Gilles Lagniel

A Proteome Analysis of the Cadmium Response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.

Sulfur Sparing in the Yeast Proteome in Response to Sulfur Demand

Genome-wide studies have recently revealed the unexpected complexity of the genetic response to apparently simple physiological changes. Here, we show that when yeast cells are exposed to Cd(2+), most of the sulfur assimilated by the cells is converted into glutathione, a thiol-metabolite essential for detoxification. Cells adapt to this vital metabolite requirement by modifying globally their proteome to reduce the production of abundant sulfur-rich proteins. In particular, some abundant glycolytic enzymes are replaced by sulfur-depleted isozymes. This global change in protein expression allows an overall sulfur amino acid saving of 30%. This proteomic adaptation is essentially regulated at the mRNA level. The main transcriptional activator of the sulfate assimilation pathway, Met4p, plays an essential role in this sulfur-sparing response.

The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons

The heat shock transcription factor Hsf1p and the stress‐responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild‐type strains using [35S]‐methionine labelling and two‐dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25°C to 38°C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.

Involvement of Superoxide Dismutases in the Response of Escherichia coli to Selenium Oxides

Selenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response of Escherichia coli cells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions of sodA and sodB were controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.

Endoplasmic reticulum is a major target of cadmium toxicity in yeast

Cadmium (Cd2+) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd2+ cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Δire1 and Δhac1) unable to induce the UPR are hypersensitive to Cd2+, but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd2+ tolerance. The data also suggest that Cd2+‐induced ER stress and Cd2+ toxicity are a direct consequence of Cd2+ accumulation in the ER. Cd2+ does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd2+ activates the calcium channel Cch1/Mid1, which also contributes to Cd2+ entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.

Chromate Causes Sulfur Starvation in Yeast

Chromate is a widespread pollutant as a waste of human activities. However, the mechanisms underlying its high toxicity are not clearly understood. In this work, we used the yeast Saccharomyces cerevisiae to analyse the physiological effects of chromate exposure in a eukaryote cell model. We show that chromate causes a strong decrease of sulfate assimilation and sulfur metabolite pools suggesting that cells experience sulfur starvation. As a consequence, nearly all enzymes of the sulfur pathway are highly induced as well as enzymes of the sulfur-sparing response such as Pdc6, the sulfur-poor pyruvate decarboxylase. The induction of Pdc6 was regulated at the mRNA level and dependent upon Met32, a coactivator of Met4, the transcriptional activator of the sulfur pathway. Finally, we found that chromate enters the cells mainly through sulfate transporters and competitively inhibits sulfate uptake. Also consistent with a competition between the two substrates, sulfate supplementation relieves chromate toxicity. However, the data suggest that the chromate-mediated sulfur depletion is not simply due to this competitive uptake but would also be the consequence of competitive metabolism between the two compounds presumably at another step of the sulfur assimilation pathway.

H2O2 activates the nuclear localization of Msn2 and Maf1 through thioredoxins in Saccharomyces cerevisiae.

ABSTRACT The cellular response to hydrogen peroxide (H2O2) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H2O2 treatment. Contrary to the case with many stress conditions previously described for yeast, the H2O2-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H2O2 treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H2O2 signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Background: Intracellular concentration of glutathione, an essential sulfur compound, is tightly controlled. Results: In yeast, glutathione degradation is faster than previously published, and glutathione intracellular concentration does not affect its synthesis. Conclusion: Glutathione degradation is a key determinant of glutathione homeostasis. Significance: This work challenges notions on glutathione synthesis and degradation, which were considered as established. Glutathione (GSH) has several important functions in eukaryotic cells, and its intracellular concentration is tightly controlled. Combining mathematical models and 35S labeling, we analyzed Saccharomyces cerevisiae sulfur metabolism. This led us to the observation that GSH recycling is markedly faster than previously estimated. We set up additional in vivo assays and concluded that under standard conditions, GSH half-life is around 90 min. Sulfur starvation and growth with GSH as the sole sulfur source strongly increase GSH degradation, whereas cadmium (Cd2+) treatment inhibits GSH degradation. Whatever the condition tested, GSH is degraded by the cytosolic Dug complex (composed of the three subunits Dug1, Dug2, and Dug3) but not by the γ-glutamyl-transpeptidase, raising the question of the role of this enzyme. In vivo, both DUG2/3 mRNA levels and Dug activity are quickly induced by sulfur deprivation in a Met4-dependent manner. This suggests that Dug activity is mainly regulated at the transcriptional level. Finally, analysis of dug2Δ and dug3Δ mutant cells shows that GSH degradation activity strongly impacts on GSH intracellular concentration and that GSH intracellular concentration does not affect GSH synthesis rate. Altogether, our data led us to reconsider important aspects of GSH metabolism, challenging notions on GSH synthesis and GSH degradation that were considered as established.

Identification in Saccharomyces cerevisiae of a New Stable Variant of Alkyl Hydroperoxide Reductase 1 (Ahp1) Induced by Oxidative Stress

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern ofSaccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide.In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was activein vitro and was more resistant to inactivation bytert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.

Development of a new method for absolute protein quantification on 2-D gels

With the development of systems biology projects aimed at modeling the cell, accurate and absolute measurements of cellular protein concentrations are increasingly important. However, methods for absolute quantification at the proteomic level remain rare. Using the yeast Saccharomyces cerevisiae, we propose a new method based on the radioactive labeling with an 35S compound and 2‐D PAGE. The principle is simple: cells are grown for more than four generations in the presence of a unique sulfur source labeled at a defined specific radioactivity, ensuring that more than 90% of the proteins are labeled at the same specific radioactivity as the sulfur source. After separation of 35S‐labeled proteins on 2‐D gels, each protein is counted. The amount of each protein present in the gel is then calculated, from which is deduced the amount of each protein per cell. The method, limited to soluble and abundant proteins visible on 2‐D gels, is simple, precise and reproducible and does not require an internal standard. We use it to compare the amounts of proteins in two growth conditions: 100 μM sulfate or 500 μM methionine. Up to now, we only had transcriptional data on the expression of these proteins in both conditions.