Mitochondrial dysfunction reduces yeast replicative lifespan by elevating RAS-dependent ROS production by the ER-localized NADPH oxidase Yno1

<div><p>Mitochondrial dysfunction leads to the accumulation of reactive oxygen species (ROS) which is associated with cellular dysfunction, disease etiology, and senescence. Here, we used the eukaryotic model <i>Saccharomyces cerevisiae</i>, commonly studied for cellular aging, to demonstrate how defective mitochondrial function affects yeast replicative lifespan (RLS). We show that RLS of respiratory-deficient cells decreases significantly, indicating that the maintenance of RLS requires active respiration. The shortening of RLS due to mitochondrial dysfunction was not related to the accumulation of extrachromosomal ribosomal DNA circles, a well-known cause of aging in yeast. Instead, intracellular ROS and oxidatively damaged proteins increased in respiratory-deficient mutants. We show that, while the protein kinase A activity is not elevated, ROS generation in respiratory-deficient cells depends on RAS signaling pathway. The ER-localized NADPH oxidase Yno1 also played a role in producing ROS. Our data suggest that a severe defect in mitochondrial respiration accelerates cellular aging by disturbing protein homeostasis in yeast.</p></div

Transcriptional silencing is not a major cause for shortening of RLS in respiratory-deficient cells.

<p>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, <i>cyc3</i>Δ, and <i>shy1</i>Δ 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, <i>cox5a</i>Δ, and <i>cyc1</i>Δ cells were spotted on SC media without uracil or with FOA. (E) Total RNA was extracted from WT, rho⁰, <i>cyc3</i>Δ, <i>shy1</i>Δ, <i>cox5a</i>Δ, <i>cyc1</i>Δ, and <i>sir2</i>Δ cells. Quantitative real-time reverse transcription-PCR analysis was performed to measure the <i>mURA3</i> transcript level. Amplification efficiencies were validated and normalized against <i>ACT1</i>. The relative transcript levels of the <i>mURA3</i> gene were calculated as the ratio of the normalized transcript levels of the <i>mURA3</i> gene inside the rDNA array (<i>NTS1</i>::<i>mURA3</i> and <i>NTS2</i>::<i>mURA3</i>) to that outside the rDNA array (<i>leu2</i>::<i>mURA3</i>). 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 <i>ADE2</i> 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 <i>P</i><0.01, compared with WT cells (one-way ANOVA).</p