Yeast Replicative Lifespan Research Articles

Suppressor analysis points to the subtle role of the LAG1 ceramide synthase gene in determining yeast longevity

Individual yeast cells display a finite replicative capacity. LAG1 was identified as a gene that is differentially expressed during the yeast replicative life span and was shown to play a role in determining yeast longevity. This gene is not essential, but simultaneous deletion of LAG1 and its close homologue LAC1 is lethal. Lag1p and Lac1p have been found to be an essential component of ceramide synthase. In this study, multicopy suppressors of the lethality of a lag1Δ lac1Δ double mutant were isolated to help clarify the role of LAG1 in yeast longevity. The two multicopy suppressors YBR183w ( YPC1) and YPL087w ( YDC1) encode ceramidases unrelated to Lag1p and Lac1p, which were previously found to support the reverse reaction of ceramide synthesis. Multiple copies of YPC1 were much more efficient than YDC1 in rescuing cell growth. They were also much more effective in rescuing the life span of a lag1Δ lac1Δ double mutant, sustaining a life span approaching that obtained by the restoration of LAG1 expression. Neither deletion of LAC1 nor overexpression of YPC1 had a detectable effect on wild-type life span. However, the overexpression of LAG1 had a bimodal effect on longevity, with moderate expression resulting in increased longevity and with higher expression curtailing life span. These results suggest that subtle changes in ceramide/sphingolipid metabolism are important in determining yeast longevity. They also indicate that Lag1p plays a special role in this relationship. Homologues of Lag1p have been identified in higher eukaryotes, including human, raising the possibility that ceramide and other sphingolipid metabolites play a wider role in biological aging.

Rtg2 Protein Links Metabolism and Genome Stability in Yeast Longevity

Abstract Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromsomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

Rtg2 protein links metabolism and genome stability in yeast longevity.

Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromosomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

Calorie restriction extends yeast life span by lowering the level of NADH.

Calorie restriction (CR) extends life span in a wide variety of species. Previously, we showed that calorie restriction increases the replicative life span in yeast by activating Sir2, a highly conserved NAD-dependent deacetylase. Here we test whether CR activates Sir2 by increasing the NAD/NADH ratio or by regulating the level of nicotinamide, a known inhibitor of Sir2. We show that CR decreases NADH levels, and that NADH is a competitive inhibitor of Sir2. A genetic intervention that specifically decreases NADH levels increases life span, validating the model that NADH regulates yeast longevity in response to CR.

Plasmid Accumulation Reduces Life Span in Saccharomyces cerevisiae

Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.

Of Red Wine, Diet, and a Long Life

Caloric restriction slows aging and extends the life-span of various organisms. In the yeast Saccharomyces cerevisiae , this extension of life-span is mediated through activation of the sirtuin-family deacetylase Sir2 in what may represent a stress response. Sirtuin activity has also been implicated in Caenorhabditis elegans longevity, and the related human enzyme, SIRT1, which deacetylates the tumor suppressor p53, promotes cell survival (see Finkel). Howitz et al. screened small molecule libraries for compounds that affected sirtuin function and discovered that several plant polyphenols stimulated SIRT1 activity in an in vitro fluorescent deacetylation assay. Resveratrol, which is found in red wine and may be implicated in various health benefits associated with moderate wine drinking, was the most potent of these sirtuin-activating compounds (STACs). Low concentrations of resveratrol stimulated yeast Sir2, and resveratrol and other STACs prolonged yeast replicative life-span. The effects of resveratrol on life-span were dependent on Sir2, and, in the presence of resveratrol, glucose deprivation produced no further life-span extension. Low concentrations of resveratrol extended the survival of human embryonic kidney cells (HEK 293 cells) exposed to ionizing radiation and decreased p53 acetylation in irradiated HEK 293 and osteosarcoma cells. (The concentration dependence of resveratrol's effects was complex, and high concentrations of resveratrol increased p53 acetylation in the osteosarcoma cells.) The authors proposed a model in which plants under stress synthesize polyphenols in order to regulate sirtuin-mediated responses, and animals and fungi are able to respond to these plant stress metabolites. K. T. Howitz, K. J. Bitterman, H. Y. Cohen, D. W. Lamming, S. Lavu, J. G. Wood, R. E. Zipkin, P. Chung, A. Kisielewski, L.-L. Zhang, B. Scherer, D. A. Sinclair, Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425 , 191-196 (2003). [Online Journal] T. Finkel, A toast to long life. Nature 425 , 132-133 (2003). [Online Journal]

Mnsod overexpression extends the yeast chronological (G(0)) life span but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells.

Studies in Drosophila and Caenorhabditis elegans have shown increased longevity with the increased free radical scavenging that accompanies overexpression of oxidant-scavenging enzymes. This study used yeast, another model for aging research, to probe the effects of overexpressing the major activity protecting against superoxide generated by the mitochondrial respiratory chain. Manganese superoxide dismutase (MnSOD) overexpression increased chronological life span (optimized survival of stationary (G(0)) yeast over time), showing this is a survival ultimately limited by oxidative stress. In contrast, the same overexpression dramatically reduced the replicative life span of dividing cells (the number of daughter buds produced by each newly born mother cell). This reduction in the generational life span by MnSOD overexpression was greater than that generated by loss of the major redox-responsive regulator of the yeast replicative life span, NAD+-dependent Sir2p histone deacetylase. It was also independent of the latter activity. Expression of a mitochondrially targeted green fluorescent protein in the MnSOD overexpressor revealed that the old mother cells of this overexpressor, which had divided for a few generations, were defective in segregation of the mitochondrion from the mother to daughter. Mitochondrial defects are, therefore, the probable reason that MnSOD overexpression shortens replicative life span.

Manipulation of a Nuclear NAD+ Salvage Pathway Delays Aging without Altering Steady-state NAD+ Levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.

Profiles of Random Change During Aging Contain Hidden Information about Longevity and the Aging Process

Many different morphological and physiological changes occur during the yeast replicative lifespan. It has been proposed that change is a cause rather than an effect of aging. It is difficult to ascribe causality to processes that manifest themselves at the level of the entire organism, because of their global nature. Although causal connections can be established for processes that occur at the molecular level, their exact contributions are obscured, because they are immersed in a highly interactive network of processes. A top–down approach that can isolate crucial features of aging processes for further study may be a productive avenue. We have mathematically depicted the complicated and random changes that occur in cellular spatial organization during the lifespan of individual yeast cells. We call them budding profiles. This has allowed us to demonstrate that budding profiles are a highly individual characteristic, and that they are correlated with an individual cell's longevity. Additional information can be extracted from our model, indicating that random budding is associated with longevity. This expectation was confirmed, providing new avenues for exploring causal factors in yeast aging. The methodology described here can be readily applied to other aspects of aging in yeast and in higher organisms.

Impact of Carbohydrate Composition of Media on Lager Yeast Replicative Lifespan

The relationship between replicative lifespan, wort carbohydrate, and specific growth rate for four lager production brewing yeast strains was studied. Lifespan in brewing yeast was affected by media composition, and the effect of carbon source on the longevity of the four strains appeared to be strain dependent. No relationship between replicative lifespan and specific growth rate in laboratory media or wort could be demonstrated.

The RAS genes: a homeostatic device in Saccharomyces cerevisiae longevity☆

The genetic analysis of the yeast replicative life span has revealed the importance of metabolic control and resistance to stress. It has also illuminated the pivotal role in determining longevity that the RAS genes play by the maintenance of homeostasis. This role appears to be performed by the coordination of a variety of cellular processes. Metabolic control seems to occupy a central position among these cellular processes that include stress resistance. Some of the features of metabolic control in yeast resemble the effects of the daf pathway for adult longevity in Caenorhabditis elegans and the metabolic consequences of selection for extended longevity in Drosophila melanogaster, as well as some of the features of caloric restriction in mammals. The distinction between dividing and nondividing cells is proposed to be less important for the aging process than generally believed because these cell types are part of a metabolic continuum in which the total metabolic capacity determines life span. As a consequence, the study of yeast aging may be helpful in understanding processes occurring in the aging brain.

Differential response to UV stress and DNA damage during the yeast replicative life span.

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.