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dc.contributor.advisorTrush, Michael A.en_US
dc.creatorBaron, James Allenen_US
dc.date.accessioned2014-12-23T04:40:15Z
dc.date.available2014-12-23T04:40:15Z
dc.date.created2013-12en_US
dc.date.issued2013-10-18en_US
dc.date.submittedDecember 2013en_US
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/37081
dc.description.abstractpH 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.en_US
dc.format.mimetypeapplication/pdfen_US
dc.languageen
dc.publisherJohns Hopkins University
dc.subjectreactive oxygen speciesen_US
dc.subjectsuperoxideen_US
dc.subjectsuperoxide dismutaseen_US
dc.subjectoxidative stressen_US
dc.subjectpHen_US
dc.subjectSOD1en_US
dc.subjectSOD2en_US
dc.subjectPMA1en_US
dc.subjectALD4en_US
dc.subjectSDHen_US
dc.subjectACO1en_US
dc.subjectTCA cycleen_US
dc.subjectnutrient starvationen_US
dc.subjectglucoseen_US
dc.titleThe Dual Nature of Reactive Oxygen Species: Regulation of pH Homeostasis and Survival in Saccharomyces cerevisiae by ROS Damage and Signalingen_US
dc.typeThesisen_US
thesis.degree.disciplineBiologyen_US
thesis.degree.grantorJohns Hopkins Universityen_US
thesis.degree.grantorBloomberg School of Public Healthen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePh.D.en_US
dc.type.materialtexten_US
thesis.degree.departmentBiochemistry and Molecular Biologyen_US
dc.contributor.committeeMemberCulotta, Valeria L.en_US
dc.contributor.committeeMemberWang, Jiouen_US
dc.contributor.committeeMemberRao, Rajinien_US


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