Figure 3.

Yeast studies. A: Growth of Δmto1 strain transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid on YP medium supplemented with 2% glucose (left panel) or 2% glycerol (left panel). Cells were pregrown on YP+glucose and plated after serial dilutions to obtain spots of 5 × 10⁴, 5 × 10³, 5 × 10², and 5 × 10¹ cells/spot. Pictures were taken after 2 days of growth. B: Respiratory activity of Δmto1 strains transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid. Respiratory rates were normalized to the strain transformed with wt MTO1, for which the respiratory rate was 34.7 nmol min⁻¹ mg⁻¹. Values are the mean of three independent experiments, each with an independent clone. Two-tail, paired t-test was applied for statistical significance. ^***P < 0.001. C: In vivo mitochondrial translation of Δmto1 strain transformed with MTO1 wt allele, mto1^R481H, and mto1^T414I mutant alleles or empty plasmid. Mitochondrial gene products were labeled with [³⁵S]-methionine in whole cells in the presence of cycloheximide for 10 min at 28°C. Cox: cytochrome c oxidase; Cob: cytochrome b; Atp: ATP synthase; Var1: small mitochondrial ribosome subunit. D: Cytochrome c oxidase (CIV) activity of Δmto1 strain transformed with MTO1 wt allele, mto1^T414I, mto1^A431T, mto1^R481H, and mto1^P622X mutant alleles or empty plasmid. Cytochrome c oxidase activities were normalized to the strain transformed with wt MTO1, for which the activity was 368.8 units per mg of mitochondrial proteins. Values are the mean of three independent experiments, each with an independent clone. Two-tail, unpaired t-test was applied for statistical significance. ^**P < 0.01.