Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging

Abstract

Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long-lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long-lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2-independent but Gcn4-dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.

Figure 1. Growth inhibition of long lived strains in response to tunicamycin

(a) Long lived strains which showed significant (p < 0.05) changes in relative growth inhibition compared to wild type BY4742 are shown. The control sensitive strain hac1Δ did not grow in the media. Error bars are s.e.m. Data for all strains tested can be found in Supplemental Table 1. (b–d) Representative outgrowth curves for wild type, rpl20bΔ, and afg3Δ strains in 0, 1, or 5 µg/mL tunicamycin. (e) Scatter plot comparing the percent change in replicative lifespan to percent change in growth rate for each long-lived mutant.

Figure 2. Growth inhibition of long lived strains in response to paraquat

(a) Long lived strains which showed significant (p < 0.05) changes in relative growth inhibition compared to wild type BY4742 are shown. The control sensitive strains ctt1 Δ,cta1 Δ, and sod1Δ are shown for comparison. Error bars are s.e.m. Data for all strains tested can be found in Supplemental Table 2. (b–d) Representative outgrowth curves for wild type, rpl20bΔ, and afg3Δ strains in 0, 5, or 10 mM paraquat. (c) Scatter plot comparing the percent change in replicative lifespan to percent change in growth rate for each long-lived mutant.

Figure 3. Growth inhibition of long lived strains in response to MMS

(a) Long lived strains which showed significant (p < 0.05) changes in relative growth inhibition compared to wild type BY4742 are shown. The control sensitive rad52Δ strain is shown for comparison. Error bars are s.e.m. Data for all strains tested can be found in Supplemental Table 3. (b–d) Representative outgrowth curves for wild type, rpl20bΔ, and afg3Δ strains in 0, 0.005%, or 0.01% MMS. (c) Scatter plot comparing the percent change in replicative lifespan to percent change in growth rate for each long-lived mutant.

Figure 4. Clustering of long lived mutants based on their stress response profiles

Clusters were formed using Euclidean distance calculations of vectors constructed from the percent difference in sensitivity of a mutant strain compared to wild type, as described in Methods.

Figure 5. Hac1-independent resistance to tunicamycin in afg3Δ and rpl20bΔ cells

(a) Average doubling times for wild type BY4742, afg3Δ, hac1Δ, and afg3Δhac1Δ strains at the indicated concentrations of tunicamcycin. Note how afg3Δhac1Δ double mutant cells can grow, albeit very slowly, at 0.25µg/ml tunicamycin, while hac1Δ cells cannot, indicating Hac1-independent tunicamycin resistance. (b) Replicative lifespan curves of the indicated strains. Parentheses denote mean lifespan. afg3Δ does not require Hac1 for lifespan extension. (c) Spot assays of the indicated strains across tunicamycin concentrations. Spots were diluted 1:10. Panels were grown for 3 days at 30°C.

Figure 6. Deletion of AFG3 reduces cytoplasmic translation and extends life span by a Sir2-independent, Gcn4-dependent mechanism

(a) Representative polysome profiles of log phase wild type BY4742 yeast and afg3Δ mutant yeast grown in YPD at 30°C. Deletion of AFG3 causes a shift of ribosomes from highly translated mRNA to unbound forms. Curves are normalized by the minima between 80S free subunits and 2R disomes. (b) Quantitation of peak areas from triplicate polysomes. *p<0.05, ns= not significant, error bars are s.e.m. (c–f) Replicative lifespan curves of the indicated strains. Parentheses denote mean lifespan. afg3Δ requires Gcn4 for lifespan extension but does not require Sir2 or Fob1. afg3Δ is not additive with rpl20bΔ nor DR (0.05% glucose) for lifespan extension. (g) Flow cytometric analysis of log phase genomically tagged Gcn4-GFP strains.