Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan

Abstract

Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal.

Keywords: Chronological lifespan; DNA damage response; Rad53p; Reactive oxygen species; mtDNA.

Figure 1. Lack of Ntg1p prevents adaptive mtROS-mediated lifespan extension and elicits mild and conditional effects on respiration

A. Chronological lifespan (CLS) analysis of wild-type DBY2006 and ntg1Δ following treatment with 50 μM menadione (MD) or ethanol (nt) during exponential growth. In all graphs, data points represent the average viability of three biological replicates and error bars represent the standard error of the mean. B. CLS of wild-type and ntg2Δ as in A. C. CLS of wild-type, tor1Δ, and ntg1Δ/tor1Δ. D. CLS of wild-type and ntg1Δ grown in 20% glucose (nt) or 0.5% glucose (CR). E-F. Analysis of mitochondrial oxygen consumption in wild-type DBY2006 and ntg1Δ strains during exponential growth (OD=0.5, E) and at day one of CLS (48 hours after inoculation, F). Oxygen consumption was measured as %O₂/min/OD600, and the wild-type values were set to one. In all graphs, data points represent the mean of three biological replicates and error bars the SEM.

Figure 2. Lack of Ntg1p causes mtDNA depletion and Rad53p activation

A. Analysis of mtDNA copy number in wild-type DBY2006 and ntg1Δ during exponential growth (OD=0.5) and at day one of CLS (48 hours after inoculation). The copy number of the wild-type samples was set to one. B. Western blot of Rad53p phosphorylation in wild-type DBY2006 and ntg1Δ strains treated with 0.01% methylmethane sulfate (MMS, +) or water (-) for 30 minutes during exponential growth. C. Expression of two Rad53p-regulated genes in wild-type DBY2006 and ntg1Δ strains treated with MMS as described in B.

Figure 3. Restoring mtDNA copy number in ntg1Δ alleviates Rad53p activation

A. Mitochondrial DNA copy number in wild-type DBY2006, ntg1Δ, sml1Δ, and ntg1Δ/sml1Δ measured at OD=0.5. B. Cell cycle analysis by FACS of asynchronous cultures in early exponential growth (OD=0.2-0.3). The first peak indicates cells in G1, and the second indicates cells in G2. Representative histograms of three biological replicates are shown C. Quantification of cell cycle profiles determined by FACS. D. Western blot of Rad53p phosphorylation in sml1Δ and ntg1Δ/sml1Δ strains treated with 0.01% methylmethane sulfate (MMS, +) or water (-) for 30 minutes during exponential growth.

Figure 4. An mtDNA copy number threshold supports mtROS adaptation and longevity

A. RT-PCR analysis of subtelomeric genes at day one of CLS, or 24 hours after treatment with menadione (MD+) or ethanol (-) during exponential growth. Statistically significant changes between ethanol treated and menadione treated samples are indicated. B. CLS of ntg1Δ and ntg1Δ/sml1Δ following treatment with menadione (MD) or ethanol (nt) during exponential growth.

Figure 5. DNA damage response kinases regulate Ntg1p localization

A. Fluorescence microscopy of Ntg1p-GFP in strains lacking DNA damage response kinases during exponential growth. Mitochondria were visualized with MitoTracker Red (MTR), and DAPI staining identifies nuclear DNA (indicated by arrows labeled “n”) and mitochondrial DNA nucleoids (“m”). B. Quantification of Ntg1-GFP mitochondrial and nuclear co-localization. The percentage of cell with exclusively nuclear GFP (open circles), exclusively mitochondrial (filled squares), or mixed nuclear and mitochondrial (“X”) localization was determined by counting at least 100 cells for each condition from two experiments. Data points represent the mean and error bars the range of percentages. C. Western blot of nuclear and mitochondrial fractions of the indicated strains expressing Ntg1p-HA. Rad53Δ and mec1Δ are also lacking SML1. Porin and Histone 3 serve as mitochondrial and nuclear loading controls, respectively.