The control of translational accuracy is a determinant of healthy ageing in yeast.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Introduction.

Aging in yeast is now a well researched area with hundreds of new research and review papers appearing every year. The chapters following in this book written by some of the leading experts in the field will give an overview of the most relevant areas of yeast aging. The purpose of this chapter is to give the newcomer an introduction to the field including some basic technical questions.

The role of mitochondria in the aging processes of yeast.

This chapter reviews the role of mitochondria and of mitochondrial metabolism in the aging processes of yeast and the existing evidence for the "mitochondrial theory of aging mitochondrial theory of aging ". Mitochondria are the major source of ATP in the eukaryotic cell but are also a major source of reactive oxygen species reactive oxygen species (ROS) and play an important role in the process of apoptosis and aging. We are discussing the mitochondrial theory of aging mitochondrial theory of aging (TOA), its origin, similarity with other TOAs, and its ramifications which developed in recent decades. The emphasis is on mother cell-specific aging mother cell-specific aging and the RLS (replicative lifespan) with only a short treatment of CLS (chronological lifespan). Both of these aging processes may be relevant to understand also the aging of higher organisms, but they are biochemically very different, as shown by the fact the replicative aging occurs on rich media and is a defect in the replicative capacity of mother cells, while chronological aging occurs in postmitotic cells that are under starvation conditions in stationary phase leading to loss of viability, as discussed elsewhere in this book. In so doing we also give an overview of the similarities and dissimilarities of the various aging processes of the most often used model organisms for aging research with respect to the mitochondrial theory of aging mitochondrial theory of aging.

Yeast aging and apoptosis.

A concerted balance between proliferation and apoptosis is essential to the survival of multicellular organisms. Thus, apoptosis per se, although it is a destructive process leading to the death of single cells, also serves as a pro-survival mechanism pro-survival mechanism that ensures healthy organismal development and acts as a life-prolonging or anti-aging anti-aging program. The discovery that yeast also possess a functional and, in many cases, highly conserved apoptotic machinery has made it possible to study the relationships between aging and apoptosis in depth using a well-established genetic system and the powerful tools available to yeast researchers for investigating complex physiological and cytological interactions. The aging process of yeast, be it replicative replicative or chronological chronological aging, is closely related to apoptosis, although it remains unclear whether apoptosis is a causal feature of the aging process or vice versa. Nevertheless, experimental results obtained during the past several years clearly demonstrate that yeast serve as a powerful and versatile experimental system for understanding the interconnections between these two fundamentally important cellular and physiological pathways.

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis.

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging.

The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.

Quantitation of (a)symmetric inheritance of functional and of oxidatively damaged mitochondrial aconitase in the cell division of old yeast mother cells.

Asymmetric segregation of oxidatively damaged proteins is discussed in the literature as a mechanism in cell division cycles which at the same time causes rejuvenation of the daughter cell and aging of the mother cell. This process must be viewed as cooperating with the cellular degradation processes like autophagy, proteasomal degradation and others. Together, these two mechanisms guarantee survival of the species and prevent clonal senescence of unicellular organisms, like yeast. It is widely believed that oxidative damage to proteins is primarily caused by oxygen radicals and their follow-up products produced in the mitochondria. As we have shown previously, old yeast mother cells in contrast to young cells contain reactive oxygen species and undergo programmed cell death. Here we show that aconitase of the mitochondrial matrix is readily inactivated by oxidative stress, but even in its inactive form is relatively long-lived and retains fluorescence in the Aco1p-eGFP form. The fluorescent protein is distributed between old mothers and their daughters approximately corresponding to the different sizes of mother and daughter cells. However, the remaining active enzyme is primarily inherited by the daughter cells. This indicates that asymmetric distribution of the still active enzyme takes place and a mechanism for discrimination between active and inactive enzyme must exist. As the aconitase remains mitochondrial during aging and cell division, our findings could indicate discrimination between active and no longer active mitochondria during the process.

Induction of autophagy by spermidine promotes longevity.

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review.

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.

Cladosporium herbarum translationally controlled tumor protein (TCTP) is an IgE-binding antigen and is associated with disease severity.

Cladosporium herbarum represents one of the most important world-wide occurring allergenic fungal species. The prevalence of IgE reactivity to C. herbarum in patients suffering from allergy varies between 5 and 30% in the different climatic zones. Since mold allergy has often been associated with severe asthma, along with other allergic symptoms, it is important to define more comprehensively the allergen repertoire of this ascomycete. In this context we are reporting our successful approach to identify, clone, produce as a recombinant protein, purify and further characterize a new C. herbarum allergen which is a close homolog of the human translationally controlled tumor protein (TCTP, also called histamine releasing factor, HRF). The immunoreactivity of both pure recombinant molecules was investigated by means of immunoblot analyses, enzyme-linked immunosorbent assays as well as histamine release studies. To summarize, IgE antibodies from five out of nine individuals recognized both the human and the fungal protein in immunoblots. The latter was able to cause histamine release from human basophils with about half the efficiency compared to its human homolog HRF. Cross-inhibition assays showed that the patients' IgEs recognize common epitopes on both the human and C. herbarum proteins, but however, only pre-incubation with C. herbarum TCTP could completely inhibit reactivity with HRF. Furthermore, it appears that patients reactive to TCTP have a higher probability to suffer from asthma than other allergic patients.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells.

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.

Evidence for ORC-dependent repression of budding yeast genes induced by starvation and other stresses.

The highly conserved origin recognition complex (ORC) is required for repressing genes in the silent mating type loci of budding yeast. Here we report that at a non-permissive temperature, the temperature-sensitive orc2-1 mutation induces the expression of more than 500 genes, the majority of which are also induced during starvation of wild-type cells. Many genes induced by starvation or by the orc2-1 mutation are also induced by inactivation of proteins required for chromatin-mediated repression of transcription. Genes induced by the orc2-1 mutation, starvation, or inactivation of repressor proteins, map near ORC-binding loci significantly more frequently compared to all genes. Genes repressed by starvation map near ORC-binding sites less frequently compared to all genes, which suggests they have been evolutionarily excluded from regions of repressive chromatin near ORC-binding sites. Deletion of sequences containing ORC-binding sites near the DAL2 and DAL4 genes in the DAL gene cluster, which are induced by either the orc2-1 mutation or by starvation, constitutively activates these genes and abolishes their activation by the orc2-1 mutation. Our findings suggest a role for ORC in the repression of a large number of budding yeast genes induced by starvation or other aspects of a deleterious environment.

MMI1 (YKL056c, TMA19), the yeast orthologue of the translationally controlled tumor protein (TCTP) has apoptotic functions and interacts with both microtubules and mitochondria.

The yeast orthologue of mammalian TCTP is here proposed to be named Mmi1p (microtubule and mitochondria interacting protein). This protein displays about 50% amino acid sequence identity with its most distantly related orthologs in higher organisms and therefore probably belongs to a small class of yeast proteins which have housekeeping but so far incompletely known functions needed for every eukaryotic cell. Previous investigations of the protein in both higher cells and yeast revealed that it is highly expressed during active growth, but transcriptionally down-regulated in several kinds of stress situations including starvation stress. In human cells, TCTP presumably has anti-apoptotic functions as it binds to Bcl-XL in vivo. TCTP of higher cells was also shown to interact with the translational machinery. It has acquired an additional function in the mammalian immune system, as it is identical with the histamine releasing factor. Here, we show that in S. cerevisiae induction of apoptosis by mild oxidative stress, replicative ageing or mutation of cdc48 leads to translocation of Mmi1p from the cytoplasm to the mitochondria. Mmi1p is stably but reversibly attached to the outer surface of the mitochondria and can be removed by digestion with proteinase K. Glutathionylation of Mmi1p, which is also induced by oxidants, is not a prerequisite or signal for translocation as shown by replacing the only cysteine of Mmi1p by serine. Mmi1p probably interacts with yeast microtubules as deletion of the gene confers sensitivity to benomyl. Conversely, the deletion mutant displays resistance to hydrogen peroxide stress and shows a small but significant elongation of the mother cell-specific lifespan. Our results so far indicate that Mmi1p is one of the few proteins establishing a functional link between microtubules and mitochondria which may be needed for correct localization of mitochondria during cell division.

The actin cytoskeleton, RAS-cAMP signaling and mitochondrial ROS in yeast apoptosis.

The release of reactive oxygen species (ROS) by mitochondria instigates the pathways of programmed cell death in eukaryotic cells. Gourlay and Ayscough present intriguing experimental evidence that mutations in the genes encoding the regulatory proteins End3p and Sla1p, which influence actin dynamics in budding yeast, lead to a loss of mitochondrial membrane potential, resulting in ROS production and apoptosis. This effect can be suppressed by downregulation of the RAS-cAMP signaling pathway, thus establishing the existence of a new and complex regulatory network.

A comparison of the aging and apoptotic transcriptome of Saccharomyces cerevisiae.

In this paper, we present the results of global transcript analysis by the microarray technique of senescent and apoptotic yeast cells. We compared young daughter and old mother cells isolated by elutriation centrifugation, and non-apoptotic and apoptotic cells induced either by a temperature shift of the cdc48(S565G) temperature-sensitive mutant or of the orc2-1 temperature-sensitive mutant. The majority of all genes found to be differentially regulated in these three physiological situations was upregulated, indicating that a cellular death process was initiated rather than an unspecific shut-down of gene expression due to immediate killing. The functional classes of genes upregulated in all three conditions were largely the same, although individual genes were in many cases not identical. The largest group of genes involved were nuclear genes coding for mitochondrial components or functions, which is understandable given the fact that apoptosis can be triggered by mitochondrially generated oxygen radicals and that mitochondria play an important role in the execution of apoptosis. Other functional classes consisted of genes involved in DNA damage response, in cell cycle regulation and checkpoints, in DNA repair, and in membrane lipid and cell wall synthesis. These functional classes represent the response of the cell to the known cellular insults, which occur during aging and apoptosis. As we have shown previously, final-stage senescent yeast mother cells (of the wild-type) are apoptotic.

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.