The Dual Nature of Reactive Oxygen Species: Regulation of pH Homeostasis and Survival in Saccharomyces cerevisiae by ROS Damage and Signaling
Baron, James Allen
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pH homeostasis is intimately linked with the metabolic state of cells. The baker’s yeast, Saccharomyces cerevisiae, is an excellent model of this as it readily switches from fermentation to respiration contingent on carbon source availability. When grown in abundant glucose, yeast obtain energy by fermentation and maintain a stable intracellular pH over a variety of environmental pH’s by activating energetically expensive H+-ATPases. When glucose is limited or absent, yeast switch to respiration, decrease the activity of H+-ATPases to conserve energy, and pH homeostasis becomes highly dependent on environmental pH and buffering by cellular metabolites. In this thesis, the regulation of pH homeostasis by reactive oxygen species (ROS) is examined at these two extremes of glucose abundance and glucose starvation using a variety of genetic and biochemical techniques. During long-term growth in the absence of glucose, yeast alternately alkalinize and acidify their environment which affects growth and survival. We demonstrate that mitochondrial ROS initiate the alkali-to-acid shift by inactivating the Fe-S cluster enzymes aconitase and succinate dehydrogenase of the tricarboxylic acid (TCA) cycle and that this shift is accelerated by deletion of the mitochondrial superoxide dismutase, SOD2. Inhibition of the TCA cycle enzymes alters metabolite flux through the cycle and leads to the buildup and secretion of the upstream metabolite acetic acid, which is generated by the aldehyde dehydrogenase Ald4p. Acetic acid secretion acidifies the environment without activating the plasma membrane H+-ATPase, Pma1p, and promotes the growth of yeast under these conditions by providing a new carbon source for survival. This work demonstrates that inhibition of Fe-S cluster enzymes by ROS can be beneficial under conditions of glucose starvation. In abundant glucose the principally cytosolic superoxide dismutase, Sod1p, is required for maximal activation of Pma1p, although the mechanism was not fully understood. In this work we discovered that deletion of SOD1 in combination with a specific mutation in the C-terminal autoinhibitory region of Pma1p, Pma1-T912D, is lethal to yeast cells. This lethality can be rescued by treatment with Mn-based antioxidants and by hypoxia. Furthermore, we identified spontaneous second-site mutations in PMA1 that reverse the aerobic lethality by activating the H+-ATPase. Thus, lethality results from profound inhibition of essential Pma1p activity. Together these results support a model in which Sod1p helps protect Pma1p from oxidative damage, particularly in cases where auto-inhibition of Pma1p is disrupted, as with Pma1-T912D.