Fig 1.
Mitochondrial respiration defect impairs the maintenance of RLS.
(A) RLS analysis was performed with wild-type (WT), rho0, cyc3Δ, and shy1Δ cells. (B) The relative changes in RLS were calculated as the ratio of the mean RLS to that of WT cells in (A). (C) RLS analysis was performed with WT, rho0, and WT cells on media containing 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM). (D) The relative changes in RLS of indicated strains were calculated as the ratio of the mean RLS to that of WT cells in (C). (E) RLS analysis was performed with WT, rho0, cox5aΔ, and cyc1Δ cells. (F) The relative changes in RLS of indicated strains were calculated as the ratio of the mean RLS to that of WT cells in (E). Mean RLS values are shown in parentheses. All asterisks indicate P<0.01, compared with WT cells (one-way ANOVA).
Fig 2.
Transcriptional silencing is not a major cause for shortening of RLS in respiratory-deficient cells.
Silencing at the rDNA region was assessed by monitoring the growth of 10-fold serial dilution of cells on SC media lacking uracil or supplemented with FOA. SC medium was used as a control. (A) 10-fold serial dilutions of wild-type (WT) and rho0 cells were spotted on SC media without uracil or with FOA. (B) 10-fold serial dilutions of WT, cyc3Δ, and shy1Δ cells were spotted on SC media without uracil or with FOA. (C) 10-fold serial dilutions of WT cells were spotted on SC media without uracil containing 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM). (D) 10-fold serial dilutions of WT, cox5aΔ, and cyc1Δ cells were spotted on SC media without uracil or with FOA. (E) Total RNA was extracted from WT, rho0, cyc3Δ, shy1Δ, cox5aΔ, cyc1Δ, and sir2Δ cells. Quantitative real-time reverse transcription-PCR analysis was performed to measure the mURA3 transcript level. Amplification efficiencies were validated and normalized against ACT1. The relative transcript levels of the mURA3 gene were calculated as the ratio of the normalized transcript levels of the mURA3 gene inside the rDNA array (NTS1::mURA3 and NTS2::mURA3) to that outside the rDNA array (leu2::mURA3). Values represent the average of three independent experiments, and error bars indicate the standard deviation. (F) rDNA recombination assay was performed to check rDNA stability of the indicated cells. rDNA recombination is represented by the frequency of loss of the ADE2 marker gene integrated at the rDNA locus in the corresponding cells. Values represent the average of three independent experiments, and error bars indicate the standard deviation. Asterisks indicate P<0.01, compared with WT cells (one-way ANOVA).
Fig 3.
Increased ROS level decreases RLS in respiratory-deficient cells.
(A) Intracellular ROS levels in wild-type (WT), rho0, cyc3Δ, shy1Δ, cox5aΔ, cyc1Δ, and WT cells treated with 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM) were detected with H2DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. (B) Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P<0.01, compared with WT cells (one-way ANOVA). (C) Carbonylated proteins in WT, rho0, cyc3Δ, shy1Δ, cox5aΔ, cyc1Δ, and WT cells treated with 3 μg/ml AA or 10 μg/ml OM were detected using Oxidized Protein Detection kit. Hexokinase was used as a loading control. The relative ratio of carbonylated proteins in the indicated strain to those of WT cells is shown below each lane. Data are representative of at least three independent experiments. (D) Total protein was extracted from WT, rho0, cyc3Δ, shy1Δ, and WT cells treated with 3 μg/ml AA or 10 μg/ml OM, and WT cells under nitrogen starvation. All cells harbor pRS416-SCH9T570A-5HA. Immunoblotting was performed using a mouse anti-HA antibody. Data are representative of at least three independent experiments. (E) Total protein was extracted from WT, rho0, cyc3Δ, shy1Δ, WT cells treated with 3 μg/ml AA or 10 μg/ml OM, WT cells expressing constitutively active RAS2val19 (19V), and WT cells under glucose starvation. All cells harbor pRS423-CUP1-6xMYC-cki12-200(S125/130A). Immunoblotting was performed using a mouse anti-Myc antibody. The relative ratio of phosphorylated to unphosphorylated forms of Cki1 is shown below each lane. Data are representative of at least three independent experiments.
Fig 4.
RAS signaling pathway and Yno1 contribute to the buildup of intracellular ROS in cells lacking mitochondrial respiration.
(A, E, and H) Intracellular ROS levels in the indicated strains were detected with H2DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P<0.01, compared with rho0 cells (one-way ANOVA). (B, F, and I) RLS analysis was performed with the indicated strains. (C, G, and J) The relative changes in RLS were calculated as the ratio of the mean RLS to that of WT cells. Mean RLS values are shown in parentheses. All asterisks indicate P<0.01, compared with rho0 cells (one-way ANOVA). (D and K) Carbonylated proteins in indicated strains were detected using Oxidized Protein Detection kit. Hexokinase was used as a loading control. The relative ratio of carbonylated proteins in the indicated strain to those of WT cells is shown below each lane. Data are representative of at least three independent experiments.