Saccharomyces cerevisiae chronological life span (CLS) is determined by a wide variety of environmental and genetic factors. Nutrient limitation without malnutrition, i.e. dietary restriction, expands CLS through the control of nutrient signaling pathways, of which TOR/Sch9 has proven to be the most relevant, particularly under nitrogen deprivation. The use of prototrophic wine yeast allows a better understanding of the role of nitrogen in longevity in natural and more demanding environments, such as grape juice fermentation. We previously showed that acetyltransferase Gcn5, a member of the SAGA complex, has opposite effects on CLS under laboratory and winemaking conditions, and is detrimental under the latter. Here we demonstrate that integrity of the SAGA complex is necessary for prolonged longevity, as its dismantling by SPT20 deletion causes a drop in CLS under both laboratory and winemaking conditions. The sch9Δ mutant is long-lived in synthetic SC medium, as expected, and the combined deletion of GCN5 partially suppresses this phenotype. However it is short-lived in grape juice, likely due to its low nitrogen/carbon ratio. Therefore, unbalance of nutrients can be more relevant for life span than total amounts of them. Deletion of RTG2, which codes for a protein associated with Gcn5 and is a component of the mitochondrial retrograde signal, and which communicates mitochondrial dysfunction to the nucleus, is detrimental under laboratory, but not under winemaking conditions, where respiration seems not so relevant for longevity. Transcription factor Rgm1 was found to be a novel CLS regulator Sch9-dependently.

Peroxiredoxins are H2O2 scavenging enzymes that also carry H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro. We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2. The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo. We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

Thioredoxins are small proteins that regulate the cellular redox state, prevent oxidative damage, and play an active role in cell repair. Oxidative stress has proven to be of much relevance in biotechnological processes when the metabolism of Saccharomyces cerevisiae is mainly respiratory. During wine yeast starter production, active dry yeast cytosolic thioredoxin Trx2p is a key player in protecting metabolic enzymes from being oxidized by carbonylation. Less is known about the role of redox control during grape juice fermentation. A mutant strain that lacked both cytosolic thioredoxins, Trx1p and Trx2p, was tested for grape juice fermentation. Its growth and sugar consumption were greatly impaired, which indicates the system’s relevance under fermentative conditions. A proteomic analysis indicated that deletion of the genes TRX1 and TRX2 caused a reduction in the ribosomal proteins and factors involved in translation elongation in addition to enzymes for glycolysis and amino acid biosynthesis. A metabolomic analysis of the trx1Δ trx2Δ mutant showed an increase in most proteogenic amino acids, phospholipids, and sphingolipids and higher fatty acid desaturase Ole1p content. Low glycolytic activity was behind the reduced growth and fermentative capacity of the thioredoxin deletion strain. All three hexokinases were downregulated in the mutant strain, but total hexokinase activity remained, probably due to posttranslational regulation. Pyruvate kinase Cdc19p presented an early level of aggregation in the trx1Δ trx2Δ mutant, which may contribute to a diminished hexose metabolism and trigger regulatory mechanisms that could influence the level of glycolytic enzymes. IMPORTANCE Oxidative stress is a common hazardous condition that cells have to face in their lifetime. Oxidative damage may diminish cell vitality and viability by reducing metabolism and eventually leading to aging and ultimate death. Wine yeast Saccharomyces cerevisiae also faces oxidative attack during its biotechnological uses. One of the main yeast antioxidant systems involves two small proteins called thioredoxins. When these two proteins are removed, wine yeast shows diminished growth, protein synthesis, and sugar metabolism under wine-making conditions, and amino acid and lipid metabolism are also affected. Altogether, our results indicate that proper redox regulation is a key factor for metabolic adaptations during grape juice fermentation.

In modern oenology, yeast starters are employed to inoculate grape juice, usually in the form of active dry yeast (ADY). The dehydration process implies stressful conditions that lead to oxidative damage. Other yeast species and interspecific hybrids other than Saccharomyces cerevisiae may be used to confer novel properties to the final product. However, these yeasts are usually more sensitive to drying. Understanding the causes of oxidative stress tolerance is therefore necessary for developing the use of these organisms in industry. This study indicates the impact of mitochondrial DNA inheritance for oxidative stress resistance in an interspecific context using isogenic Saccharomyces cerevisiae × Saccharomyces uvarum hybrids.

The thioredoxin system plays a predominant role in the control of cellular redox status. Thioredoxin reductase fuels the system with reducing power in the form of NADPH. The TORC1 complex promotes growth and protein synthesis when nutrients, particularly amino acids, are abundant. It also represses catabolic processes, like autophagy, which are activated during starvation. We analyzed the impact of yeast cytosolic thioredoxin reductase TRR1 deletion under different environmental conditions. It shortens chronological life span and reduces growth in grape juice fermentation. TRR1 deletion has a global impact on metabolism during fermentation. As expected, it reduces oxidative stress tolerance, but a compensatory response is triggered, with catalase and glutathione increasing. Unexpectedly, TRR1 deletion causes sensitivity to the inhibitors of the TORC1 pathway, such as rapamycin. This correlates with low Tor2p kinase levels and indicates a direct role of Trr1p in its stability. Markers of TORC1 activity, however, suggest increased TORC1 activity. The autophagy caused by nitrogen starvation is reduced in the trr1Δ mutant. Ribosomal protein Rsp6p is dephosphorylated in the presence of rapamycin. This dephosphorylation diminishes in the TRR1 deletion strain. These results show a complex network of interactions between thioredoxin reductase Trr1p and the processes controlled by TOR.

Cells must be able to respond and adapt to different stress conditions to maintain normal function. A common response to stress is the global inhibition of protein synthesis. Protein synthesis is an expensive process consuming much of the cell’s energy. Consequently, it must be tightly regulated to conserve resources. One of these stress conditions is oxidative stress, resulting from the accumulation of reactive oxygen species (ROS) mainly produced by the mitochondria but also by other intracellular sources. Cells utilize a variety of antioxidant systems to protect against ROS, directing signaling and adaptation responses at lower levels and/or detoxification as levels increase to preclude the accumulation of damage. In this review, we focus on the role of hydrogen peroxide, H2O2, as a signaling molecule regulating protein synthesis at different levels, including transcription and various parts of the translation process, e.g., initiation, elongation, termination and ribosome recycling.

1Peroxiredoxins are major peroxide scavenging enzymes and mediators of the beneficial effects of 2 caloric restriction on aging. By which mechanism peroxiredoxins stimulate H2O2 resistance and slow 3 down aging is, however, incompletely understood. Here we show that the yeast peroxiredoxin Tsa1 4boosts H2O2 tolerance and delays aging not by its action of scavenging H2O2, but by repressing the 5 nutrient signaling protein kinase A (PKA). Tsa1 represses PKA activity through redox modification of 6 the catalytic subunits and a significant proportion of catalytic subunits are glutathionylated on two 7 cysteine residues. Redox modification of the conserved Cys243 in the catalytic subunits inhibits the 8 phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity and 9preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results 10 support a model of aging where nutrient signaling pathways constitute hubs integrating information 11 from multiple aging-related conduits including a peroxiredoxin-dependent responding to H2O2.