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.