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 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.

SNF1-related protein kinase (snRK1) phosphorylates class I heat shock protein.

The nucleotide sequence of cBSnIP2, a cDNA that had been cloned from a barley (Hordeum vulgare) seed endosperm cDNA library by two-hybrid screening with barley SNF1-related protein kinase (SnRKI) was determined. It was found to contain a complete open reading frame encoding a class I heat shock protein. Transcripts corresponding to the cDNA (renamed cBHSP17) were detectable in RNA isolated from barley seeds harvested in mid-development but not RNA from roots or leaves. BHSP17 protein was expressed in Escherischia coli and shown to be phosphorylated by SnRKI from barley endosperm and spinach leaf. It was found to be a less effective substrate than 3-hydroxy-3-methylglutaryl-Coenzyme A, a previously identified substrate of SnRK1. However, a specific phosphorylation site at serine-35 was identified by solid phase sequencing of RP-HPLC-purified peptides after phosphorylation by spinach SnRK1.

The retroviral proteinase active site and the N-terminus of Ddi1 are required for repression of protein secretion.

The Ddi1 protein of the yeast Saccharomyces cerevisiae is involved in numerous interactions with the ubiquitin system, which may be mediated by its N-terminal ubiquitin like domain and its C-terminal ubiquitin associated domain. Ddi1 also contains a central region with all the features of a retroviral aspartic proteinase, which was shown to be important in cell-cycle control. Here we demonstrate an additional role for this domain, along with the N-terminal region, in protein secretion. These results further substantiate the hypothesis that Ddi1 functions in vivo as a catalytically-active aspartic proteinase.

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.

DNA sequences from Arabidopsis, which encode protein kinases and function as upstream regulators of Snf1 in yeast.

Sucrose nonfermenting-1 (Snf1)-related protein kinase-1 (SnRK1) of plants is a global regulator of carbon metabolism through the modulation of enzyme activity and gene expression. It is structurally and functionally related to the yeast protein kinase, Snf1, and to mammalian AMP-activated protein kinase. Two DNA sequences from Arabidopsis thaliana, previously known only by their data base accession numbers of NM_ 125448.3 (protein ID NP_200863) and NM_114393.3 (protein ID NP_566876) each functionally complemented a Saccharomyces cerevisiae elm1 sak1 tos3 triple mutant. This indicates that the Arabidopsis proteins are able to substitute for one of the missing yeast upstream kinases, which are required for activity of Snf1. Both plant proteins were shown to phosphorylate a peptide with the amino acid sequence of the phosphorylation site in the T-loop of SnRK1 and by inference SnRK1 in Arabidopsis. The proteins encoded by NM_125448.3 and NM_114393.3 have been named AtSnAK1 and AtSnAK2 (Arabidopsis thaliana SnRK1-activating kinase), respectively. We believe this is the first time that upstream activators of SnRK1 have been described in any plant species.

A transcriptome analysis of isoamyl alcohol-induced filamentation in yeast reveals a novel role for Gre2p as isovaleraldehyde reductase.

A transcriptome analysis was performed of Saccharomyces cerevisiae undergoing isoamyl alcohol-induced filament formation. In the crucial first 5 h of this process, only four mRNA species displayed strong and statistically significant increases in their levels of more than 10-fold. Two of these (YEL071w/DLD3 and YOL151w/GRE2) appear to play important roles in filamentation. The biochemical activities ascribed to these two genes (d-lactate dehydrogenase and methylglyoxal reductase, respectively) displayed similarly timed increases to those of their respective mRNAs. Mutants carrying dld3 mutations displayed reduced filamentation in 0.5% isoamyl alcohol and needed a higher concentration of isoamyl alcohol to effect more complete filament formation. Hence, DLD3 seems to be required for a full response to isoamyl alcohol, but is not absolutely essential for it. Mutants carrying gre2 mutations were derepressed for filament formation and formed large, invasive filaments even in the absence of isoamyl alcohol. These results indicate a previously unsuspected and novel role for the GRE2 gene product as a suppressor of filamentation by virtue of encoding isovaleraldehyde reductase activity.

Methionine catabolism in Saccharomyces cerevisiae.

The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol.

Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

'Fusel' alcohols induce hyphal-like extensions and pseudohyphal formation in yeasts.

At a concentration of 0.5% (v/v), isoamyl alcohol induced the formation of hyphal-like extensions in haploid and diploid strains of Saccharomyces cerevisiae in liquid complex medium. These extensions, which develop via bud initiation and elongation, undergo DNA replication and nuclear division and appear similar in many respects to an aberrant form of the cell division cycle. However, in 0.25% (v/v) isoamyl alcohol, S. cerevisiae formed pseudohyphae. Other 'fusel' alcohols (which are the products of amino acid catabolism) also induced hyphal-like extensions in this yeast, with n-amyl alcohol being as equally effective as isoamyl alcohol. Isoamyl alcohol induced the formation of pseudohyphae in two species of Candida and both hyphal-like extensions and pseudohyphae in Brettanomyces anomalus, suggesting a close relationship or a common basis to the development of the two morphologies.

Biotransformation of hop aroma terpenoids by ale and lager yeasts.

Terpenoids are important natural flavour compounds, which are introduced to beer via hopping. It has been shown recently that yeasts are able to biotransform some monoterpene alcohols. As a first step towards examining whether yeasts are capable of altering hop terpenoids during the brewing of beer, we investigated whether they were transformed when an ale and lager yeast were cultured in the presence of a commercially available syrup. Both yeasts transformed the monoterpene alcohols geraniol and linalool. The lager yeast also produced acetate esters of geraniol and citronellol. The major terpenoids of hop oil, however, were not biotransformed. Oxygenated terpenoids persisted much longer than the alkenes.

In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint.

Isoamyl alcohol (IAA) induces a phenotype that resembles pseudohyphae in the budding yeast Saccharomyces cerevisiae. We show here that IAA causes the rapid formation of linear chains of anucleate buds, each of which is accompanied by the formation of a septin ring at its neck. This process requires the activity of Swe1 and Slt2 (Mpk1). Cdc28 is phosphorylated on tyrosine 19 in a Swe1-dependent manner, while Slt2 becomes activated by dual tyrosine/threonine phosphorylation. Tyrosine 19 phosphorylation of Cdc28 is not dependent on Slt2. However, the defective response in the slt2Delta mutant is rescued by an mih1Delta mutation. The IAA response still occurs in a cell containing a dominant non-phosphorylatable form of Cdc28, but no longer occurs in an mih1Delta slt2Delta mutant containing this form of Cdc28. These observations show that IAA induces the Swe1-dependent morphogenesis checkpoint and so the resulting pseudohyphal phenotype arises in an entirely different way from the formation of pseudohyphae induced by nitrogen-limited growth.

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content.

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.

Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation.

DNA homologous to the yeast ( Saccharomyces cerevisiae) protein kinase gene, GCN2, was amplified from arabidopsis [ Arabidopsis thaliana (L.) Heynh.] RNA and given the name AtGCN2. The AtGCN2 peptide sequence included adjacent protein kinase and histidyl tRNA synthetase-like domains and showed 45% sequence identity with the GCN2 peptide sequence in the protein kinase domain. AtGCN2 transcripts were detectable in RNA from roots, leaves, stems, buds, flowers, siliques and seedlings. GCN2 is required for yeast cells to respond to amino acid starvation. Expression of AtGCN2 in yeast gcn2 mutants complemented the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched-chain amino acid biosynthesis, and 3-aminotriazole, an inhibitor of histidine biosynthesis.

The catabolism of amino acids to long chain and complex alcohols in Saccharomyces cerevisiae.

The catabolism of phenylalanine to 2-phenylethanol and of tryptophan to tryptophol were studied by (13)C NMR spectroscopy and gas chromatography-mass spectrometry. Phenylalanine and tryptophan are first deaminated (to 3-phenylpyruvate and 3-indolepyruvate, respectively) and then decarboxylated. This decarboxylation can be effected by any of Pdc1p, Pdc5p, Pdc6p, or Ydr380wp; Ydl080cp has no role in the catabolism of either amino acid. We also report that in leucine catabolism Ydr380wp is the minor decarboxylase. Hence, all amino acid catabolic pathways studied to date use a subtly different spectrum of decarboxylases from the five-membered family that comprises Pdc1p, Pdc5p, Pdc6p, Ydl080cp, and Ydr380wp. Using strains containing all possible combinations of mutations affecting the seven AAD genes (putative aryl alcohol dehydrogenases), five ADH genes, and SFA1, showed that the final step of amino acid catabolism (conversion of an aldehyde to a long chain or complex alcohol) can be accomplished by any one of the ethanol dehydrogenases (Adh1p, Adh2p, Adh3p, Adh4p, Adh5p) or by Sfa1p (formaldehyde dehydrogenase.)

Identification of SnIP1, a novel protein that interacts with SNF1-related protein kinase (SnRK1).

Plant SNF1-related protein kinase (SnRK1) phosphorylates 3-hydroxy-3-methylglutaryl-Coenzyme A, nitrate reductase and sucrose phosphate synthase in vitro, and is required for expression of sucrose synthase in potato tubers and excised leaves. In this study, a barley (Hordeum vulgare) endosperm cDNA, SnIP1, was isolated by two-hybrid screening with barley SnRK1b, a seed-specific form of SnRK1. The protein encoded by the SnIP1 cDNA was found to interact with barley SnRK1b protein in vitro. Southern analysis suggested that barley contains a single SnIP1 gene or small gene family. SnIP1 transcripts were detected in RNA isolated from leaf, root and mid-maturation seed. Sequence similarity searches against protein, nucleotide and expressed sequence tag databases identified hitherto uncharacterized sequences related to SnIP1 from maize (Zea mays, accession number AI691404), arabidopsis (Arabidopsis thaliana. AC079673 and AB016886) and poplar (Populus balsamifera, AI166543). No homologous sequences were identified from outside the plant kingdom, but weak sequence similarity was found between the SnIP1 peptide and yeast (Saccharomyces cerevisiae) SNF4 and its mammalian homologue AMPKy. Nevertheless, SnIP1 failed to complement a yeast snf4 mutant. SnIP1 was found to have little overall sequence similarity with the PV42 family of SNF4-like plant proteins, but proteins of both the SnIP1 and PV42 families contain a conserved hydrophobic sequence we named the SnIP motif.

A WEE1 homologue from Arabidopsis thaliana.

Little is known about the genes that regulate cyclinB-Cdc2 complexes at the G2/M transition of the plant cell cycle although in yeast and animals cdc25 and wee1 are central regulators of cdc2. Here we describe the isolation, by reverse transcription polymerase chain reaction (RT-PCR), of a WEE1 cDNA (AtWEE1) in Arabidopsis thaliana (L.) Heynh. Semi-quantitative RT-PCR showed that AtWEE1 expression was confined to actively dividing regions of the plant. The overexpression of AtWEE1 in fission yeast (Schizosaccharomyces pombe) caused cells to arrest, and to grow but not divide, resulting in very elongated cells. Our data provide evidence for a functional WEE1 in A. thaliana.

Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions.

Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1, PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the PGK1 promoter (PGK1(Delta767)) expressed Pyk1 and Pf1k at about 2.5-fold lower levels than normal. Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1(Delta767)-PYK1 mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.

The Arabidopsis 14-3-3 protein, GF14omega, binds to the Schizosaccharomyces pombe Cdc25 phosphatase and rescues checkpoint defects in the rad24- mutant.

The fission yeast (S. pombe) mitotic inducer gene, Spcdc25, interacts with the plant cell cycle to establish a small cell size phenotype compared with wild-type cells. We have investigated the nature of this interaction by yeast two-hybrid screening using Spcdc25 as bait in a cDNA library prepared from root tips of Arabidopsis thaliana (L.) Heynh. Three 14-3-3 proteins were detected: G-box Factor-like (GF)14kappa, lambda and omega; binding with Spcdc25 was confirmed by an independent immunoprecipitation assay. To test for cell cycle checkpoint function, GF14kappa, lambda and omega were transformed independently, using the strong nmt1+ promoter, into rad24-, a fission yeast mutant deficient in a 14-3-3 checkpoint protein. When exposed to UV irradiation or in the presence of 10 mM hydroxyurea, only cells transformed with GF14omega could fully rescue the defects in the DNA-damage and DNA-replication checkpoints of this mutant. Supporting evidence for a GF14omega cell cycle function was provided by semi-quantitative reverse transcription-polymerase chain reaction indicating that expression of this gene was elevated in regions of the plant that comprise dividing cells whereas GF14kappa and lambda expression was more evenly detected in all tissues examined. The data are consistent with the hypothesis that interaction between Spcdc25 and the plant cell cycle occurs at the level of a 14-3-3 protein with distinct checkpoint properties.