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.
Heeren
Gino
G
Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Jarolim
Stefanie
S
Laun
Peter
P
Rinnerthaler
Mark
M
Stolze
Klaus
K
Perrone
Gabriel G
GG
Kohlwein
Sepp D
SD
Nohl
Hans
H
Dawes
Ian W
IW
Breitenbach
Michael
M
D015152:Blotting, Northern; D004578:Electron Spin Resonance Spectroscopy; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D005978:Glutathione / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D016296:Mutagenesis; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012235:Rhodamines / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D012331:RNA, Fungal; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; C058319:dihydrorhodamine 123; D013481:Superoxides; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D005978:Glutathione