Elucidation of the enzyme involved in 2,3,5-triphenyl tetrazolium chloride (TTC) staining activity and the relationship between TTC staining activity and fermentation profiles in Saccharomyces cerevisiae

2,3,5-Triphenyl tetrazolium chloride (TTC) staining is a method to distinguish the mitochondrial activity of cells based on the color: colorless TTC turns red when under reducing conditions. Although the assay reflects the mitochondrial activity of cells, which enzyme(s) in the electron transport system contribute to TTC reduction has been unclear. TTC staining assays using gene disruptants related to the electron transport system in Saccharomyces cerevisiae revealed those disruptants related to electron transport from each electron donor to ubiquinone (red colonies) and disruptants that were related to ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase (white colonies). In addition, when the enzyme activities of ubiquinol-cytochrome c oxidoreductase and cytochrome c oxidase were measured using TTC as the electron acceptor, only ubiquinol-cytochrome c oxidoreductase showed TTC reduction activity, and the activity was enhanced by potassium cyanide, an inhibitor of cytochrome c oxidase. These results indicated that ubiquinol-cytochrome c oxidoreductase is involved in TTC reduction in S. cerevisiae. The fermentation profiles of BY4741UΔcor1 and BY4741UΔcox4, which exhibited no TTC staining activity, were almost identical to that of the parental strain BY4741U. However, cell growth and ethanol and succinate production of the ura3-mutated strain BY4741, which also exhibited no TTC staining activity, was altered compared to those of BY4741U, indicating that the fermentation profile varies among strains that show no TTC staining activity. The relationship between uracil metabolism and TTC staining activity was also determined based on metabolome analysis.