<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

Induction of Stress Genes in Saccharomyces cerevisiae during Starvation

In order to analyze the response of Saccharomyces cerevisiae to starvation on a gene expression level, microarray experiments were performed using a yeast whole genome array. It is well known that under stress conditions like heat, high salt concentrations, pressure or the presence of toxins, special stress response genes are induced in Saccharomyces cerevisiae. This includes the genes encoding the typical heat shock proteins as well as numerous genes concerning cell membrane composition, central carbon metabolism or cell cycle. In this contribution, the Saccharomyces cerevisiae starvation‐stress response is analyzed. Starvation is a living condition often experienced by yeast in natural surroundings. As Saccharomyces cerevisiae is an eukaryote, many results from the gene expression analysis are valid for mammalians as well. The understanding of response of the yeast to the absence of a nutrient is also important for the development of feeding strategies in cultivations. Therefore, knowledge about the gene expression during starvation is important for both research and industrial applications. The regulation of 233 genes, which are involved in the stress response according to the literature, was examined via microarray experiments. In addition, a screening was carried out identifying 115 genes, which are hitherto not known to be comprised in the stress response, but which were significantly up‐regulated during starvation.     

Regulation of genes encoding subunits of the trehalose synthase complex inSaccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex,GGS1/TPS1, TPS2, TPS3 andTSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth onlyTSL1 shows an expression pattern like that of the STRE-controlled genesCTT1 andSSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.     

Yeast Colony Survival Depends on Metabolic Adaptation and Cell Differentiation Rather Than on Stress Defense

Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Δ and ctt1Δ colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling.     

Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

Induction of stress resistance and extension of lifespan in Chaenorhabditis elegans serotonin-receptor knockout strains by withanolide A

IntroductionApproximately 300 million people worldwide suffer from depression. The COVID-19 crisis may dramatically increase these numbers. Severe side effects and resistance development limit the use of standard antidepressants. The steroidal lactone withanolide A (WA) from Withania somnifera may be a promising alternative. Caenorhabditis elegans was used as model to explore WA's anti-depressive and anti-stress potential.MethodsC. elegans wildtype (N2) and deficient strains (AQ866, DA1814, DA2100, DA2109 and MT9772) were used to assess oxidative, osmotic or heat stress as measured by generation of reactive oxygen species (ROS), determination of lifespan, and mRNA expression of serotonin receptor (ser-1, ser-4, ser-7) and serotonin transporter genes (mod-5). The protective effect of WA was compared to fluoxetine as clinically established antidepressant. Additionally, WA's effect on lifespan was determined. Furthermore, the binding affinities and pKi values of WA, fluoxetine and serotonin as natural ligand to Ser-1, Ser-4, Ser-7, Mod-5 and their human orthologues proteins were calculated by molecular docking.ResultsBaseline oxidative stress was higher in deficient than wildtype worms. WA and fluoxetine reduced ROS levels in all strains except MT9772. WA and fluoxetine prolonged survival times in wildtype and mutants under osmotic stress. WA but not fluoxetine increased lifespan of all heat-stressed C. elegans strains except DA2100. Furthermore, WA but not fluoxetine extended lifespan in all non-stressed C. elegans strains. WA also induced mRNA expression of serotonin receptors and transporters in wildtype and mutants. WA bound with higher affinity and lower pKi values to all C. elegans and human serotonin receptors and transporters than serotonin, indicating that WA may competitively displaced serotonin from the binding pockets of these proteins.ConclusionWA reduced stress and increased lifespan by ROS scavenging and interference with the serotonin system. Hence, WA may serve as promising candidate to treat depression.     

Vacuolar H+-ATPase is involved in preventing heavy metal-induced oxidative stress in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, vacuolar H⁺-ATPase (V-ATPase) involved in the regulation of intracellular pH homeostasis has been shown to be important for tolerances to cadmium, cobalt and nickel. However, the molecular mechanism underlying the protective role of V-ATPase against these metals remains unclear. In this study, we show that cadmium, cobalt and nickel disturbed intracellular pH balance by triggering cytosolic acidification and vacuolar alkalinization, likely via their membrane permeabilizing effects. Since V-ATPase plays a crucial role in pumping excessive cytosolic protons into the vacuole, the metal-sensitive phenotypes of the Δvma2 and Δvma3 mutants lacking V-ATPase activity were supposed to result from highly acidified cytosol. However, we found that the metal-sensitive phenotypes of these mutants were caused by increased production of reactive oxygen species, likely as a result of decreased expression and activities of manganese superoxide dismutase and catalase. In addition, the loss of V-ATPase function led to aberrant vacuolar morphology and defective endocytic trafficking. Furthermore, the sensitivities of the Δvma mutants to other chemical compounds (i.e. acetic acid, H₂O₂, menadione, tunicamycin and cycloheximide) were a consequence of increased endogenous oxidative stress. These findings, therefore, suggest the important role of V-ATPase in preventing endogenous oxidative stress induced by metals and other chemical compounds.     

Oxidative stress and aging: Learning from yeast lessons

The yeast Saccharomyces cerevisiae has played a vital role in the understanding of the molecular basis of aging and the relationship of aging process with oxidative stress (non-homeostatic accumulation of Reactive Oxygen Species, ROS). The mammalian and yeast antioxidant responses are similar and over 25 % of human-degenerative disease related genes have close homologues in yeast. The reduced genetic redundancy of yeast facilitates visualization of the effect of a deleted or mutated gene. By manipulating growth conditions, yeast cells can survive only fermenting (low ROS levels) or respiring (increased ROS levels), which facilitates the elucidation of the mechanisms involved with acquisition of tolerance to oxidative stress. Furthermore, the yeast databases are the most complete of all eukaryotic models. In this work, we highlight the value of S. cerevisiae as a model to investigate the oxidative stress response and its potential impact on aging and age-related diseases.     

Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca²⁺ mimicked this increase in PB number. Conversely, intracellular Ca²⁺-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca²⁺ required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of ∼40–100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

Caloric restriction and redox state: Does this diet increase or decrease oxidant production?

Abstract

Calorie restriction (CR) is well established to enhance the lifespan of a wide variety of organisms, although the mechanisms are still being uncovered. Recently, some authors have suggested that CR acts through hormesis, enhancing the production of reactive oxygen species (ROS), activating stress response pathways, and increasing lifespan. Here, we review the literature on the effects of CR and redox state. We find that there is no evidence in rodent models of CR that an increase in ROS production occurs. Furthermore, results in Caenorhabditis elegans and Saccharomyces cerevisiae suggesting that CR increases intracellular ROS are questionable, and probably cannot be resolved until adequate, artifact free, tools for real-time, quantitative, and selective measurements of intracellular ROS are developed. Overall, the largest body of work indicates that CR improves redox state, although it seems improbable that a global improvement in redox state is the mechanism through which CR enhances lifespan.     

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O^{⋅ –}), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.     

线粒体自噬基因对酿酒酵母抗氧化性能的影响

线粒体自噬是指细胞通过自噬的机制选择性地清除线粒体的过程,对维持细胞内稳态具有重要作用。为探究线粒体自噬基因对酿酒酵母(Saccharomyces cerevisiae)细胞抗氧化性能的影响,本研究分别构建了线粒体自噬相关基因ATG8、ATG11、ATG32的缺失和过表达菌株,发现在过氧化氢(H₂O₂)胁迫6 h后,过表达ATG8、ATG11基因显著降低了细胞内活性氧(reactive oxygen species,ROS)含量,分别仅为初始状态的61.23%和46.35%,并显著提高了菌株线粒体膜电位(mitochondrial membrane potential,MMP)和腺嘌呤核苷三磷酸(adenosine-triphosphate,ATP)含量,有助于提高菌株的抗氧化性能。另一方面,基因ATG8、ATG11、ATG32的缺失会导致线粒体损伤及细胞活力显著下降,同时造成胞内ROS失衡,H₂O₂胁迫6 h后,其胞内ROS含量显著升高至初始状态的174.27%、128.68%和200.92%。结果表明,ATG8、ATG11和ATG32可能是调控酵母抗氧化能力的潜在靶点。本研究为进一步研究通过调节线粒体自噬提高酵母抗氧化活性提供了新的线索。