Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.
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 replicative lifespan of Saccharomyces cerevisiae is determined by both genetic and environmental factors. Many of the same factors determine the lifespan of metazoan animals. The lack of fast and reliable lifespan assays has limited the pace of yeast aging research. In this study we describe a novel strategy for assaying replicative lifespan in yeast, and apply it in a screening of mutants that are resistant to pro-oxidants. The assay reproduces the lifespan-shortening effects of deleting SIR2 and of growth in the presence of paraquat, a pro-oxidant. The lifespan-increasing activity of resveratrol is also reproduced. Compared to current assays, this new strategy promises to significantly increase the possible number of replicative-lifespan determinations.
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.
The heat-shock response in cells, involving increased transcription of a specific set of genes in response to a sudden increase in temperature, is a highly conserved biological response occurring in all organisms. Despite considerable attention to the processes activated during heat shock, less is known about the role of genes in survival of a sudden temperature increase. Saccharomyces cerevisiae genes involved in the maintenance of heat-shock resistance in exponential and stationary phase were identified by screening the homozygous diploid deletants in nonessential genes and the heterozygous diploid mutants in essential genes for survival after a sudden shift in temperature from 30 to 50°. More than a thousand genes were identified that led to altered sensitivity to heat shock, with little overlap between them and those previously identified to affect thermotolerance. There was also little overlap with genes that are activated or repressed during heat-shock, with only 5% of them regulated by the heat-shock transcription factor. The target of rapamycin and protein kinase A pathways, lipid metabolism, vacuolar H+-ATPase, vacuolar protein sorting, and mitochondrial genome maintenance/translation were critical to maintenance of resistance. Mutants affected in l-tryptophan metabolism were heat-shock resistant in both growth phases; those affected in cytoplasmic ribosome biogenesis and DNA double-strand break repair were resistant in stationary phase, and in mRNA catabolic processes in exponential phase. Mutations affecting mitochondrial genome maintenance were highly represented in sensitive mutants. The cell division transcription factor Swi6p and Hac1p involved in the unfolded protein response also play roles in maintenance of heat-shock resistance.
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