Ecl1, a regulator of the chronological lifespan of Schizosaccharomyces pombe, is induced upon nitrogen starvation

In fission yeast, ecl1(+) was identified as a novel factor that extends chronological lifespan when overexpressed. Ecl1 is a small protein consisting of 80 amino acids localized mainly in the nucleus. However, the mechanism by which it affects chronological lifespan has not been elucidated clearly. Here we analyzed the expression profile of Ecl1, especially as to cell cycle and growth phase, and found that it is induced upon nitrogen starvation. Then we analyzed the relevance of factors, Atf1, Ste11, and Tor1, which are known to be involved in the signaling of nitrogen starvation. Though the nitrogen starvation-induced expression of Ecl1 did not change in the atf1Δ mutant, induction in both the ste11Δ mutant and the tor1Δ mutant showed a delay. Based on these observations, the regulation of Ecl1 is discussed.     

Regulation of chronological aging in Schizosaccharomyces pombe by the protein kinases Pka1 and Sck2

Budding yeast shows a progressive decline in viability after entering stationary phase, a phenomenon known as chronological aging. We show here that the fission yeast Schizosaccharomyces pombe also undergoes chronological aging and that the process is regulated by genes controlling two related nutrient signalling pathways. The first pathway includes the serine/threonine cAMP-activated protein kinase Pka1 and the second pathway comprises the serine/threonine kinase Sck2, a homologue of Saccharomyces cerevisiae SCH9. A double mutant for pka1 and sck2 displayed an additive effect on prolonging the fission yeast lifespan, suggesting that these genes regulate related but independent pathways. These long-lived mutants also accumulated less reactive oxygen species and had a delayed initiation of apoptosis compared with wild-type cells. We also found that strains carrying pka1 deletion but not those with sck2 deletion gained resistance to oxidative stress due to exposure to H(2)O(2) or menadione. On the other hand, the additional increase in lifespan shown by the Deltapka1Deltasck2 double-mutant strain correlated with an increased resistance to both oxidative stress and heat shock. These results underscore the importance of nutrient signalling pathways and reactive oxygen species on organismal lifespan and establish S. pombe as a new model organism to study the molecular mechanisms underlying aging.     

Proteasome synthesis and assembly are required for survival during stationary phase

We examined the alterations in 20S proteasome homeostasis, protein oxidation, and cell viability that occur during the stationary phase or chronological model of yeast aging. Data in this report demonstrate that proteasome subunit expression is increased, proteasome composition is altered, and levels of individual proteasome proteolytic activities are elevated during stationary phase-induced aging in Saccharomyces cerevisiae. Despite such alterations, a progressive loss of proteasome-mediated protein degradation and a significant increase in protein oxidation were observed in cells maintained under stationary phase conditions. Deletion of UMP1, a gene necessary for 20S proteasome biogenesis, had no effect on cellular viability under normal growth conditions, but impaired the ability of cells to survive under stationary phase conditions. During stationary phase, the levels of oxidized protein increased more rapidly and to higher levels in the mutant lacking UMP1 than in the wild-type cells. Taken together, these data implicate a role for proteasome synthesis and altered 20S proteasome composition in maintaining viability during stationary phase, and demonstrate that even with these modifications a gradual loss of proteasome-mediated protein degradation occurs during stationary phase-induced aging. These data also suggest a role for impaired proteasome-mediated protein degradation in increased protein oxidation and cell death observed during the aging of eukaryotic cells.     

Identification of a fatty acyl-CoA synthetase gene, lcf2+, which affects viability after entry into the stationary phase in Schizosaccharomyces pombe

The lcf1(+) gene, which encodes a long chain fatty acyl-CoA synthetase, is necessary for the maintenance of viability after entry into the stationary phase in Schizosaccharomyces pombe. In this study, we analyzed a paralogous gene, SPBP4H10.11c (named lcf2(+)), and we present evidence that the gene encodes a new fatty acyl-CoA synthetase. The enzyme preferentially recognized myristic acid as a substrate. A Deltalcf2 mutant showed increased viability after entry into the stationary phase in SD medium. A Deltalcf1Deltalcf2 double mutant showed a severe decrease in long-chain fatty acyl-CoA synthetase activity and a rapid loss of viability after entry into the stationary phase. These results suggest that fatty acid utilization and/or metabolism is important to determine viability in the stationary phase.     

The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway

We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.     

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.     

Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu(2+)-deficient or an active Cu,Zn-superoxide dismutase

Yeast overexpressing SOD1, the gene for Cu,Zn-superoxide dismutase (Cu,Zn-Sod), was used to determine how Sod1p overexpression influences the chronological lifespan [the survival of non-dividing stationary (G0) phase cells over time], the replicative lifespan (the number of buds produced by actively dividing yeast cells) and stress resistance. Increasing the level of active Cu,Zn-Sod in yeast was found to require either growth in the presence of high copper, or the simultaneous overexpression of both SOD1 and CCS1 (the latter being the gene that encodes the chaperone dedicated to Cu(2+)-loading of Sod1p in vivo). Dual SOD1 + CCS1 overexpression elevated the levels of Cu,Zn-Sod activity six- to eight-fold in vegetative cultures. It also increased the optimized survival of stationary cells up to two-fold, showing this chronological lifespan is ultimately limited by oxidative stress. In contrast, several detrimental effects resulted when the SOD1 gene was overexpressed in the absence of either high copper or a simultaneous overexpression of CCS1. Both the chronological and the replicative lifespans were shortened; the cells displayed an abnormally high level of endogenous oxidative stress, resulting in a high rate of spontaneous mutation. Such harmful effects were all reversed through the overexpression of CCS1. It is apparent therefore that they relate to the incomplete Cu(2+)-loading of the overexpressed Sod1p, most probably accumulation of a Cu(2+)-deficient Sod1p to appreciable levels in vivo. The same events may generate the detrimental effects that are frequently, though not universally, observed when Cu,Zn-Sod overexpression is attempted in metazoans.     

Availability of Amino Acids Extends Chronological Lifespan by Suppressing Hyper-Acidification of the Environment in Saccharomyces cerevisiae

The chronological lifespan of Saccharomyces cerevisiae represents the duration of cell survival in the postdiauxic and stationary phases. Using a prototrophic strain derived from the standard auxotrophic laboratory strain BY4742, we showed that supplementation of non-essential amino acids to a synthetic defined (SD) medium increases maximal cell growth and extends the chronological lifespan. The positive effects of amino acids can be reproduced by modulating the medium pH, indicating that amino acids contribute to chronological longevity in a cell-extrinsic manner by alleviating medium acidification. In addition, we showed that the amino acid-mediated effects on extension of chronological longevity are independent of those achieved through a reduction in the TORC1 pathway, which is mediated in a cell-intrinsic manner. Since previous studies showed that extracellular acidification causes mitochondrial dysfunction and leads to cell death, our results provide a path to premature chronological aging caused by differences in available nitrogen sources. Moreover, acidification of culture medium is generally associated with culture duration and cell density; thus, further studies are required on cell physiology of auxotrophic yeast strains during the stationary phase because an insufficient supply of essential amino acids may cause alterations in environmental conditions.     

Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins

Calorie restriction (CR) extends the mean and maximum lifespan of a wide variety of organisms ranging from yeast to mammals, although the molecular mechanisms of action remain unclear. For the budding yeast Saccharomyces cerevisiae reducing glucose in the growth medium extends both the replicative and chronological lifespans (CLS). The conserved NAD(+)-dependent histone deacetylase, Sir2p, promotes replicative longevity in S. cerevisiae by suppressing recombination within the ribosomal DNA locus and has been proposed to mediate the effects of CR on aging. In this study, we investigated the functional relationships of the yeast Sirtuins (Sir2p, Hst1p, Hst2p, Hst3p and Hst4p) with CLS and CR. SIR2, HST2, and HST4 were not major regulators of CLS and were not required for the lifespan extension caused by shifting the glucose concentration from 2 to 0.5% (CR). Deleting HST1 or HST3 moderately shortened CLS, but did not prevent CR from extending lifespan. CR therefore works through a Sirtuin-independent mechanism in the chronological aging system. We also show that low temperature or high osmolarity additively extends CLS when combined with CR, suggesting that these stresses and CR act through separate pathways. The CR effect on CLS was not specific to glucose. Restricting other simple sugars such as galactose or fructose also extended lifespan. Importantly, growth on nonfermentable carbon sources that force yeast to exclusively utilize respiration extended lifespan at nonrestricted concentrations and provided no additional benefit when restricted, suggesting that elevated respiration capacity is an important determinant of chronological longevity.     

Effect of hydrostatic pressure on a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0–200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.     

The Fission Yeast php2 Mutant Displays a Lengthened Chronological Lifespan

The Schizosaccharomyces pombe php2 ⁺ gene encodes a subunit of the CCAAT-binding factor complex. We found that disruption of the php2 ⁺ gene extended the chronological lifespan of the fission yeast. Moreover, the lifespan of the Δphp2 mutant was barely extended under calorie restricted (CR) conditions. Many other phenotypes of the Δphp2 mutant resembled those of wild-type cells grown under CR conditions, suggesting that the Δphp2 mutant might undergo CR. The mutant also showed low respiratory activity concomitant with decreased expression of the cyc1 ⁺ and rip1 ⁺ genes, both of which are involved in mitochondrial electron transport. On the basis of a chromatin immunoprecipitation assay, we determined that Php2 binds to a DNA region upstream of cyc1 ⁺ and rip1 ⁺ in S. pombe. Here we discuss the possible mechanisms by which the chronological lifespan of Δphp2 mutant is extended.     

Influence of chronological aging on the survival and nucleotide content of Saccharomyces cerevisiae cells grown in different conditions: occurrence of a high concentration of UDP-N-acetylglucosamine in stationary cells grown in 2% glucose

Saccharomyces cerevisiae cells (strain W303) grown in a minimal medium (containing 2% or 0.1% glucose) until exponential or stationary phase, were subjected to chronological aging in water, and yeast viability and nucleotide content were analyzed along several days of nutrient starvation. Cells collected in exponential phase (whether grown in the presence of 0.1% or 2% glucose) were viable up to five days and thereafter the viability decreased linearly with a half-survival rate of around eight days. ATP and other nucleoside triphosphates decreased similarly in both cases. Cells collected in stationary phase, and transferred to water, behaved differently whether grown in 0.1% or in 2% glucose, with a half-survival life of around nine and 28 days respectively. A double mutant in glycogen synthase (gsy1delta gsy2delta) and its isogenic wild-type strain, grown to stationary phase in 2% glucose, presented a similar half-survival life of around eight days. The W303 cells grown to stationary phase in the presence of 2% glucose showed a 7-fold increase of UDP-N-acetylglucosamine (UDP-GlcNAc) as compared with the level present in the cells grown in any of the other three metabolic situations. The nature of UDP-GlcNAc was established by MALDI-TOF ionization analysis. It is also worth noting that the rate of decay of NAD+ was lower than that of ATP in any of the situations here considered.     

NQR1 controls lifespan by regulating the promotion of respiratory metabolism in yeast

The activity and expression of plasma membrane NADH coenzyme Q reductase is increased by calorie restriction (CR) in rodents. Although this effect is well-established and is necessary for CR's ability to delay aging, the mechanism is unknown. Here we show that the Saccharomyces cerevisiae homolog, NADH-Coenzyme Q reductase 1 (NQR1), resides at the plasma membrane and when overexpressed extends both replicative and chronological lifespan. We show that NQR1 extends replicative lifespan in a SIR2-dependent manner by shifting cells towards respiratory metabolism. Chronological lifespan extension, in contrast, occurs via an SIR2-independent decrease in ethanol production. We conclude that NQR1 is a key mediator of lifespan extension by CR through its effects on yeast metabolism and discuss how these findings could suggest a function for this protein in lifespan extension in mammals.     

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.     

Fission yeast Rho5p GTPase is a functional paralogue of Rho1p that plays a role in survival of spores and stationary-phase cells

The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-beta-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5(+) but no other rho genes is able to rescue the lethality of rho1Delta cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5(+) expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Delta cells are viable and do not display any defects during logarithmic growth. However, when rho1(+) expression is repressed during stationary phase, rho5Delta cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h(90) mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.