The oxidation state of the cytoplasmic glutathione redox system does not correlate with replicative lifespan in yeast

What is cause and what is consequence of aging and whether reactive oxygen species (ROS) contribute to this phenomenon is debated since more than 50 years. Notwithstanding, little is known about the cellular buffer and redox systems in aging Saccharomyces cerevisiae, which is a model for aging stem cells. Using genetically encoded fluorescent sensors, we measured pH, H2O2 levels and the glutathione redox potential compartment-specific in the cytosol of living, replicatively aging yeast cells, growing under fermenting and respiratory conditions until the end of their lifespan. We found that the pH decreases under both conditions at later stages of the replicative lifespan. H2O2 levels increase in fermenting cells in the post-replicative stage, but increase continuously with age in respiring cells. The glutathione redox couple becomes also more oxidizing in respiring cells but surprisingly more reducing under fermenting conditions. In strains deleted for the gene encoding glutathione reductase Glr1, such a reduction of the glutathione redox couple with age is not observed. We demonstrate that in vivo Glr1 is activated at lower pH explaining the reduced glutathione potential. The deletion of glr1 dramatically increases the glutathione redox potential especially under respiratory conditions but does not reduce lifespan. Our data demonstrate that pH and the glutathione redox couple is linked through Glr1 and that yeast cells can cope with a high glutathione redox potential without impact on longevity. Our data further suggest that a breakdown of cellular energy metabolism marks the end of replicative lifespan in yeast. Accumulating reactive oxygen species were proposed as leading cause of aging. Moreover, increasing pH in vacuole and cytosol was suggested to contribute to replicative aging in yeast, considered to be a model for aging stem cells. Here we investigated how cytosolic pH, H2O2 levels, and the glutathione redox buffer changes in aging yeast, using genetically encoded fluorescent probes and a newly developed flow-cytometry based aging assay. We found that pH decreases and H2O2 increases at the end of the replicative lifespan; but, surprisingly, the glutathione redox potential became more reducing in fermenting aged yeast cells in a glutareductase Glr1 dependent manner. Glr1 deletion leads to a highly oxidized cytosol but does not influence the replicative lifespan in liquid culture. Instead the end of lifespan seems to be marked by a break down of energy metabolism.