Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Abstract

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H 2 O 2 in the yeast Saccharomyces cerevisiae. Our data show that H 2 O 2 causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the ␣-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H 2 O 2 in a gcn2 mutant. Polysome analysis indicated that H 2 O 2 causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome-and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H 2 O 2 stress. For some candidate mRNAs, we show that a low concentration of H 2 O 2 results in increased protein production. In contrast, a high concentration of H 2 O 2 promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

Using Quantitative Redox Proteomics to Dissect the Yeast Redoxome

“…This finding is in excellent agreement with recent studies in yeast cells, in which the authors reported the inhibition of protein translation during peroxide stress, caused in part by defects in protein elongation and/or termination (31). By stalling elongating ribosomes, cells not only prevent formation of erroneous proteins but increase their ability to rapidly resume protein synthesis upon return to non-stress conditions (31). As such, down-regulation of protein translation during oxidative stress might provide an adaptive mechanism to increase oxidative stress resistance of cells (32).…”

“…We also detected oxidation-sensitive cysteines in the elongation factors Eft1 and Eft2, which previously have been characterized to be redox-sensitive (17,20). This finding is in excellent agreement with recent studies in yeast cells, in which the authors reported the inhibition of protein translation during peroxide stress, caused in part by defects in protein elongation and/or termination (31). By stalling elongating ribosomes, cells not only prevent formation of erroneous proteins but increase their ability to rapidly resume protein synthesis upon return to non-stress conditions (31).…”

Guanine Nucleotide Pool Imbalance Impairs Multiple Steps of Protein Synthesis and Disrupts GCN4 Translational Control in Saccharomyces cerevisiae

“…translation initiation (Ashe et al 2000). This approach allows the detection of an effect on ribosomal transit without the added complication of de novo translation initiation (Shenton et al 2006). WT and gua1-G388D cells were grown at 28°for 2 or 5 min after glucose withdrawal, cycloheximide was added for 5 min to fix the positions of elongating ribosomes, and the P/M ratios were quantified from polysome profiles.…”

Comprehensive Transcriptional Analysis of the Oxidative Response in Yeast

“…Clusters 6 and 7 (1391 genes) exhibit a large decrease of RA accompanied by TR down-regulation, which is more dramatic in the genes of cluster 6. The two clusters include most of the genes implicated in ribosome structure and biogenesis (RP and rRNA-processing enzymes), and this reflects the inhibition of protein synthesis after application of oxidative stress (21). However, TR inhibition alone is not sufficient to explain the decrease in RA in clusters 6 and 7 genes.…”

“…This proteome pattern, although limited to about 20% of all expressed proteins in yeast cells, approximately parallels the transcriptome pattern (1), which indicates that the oxidative stress response mainly occurs through regulation of mRNA level. However, recent studies demonstrate that inhibition of protein synthesis occurring after an oxidative stress is not only caused by transcriptional down-regulation of the translation machinery but also by inhibition of translation initiation due to dissociation of ribosomes from mRNA and a slower rate of ribosomal runoff along mRNA molecules (20,21). This illustrates the importance of the post-transcriptional level of regulation (22), which may also involve the regulation of mRNA translation efficiency by 3Ј-AU-rich elements, as is the case of the MFA2 mRNA in S. cerevisiae (23).…”