Genetic and environmental factors influencing redox homeostasis in Saccharomyces cerevisiae

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is unclear how the optimal redox potential is sensed and set, and the processes that impact redox state on a cellular/organellar level are poorly understood. Genetic and environmental factors affecting organellar redox homeostasis were investigated using a recently developed redox probe, roGFP2 (an indicator of the GSSG + 2H+ + 2e-  2GSH couple; Hanson et al., 2004), targeted to the cytosol, mitochondrial matrix and peroxisome of Saccharomyces cerevisiae. Used in conjunction with organelle-targeted pHluorin (a pH-sensitive GFP; (Miesenböck et al., 1998), both steady state and dynamic changes in redox potential were studied at the sub-cellular level.Wild-type cells were subjected to various physiological and stress conditions and compartmental redox state examined. This resulted in the identification of the redox and pH changes that occur during common environmental stress conditions. To complement this a genome-wide screen to identify mutants affected in cytosolic redox state AND a targeted-screen of mitochondrial mutants were conducted with an aim of understanding the genetic basis of the setting, sensing and maintenance of redox homeostasis. From the genome-wide screen 89 mutants were identified as having altered cytosolic redox state. It was found that processes involved in antioxidant defence and oxidative stress responses were significantly enriched in the data genome-wide screen data set. Subsequently NADPH regeneration and glutathione cycling were identified as the most important processes for the maintenance of redox state across the organelles tested. From the targeted mitochondrial screen, mutants in subunits of cytochrome C oxidase and mitochondrial fusion exhibited a more oxidised mitochondrial matrix redox potential compared to wild type indicating a role for these genes as potential regulators or sensors of matrix redox homeostasis.Finally, to understand the physiological role of glutathione, glutathione-depleted cells were studied. It was found that glutathione homeostasis is intimately linked with iron homeostasis and mitochondrial DNA maintenance implicating a greater role for glutathione in cell metabolism