Involvement of oxidative stress response genes in redox homeostasis, the level of reactive oxygen species, and ageing in Saccharomyces cerevisiae.
Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.
Drakulic
Tamara
T
Ramaciotti Centre for Gene Function Analysis and School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Sydney, Australia.
Temple
Mark D
MD
Guido
Ron
R
Jarolim
Stefanie
S
Breitenbach
Michael
M
Attfield
Paul V
PV
Dawes
Ian W
IW
D002374:Catalase / Q000235:genetics / Q000378:metabolism; D016922:Cellular Senescence; D053938:DNA Fragmentation; D004996:Ethidium / Q000031:analogs & derivatives; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005800:Genes, Fungal; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020112:Rhodamine 123; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D013194:Staining and Labeling; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D005456:Fluorescent Dyes; D007211:Indoles; D017382:Reactive Oxygen Species; C067883:dihydroethidium; D020112:Rhodamine 123; C007293:DAPI; D013879:Thioredoxins; D002374:Catalase; D013482:Superoxide Dismutase; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D004996:Ethidium; D005978:Glutathione