The response to heat shock and oxidative stress in Saccharomyces cerevisiae

Abstract

A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.

Figure 1

Regulation of Msn2/4 and Hsf1. Regulation of Msn2 by growth control proteins and oxidative stress is diagrammed. H₂O₂ triggers the oxidation of cytoplasmic thioredoxin proteins (Trx^ox) from their normally reduced status (Trx^red). This induces the recruitment of Msn2 into the nucleus where it can interact with its cognate binding site (stress response element, STRE) and activate expression of target gene transcription. Hsf1 is constitutively nuclear and prebound to many target genes containing heat shock elements (HSEs) in their promoters. Hsf1 is also regulated by the nutrient-sensing kinases, Snf1 and PKA, and by oxidative stress through unknown mechanisms.

Figure 2

Divergence in HSE architecture. Three different types of HSE have been described on the basis of spacing and positioning, as described in the text. Many genes within the Hsf1 regulon have annotated HSEs within the promoters; representative examples are shown (Yamamoto et al. 2005). n, any nucleotide.

Figure 3

CLIPs and HSPS. Venn diagram depicting the intersection between chaperone networks, based on the work of Frydman and coworkers, is shown (Albanese et al. 2006). See text for details.

Figure 4

Generation of ROS. The superoxide anion (O₂⁻) can be formed via electron leakage to oxygen from electron transport chains. Hydrogen peroxide (H₂O₂) is generated by the breakdown of superoxide catalyzed by superoxide dismutases (SODs). Hydrogen peroxide can be reduced by iron (Fe²⁺) in the Fenton reaction to produce the highly reactive hydroxyl radical. In the Haber-Weiss reaction, superoxide can donate an electron to iron (Fe³⁺), generating the hydroxyl radical and Fe²⁺, which can further reduce hydrogen peroxide. Various antioxidant enzymes, including catalases and peroxidases, detoxify hydrogen peroxide to prevent such ROS generation.

Figure 5

Oxidative stress. All organisms can be exposed to ROS during the course of aerobic metabolism or following exposure to ionizing radiation and radical-generating compounds. Antioxidant defense systems protect against ROS by detoxifying ROS as they are generated and by maintaining the intracellular redox environment in a reduced state. An oxidative stress occurs when the antioxidant and cellular survival mechanisms are unable to cope with the ROS or the damage caused by them. Oxidative stress can damage a wide variety of cellular components resulting in lipid peroxidation, protein oxidation, and genetic damage through the modification of DNA.

Figure 6

Yap1 folding and trafficking. A scheme for oxidant-specific folding and nuclear import of Yap1 is shown. The four key regulatory cysteine residues in their reduced conditions are indicated by the purple circles. Reduced Yap1 is imported into the nucleus at a basal rate but interacts with the exportin Crm1 and is returned to the cytoplasm in the absence of oxidative stress. In the presence of oxidants that directly act on Yap1 (like diamide), C-terminal cysteine residues are oxidized or modified (yellow circle) in a manner that prevents Crm1 from recognizing the nuclear export signal (blue triangle) in the Yap1 C terminus. Yap1 accumulates in the nucleus and activates gene expression. Finally, during challenge by peroxides that engage the Gpx3/Ybp1 folding pathway, Gpx3 is covalently linked to cysteine 598 of Yap1 by a disulfide bond (linked red circles). This modification, along with the participation of Ybp1, catalyzes an intramolecular folding reaction that leads to a dually disulfide bonded Yap1 form. This species also accumulates in the nucleus and can activate expression of genes required for the response to peroxide stress.

Figure 7

Comparison of cytoplasmic and mitochondrial thioredoxin systems. The yeast cytoplasmic thioredoxin system comprises two thioredoxins (Trx1-2) and a thioredoxin reductase (Trr1). The oxidized disulphide form of thioredoxin is reduced directly by NADPH and thioredoxin reductase (Trr1). The yeast cytosol contains three typical 2-Cys Prx’s (Tsa1, Tsa2, and Ahp1) but only Tsa1 is shown for simplicity. 2-Cys Prx’s are active as a dimer and contain two redox active Cys residues that are directly involved in enzyme activity. During reduction of hydroperoxides, a disulphide bond is formed between the peroxidatic cysteine (S_P) of one subunit and the resolving cysteine (S_R) from the other subunit of the dimer. This disulphide is reduced by thioredoxin. Yeast contains a complete mitochondrial thioredoxin system including a thioredoxin (Trx3) and thioredoxin reductase (Trr2). The substrate(s) of Trx3 is currently unknown. Mitochondria contain a single 1-Cys Prx (Prx1). The peroxidatic Cys residue of Prx1 is oxidized to the sulphenic form by hydroperoxides. Oxidized Prx1 is glutathionylated and reduced by Grx2 or Trr2 to regenerate the active enzyme. Reduced components are shown in blue.

Figure 8

The glutathione system. GSH is synthesized from its constituent amino acids via two ATP-dependent steps. In the first step, Gsh1 (γ-glutamylcysteine synthetase) catalyses the formation of the dipeptide γ-glutamylcysteine (γ-Glu-Cys) from glutamic acid and cysteine. In the second step, Gsh2 (glutathione synthetase) catalyses the ligation of γ-Glu-Cys with glycine. GSH can be oxidized to GSSG by ROS or in reactions catalyzed by Grx1–8 and Gto1–3. Reduced GSH is regenerated in an NADPH-dependent reaction catalyzed by Glr1 (glutathione reductase). GSH can be conjugated to xenobiotics (RX) by GSTs, including Gtt1–2 and Grx1–2. GSH conjugates are transported to the vacuole by the Ycf1 GS-X pump.

Figure 9

Control of translation initiation by Gcn2. Gcn2 is activated in response to diverse oxidative stress conditions that may occur via an accumulation of uncharged tRNA. Gcn2 phosphorylates eIF2, which converts it into a competitive inhibitor of the eIF2B guanine nucleotide exchange factor. Decreased eIF2B activity generates less eIF2 in the GTP-bound form, resulting in decreased ternary complex levels and inhibition of translation initiation.