microarray analyses. Black bars denote data from the RT-PCR, and gray bars denote data from the microarray analysis.

Figure 10.

Comparison of peroxide-regulated genes with the Gcn4-dependent response to amino acid starvation. The peroxide expression data were compared with previous amino acid starvation expression data (Natarajan et al., 2001). Scatter plots show a comparison of changes in gene expression due to H₂O₂ exposure and 3-AT treatment in wild-type (left) and gcn4 mutant (right) strains. Genes denoted with a square symbol indicate 64 genes whose expression was altered by greater than twofold in the wild-type strain, but not in the gcn4 mutant.