Fig. 3: Put6 and Put7 are not required for MRC function or formation.

a, b Put6 and Put7 are not required for respiratory growth. a Growth analysis of WT, put6Δ, and put7Δ yeast cells in YPD, YPG, YPE, and YPGE at 30 °C. Absorbance at 600 nm was measured at the indicated time intervals. Data are representative of three independent experiments. b Ten-fold serially diluted WT, put6Δ, put7Δ, and put6Δput7Δ yeast cells were seeded on the indicated plates at 30 °C (upper panel) and 37 °C (lower panel). Pictures were taken after 2 (YPD) or 5 (YPG, YPE, and YPGE) days of growth. Data are representative of at least three independent experiments. c Mitochondrial proteins were extracted from WT and put6Δ yeast cells and analyzed by SDS-PAGE/western blotting using antibodies specific for MRC complexes II–V. Por1 was used as a loading control. Data are representative of three independent experiments. d Mitochondria were solubilized in 1% digitonin and analyzed by BN-PAGE immunoblotting. The molecular weights and stoichiometry of the supercomplexes are indicated. Data are representative of three independent experiments. e Digitonin-solubilized mitochondrial complexes from WT and put6Δ cells were resolved by CN-PAGE, followed by in-gel activity staining for complexes II–V. Data are representative of three independent experiments. f Oxygen consumption of YPGE grown WT and put6Δ cells was measured using high-resolution O2K Fluorespirometer at 30 °C. Mitochondrial respiration refers to antimycin sensitive respiration, non-mitochondrial respiration refers to antimycin resistant respiration, and maximal respiratory rates were calculated after addition of 5 μM CCCP. g Measurement of mitochondrial membrane potential using TMRM fluorescence in WT, put6Δ, and put7Δ cells grown in YPGE media. Data were normalized to WT control and are expressed as mean ± SD (n = 3 biologically independent experiments). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. WT and mutants used in this panel are from BY4741 genetic background (see Supplementary Table 1). Source data are provided as a Source data file.