Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Organisms growing in aerobic environments must cope with Reactive Oxygen Species (ROS). Although ROS damage all the cellular macromolecules, they play a central role in a range of biological processes requiring a tight control of redox homeostasis. It is achieved by antioxidant systems involving a large collection of enzymes that scavenge or degrade the ROS produced endogenously during cell growth. In addition to this enzymatic protection against ROS, cells also contain small antioxidant molecules, such as glutathione (GSH). With an intracellular concentration between 1 and 10mM, GSH is the most abundant non-protein thiol in the cell and is considered as the major redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing different amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear DNA and nuclear functions are protected against oxidative injury, as exemplified by low mutation frequency, moderate histone carbonylation, activation of the checkpoint kinase Rad53 and of the H2O2 transcriptional response. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Glutathione (GSH) is considered the most important redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing various amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear components and activities are protected against oxidative injury. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Glutathione degradation is a key determinant of glutathione homeostasis.

Glutathione (GSH) has several important functions in eukaryotic cells, and its intracellular concentration is tightly controlled. Combining mathematical models and (35)S 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 (Cd(2+)) 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.

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 (35)S 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 (35)S-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 microM sulfate or 500 microM methionine. Up to now, we only had transcriptional data on the expression of these proteins in both conditions.

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.

[Stoichiometric, my dear Watson!]. / Stoechiométrique, mon cher Watson!

Living organisms can be seen as complex chemicals interacting with their environment through chemical reactions. As such, they are subjected to the laws of stoichiometry: their constitutive elements (atoms) cannot be created (they must be found in their environment) nor destroyed. Acknowledging these rules led ecologists to the concept of "biological stoichiometry". In this review, I want to show that combining (1) the study of the elemental composition of biopolymers and (2) the ecologist's point of view, particularly the concept of biological stoichiometry, benefits molecular biology. In particular, this coupled approach unveils parts of the history of organisms, helps interpreting transcriptional profiles and sheds a different light on the growth of carcinogenic tumors.

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription.

We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10(3) over the error rate determined solely by selectivity (1.8 x 10(-4)). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.

Variation among species in proteomic sulphur content is related to environmental conditions.

The elemental composition of proteins influences the quantities of different elements required by organisms. Here, we considered variation in the sulphur content of whole proteomes among 19 Archaea, 122 Eubacteria and 10 eukaryotes whose genomes have been fully sequenced. We found that different species vary greatly in the sulphur content of their proteins, and that average sulphur content of proteomes and genome base composition are related. Forces contributing to variation in proteomic sulphur content appear to operate quite uniformly across the proteins of different species. In particular, the sulphur content of orthologous proteins was frequently correlated with mean proteomic sulphur contents. Among prokaryotes, proteomic sulphur content tended to be greater in anaerobes, relative to non-anaerobes. Thermophiles tended to have lower proteomic sulphur content than non-thermophiles, consistent with the thermolability of cysteine and methionine residues. This work suggests that persistent environmental growth conditions can influence the evolution of elemental composition of whole proteomes in a manner that may have important implications for the amount of sulphur used by living organisms to build proteins. It extends previous studies that demonstrated links between transient changes in environmental conditions and the elemental composition of subsets of proteins expressed under these conditions.

Determinants of the ubiquitin-mediated degradation of the Met4 transcription factor.

In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCF(Met30) ubiquitin ligase. The mechanism whereby SCF(Met30)-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Delta strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCF(Met30) activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations.

Inducible dissociation of SCF(Met30) ubiquitin ligase mediates a rapid transcriptional response to cadmium.

Activity of the Met4 transcription factor is antagonized by the SCF(Met30) ubiquitin ligase by degradation-dependent and degradation-independent mechanisms, in minimal and rich nutrient conditions, respectively. In this study, we show that the heavy metal Cd2+ over-rides both mechanisms to enable rapid Met4-dependent induction of metabolic networks needed for production of the antioxidant and Cd2+-chelating agent glutathione. Cd2+ inhibits SCF(Met30) activity through rapid dissociation of the F-box protein Met30 from the holocomplex. In minimal medium, dissociation of SCF(Met30) complex is sufficient to impair the methionine-induced degradation of Met4. In rich medium, dissociation of the SCF(Met30) complex is accompanied by a deubiquitylation mechanism that rapidly removes inhibitory ubiquitin moieties from Met4. Post-translational control of SCF(Met30) assembly by a physiological stress to allow rapid induction of a protective gene expression program represents a novel mode of regulation in the ubiquitin system.

Intimate evolution of proteins. Proteome atomic content correlates with genome base composition.

Discerning the significant relations that exist within and among genome sequences is a major step toward the modeling of biopolymer evolution. Here we report the systematic analysis of the atomic composition of proteins encoded by organisms representative of each kingdoms. Protein atomic contents are shown to vary largely among species, the larger variations being observed for the main architectural component of proteins, the carbon atom. These variations apply to the bulk proteins as well as to subsets of ortholog proteins. A pronounced correlation between proteome carbon content and genome base composition is further evidenced, with high G+C genome content being related to low protein carbon content. The generation of random proteomes and the examination of the canonical genetic code provide arguments for the hypothesis that natural selection might have driven genome base composition.