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Validation of a respiratory phenotyping assay for yeast indicates that aerobic metabolism is regulated by mitochondrial thioredoxin activity (572.1)
Author(s) -
Weber Emily,
PadillaLopez Sergio,
Pearce David,
Vitiello Seasson,
Vitiello Peter
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.572.1
Subject(s) - oxidoreductase , thioredoxin , biochemistry , glutaredoxin , biology , reactive oxygen species , cellular respiration , respiratory chain , mitochondrion , saccharomyces cerevisiae , mitochondrial respiratory chain , yeast , enzyme
Thiol oxidoreductase enzymes, including thioredoxins, glutaredoxins, peroxiredoxins, and glutathione peroxiredoxins, play an important role in regulating cellular redox reactions through reduction of oxidized thiols. These enzymes regulate integral processes such as protein folding, detoxification of reactive oxygen species, and metal and sulfur metabolism; however, many thiol oxidoreductase substrates and consequential functional changes are not known. Since thiol oxidoreductase genes are highly conserved across eukaryotic species, the objective of this study was to screen a Saccharomyces cerevisiae thiol oxidoreductase deletion library for respiratory phenotypes. A novel protocol using the XF Flux Analyzer (Seahorse Biosciences) was established to simultaneously measure real‐time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) from media which respectively correlates to cellular aerobic and anaerobic respiratory activities in S. cerevisiae. This approach was validated by artificially pressuring yeast to preference metabolic pathways by restricting nutrient availability in the media and genetic deletion of critical respiratory enzymes. Simultaneous screening of 15 thiol oxidoreductase deletion strains for OCR and ECAR resulted in a single putative respiratory‐regulating enzyme. Deficiency in thioredoxin‐3 (TRX3) resulted in impaired oxygen consumption, and no other mitochondrially‐targeted thiol oxidoreductases (Grx2, Grx5) recapitulated this phenotype. This suggests that Trx3 may have substrate specificity and regulate the activity of a rate‐limiting aerobic respiratory protein in the mitochondria. Taken together, these studies demonstrate that the XF Flux Analyzer can be used to screen for respiratory phenotypes in S. cerevisiae and that aerobic respiration is regulated through mitochondrial thioredoxin status.