Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction

Abstract

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases and determining how this affects chronological life span (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity, and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS. Extension of CLS by caloric restriction also required respiration above a similar threshold during exponential growth and completely alleviated the need for respiration in the stationary phase. Finally, we show that supplementation of media with 1% trehalose, a storage carbohydrate, restores wild-type CLS to respiratory-null cells. We conclude that mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction by increasing stress resistance, an important component of which is the optimal accumulation and mobilization of nutrient stores.

Figure 1. Mitochondrial respiratory thresholds regulate yeast CLS

(A and B) Cell respiration of W303, rho⁰ and cyc3 strains during exponential (A) and stationary (B) phases of growth. (C) CLS of W303, rho⁰ and cyc3 strains. (D and E) Cell respiration of W303, shy1 and C199 strains during exponential (D) and stationary (E) phases of growth. (F) CLS of W303, shy1 and C199 strains. (G and H) Cell respiration of W303, cox5a and cyc1 strains during exponential (G) and stationary (H) phases of growth. (I) CLS of W303, cox5a and cyc1 strains. Error bars represent the mean ± SD with p values denoted by * = p <0.05 and ** = p <0.01.

Figure 2. Effect of mitochondrial respiratory inhibitors on yeast CLS

(A and B) Cell respiration during exponential (A) and stationary (B) phases of growth of W303 cells treated with 50μM antimycin A or 10μM oligomycin through the exponential and stationary phases. (C) CLS of W303 strain treated with 50μM antimycin A (AMA) or 10μM oligomycin (OLI) through the exponential and stationary phases. (D) Cell respiration during the stationary phase of growth of W303 strain treated with 50μM antimycin A or 10μM oligomycin, through only the stationary phase (E) CLS of W303 strain treated with 50μM antimycin A or 10μM oligomycin through only the stationary phase. Error bars represent the mean ± SD with p values denoted by *= p <0.05 and **= p <0.01.

Figure 3. Regulation of CLS by mitochondrial function is acetic-acid independent

(A) Determination of acetic acid concentration in the growth media of the indicated strains. (B) CLS of W303 (rho⁺) or rho⁰ strains in their original media or subjected to media swap in the stationary phase. (C) CLS of W303, cox5a and cyc1 strains in the presence of buffered SDC media. (D) CLS of W303 strains grown in buffered SDC medium, treated with 50μM antimycin A (AMA) or 10μM oligomycin (OLI) from inoculation (Exp) or stationary phase (Sta) until the termination of the experiment. Error bars represent the mean ± SD.

Figure 4. ROS production during growth does not affect yeast CLS

(A and B) ROS production determined as DHE fluorescence in the indicated strains during exponential or stationary phases of growth. (C and D) ROS production determined as DHE fluorescence during exponential and stationary phase in W303 cells treated with 50μM antimycin A (AMA) or 10μM oligomycin (OLI) (C) through the exponential and stationary phases or (D) through only the stationary phase. Error bars represent the mean ± SD with p values denoted by * = p <0.05 and ** = p <0.01.

Figure 5. Mitochondrial respiratory thresholds regulate CLS under caloric restriction

(A and B) Cell respiration during exponential (A) and stationary (B) phases of growth of W303, cox5a and W303 strains treated with 10μM oligomycin through the exponential and stationary phases. Yeast were grown in regular (Non-CR) or caloric restriction (CR) SDC media. (C) CLS of W303, rho⁰ and cox5a strains grown under caloric restriction (CR). (D) CLS of W303 strain grown under caloric restriction (CR) treated with 50μM antimycin A (AMA) or 10μM oligomycin (OLI) from inoculation (Exp) or stationary phases (Sta) until the termination of the experiment. Error bars represent the mean ± SD. (E) CLS of tor1Δ strain treated as in panel (D)

Figure 6. Mitochondrial dysfunction curtails yeast CLS by decreasing stress resistance and altering the reserve nutrients metabolism

(A) Hydrogen peroxide and heat stress resistance of W303, rho⁰, cyc3, cox5a and cyc1 strains at day 0 during the stationary phase. (B) Time-course of glycogen and trehalose content during the stationary phase in W303, rho⁰ and cox5a cells. (C and D) Time-course of glycogen and trehalose content during the stationary phase of W303 cells treated with 50μM antimycin A (AMA) or 10μM oligomycin (OLI), (C) from inoculation (Exp) or (D) stationary phases (Sta) until the termination of the experiment. (E-F-H-I) CLS of (E) W303 cells, (F) rho⁰ cells, (G) W303 cells grown under CR and (H) tor1Δ cells grown in SDC medium supplemented with trehalose at the moment of inoculation (Exp) or at the stationary phase (Sta). Error bars represent the mean ± SD. (G) Hydrogen peroxide and heat stress resistance of W303 and rho⁰ strains grown in the presence or absence of trehalose at day 0 during the stationary phase.

Figure 7. Model of CLS regulation by mitochondrial respiration

Mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction (see explanation in the text). For simplicity, the effect of cell extrinsic factors is not included.