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Abstract

Aging cells show a number of characteristic phenotypes usually summarized by the generic term: senescence. Amongst them there is accumulation of damaged proteins or DNA, changes in metabolism and a reduced replicative capacity. The lifespan of actively dividing cells is limited by replicative senescence whereas stationary cells age in a chronological way. Single-celled organisms like yeast are potentially immortal as offspring continuously rejuvenate. Pedigree-analysis, however, demonstrates that this is not the case for both product cells originating from a cell division. This becomes clearly visible when the organism divides asymmetrically. For that reason bakers yeast Saccharomyces cerevisiae has long since become an important organism for aging research. The unfavorable relative abundance of old cells in an exponentially growing culture and the separation from the rejuvenated young cells however, makes it difficult to study this model system on a molecular level. The molecular chaperone Hsp90 and its regulators play a central role in stress-resistance and adaptation and are thought to be involved in the aging process as well. The initial aim of this work was therefore the development of a system to monitor and quantify age-associated changes with fluorescent reporters. To attain single-cell resolution at high-throughput, measurement by flow-cytometry was the method of choice. For that reason it was necessary to modify the established Hsp90 reporter-systems and to eliminate many sources of error. By that I could show a progressive decline in the heat-shock response and loss of the Hsp90 dependent pheromone response in old yeast cells. In addition, the newly designed reporter systems were used in young cells for functional studies of Hsp90 mutants. During the establishment of the method, I had the opportunity to test the recently optimized redox-sensitive roGFP2-systems and the pH sensitive pHluorin reporter in old cells. As the results were promising and unexpected, the redox homeostasis in old cells became the main focus of my project. I could successfully monitor changes in the cytosolic glutathione equilibrium, hydrogen-peroxide abundance and pH in replicatively aged yeast cells. Surprisingly, the glutathione reporter was progressively more reduced in aging cells during fermentative growth whereas hydrogen-peroxide levels seem to rise at the same time. Simultaneously, the redox-pools became more resistant to oxidant treatment and the pH in old cells decreased. The reducing phenotype turned out to be dependent on the glutareductase Glr1. Using in vivo acidification measurements I could demonstrate that Glr1 is pH dependently regulated and could reproduce the glutathione-reduction phenotype in young yeast cells by lowering the cytosolic pH. Furthermore, similar pH changes occurred in a model system for telomere-induced senescence, but no distinct changes in the redox reporters could be seen, suggesting that redox homeostasis is differentially regulated in this context. Yeast cells grown under respiratory conditions have a lower cytosolic pH and a more reduced glutathione-pool, consistent with the Glr1 activation. During aging, oxidation of both the glutathione and peroxide-pools increased in this case. Interestingly, respiring cells have an extended replicative lifespan even when Glr1 is deleted and the cells are vastly oxidized. In fermenting cells Glr1 knockout also leads to a strong oxidation without affecting the replicative lifespan. This leads to the conclusion that homeostasis of the glutathione-pool is not limiting replicative lifespan in yeast. Given that the cytosolic pH is actively maintained by H+-ATPases, I speculate that the age-associated acidification may be the result of impaired ATP production.