Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina

Abstract

In Podospora anserina, inactivation of the respiratory chain results in a spectacular life-span extension. This inactivation is accompanied by the induction of the alternative oxidase. Although the functional value of this response is evident, the mechanism behind it is far from understood. By screening suppressors able to reduce the life-span extension of cytochrome-deficient mutants, we identified mutations in two zinc-cluster proteins, RSE2 and RSE3, which are conserved in other ascomycetes. These mutations led to the overexpression of the genes encoding the alternative oxidase and the gluconeogenic enzymes, fructose-1, 6 biphosphatase, and pyruvate carboxykinase. Both RSE2 and RSE3 are required for the expression of these genes. We also show that, even in the absence of a respiratory deficiency, the wild-type RSE2 and RSE3 transcription factors are involved in life-span control and their inactivation retards aging. These data are discussed with respect to aging, the regulation of the alternative oxidase, and carbon metabolism.

Figure 1.—

Mycelium aspect of the wild-type, cox5∷ble, and cox5∷ble rse2-1 strains. Petri plates of M2 medium were inoculated with an explant of each strain and incubated for 10 days at 27°. The wild-type strain exhibits a dense, aerial, colored mycelium and is fast growing whereas the cox5∷ble mutant exhibits a thin, poorly colored mycelium and is slow growing. The cox5∷ble rse2-1 revertant exhibits an intermediate phenotype.

Figure 2.—

Western blot analysis of the AOX protein. Mitochondria (10 μg of mitochondrial protein) were extracted from the wild-type (wt), cyc1-1, rse3-1, rse2-1, gpd-aox, cyc1-1 gpd-aox, and cyc1-1 rse3-1 strains and loaded onto a 12% SDS–PAGE acrylamide gel. The AOX was revealed with a mouse antiserum against S. guttatum AOX provided by T. Elthon. As an internal control, the blot was reprobed with a rabbit anti βATPase provided by J. Velours.

Figure 3.—

Relative abundance of aox transcripts. For each strain, the levels of aox and gpd transcripts were determined by quantitative RT–PCR performed on one to three different RNA preparations (one to three replicates). For each experiment, the level of aox transcripts was normalized using the level of gpd transcripts as a reference. The graph shows the level of aox transcripts relative to the level of aox transcripts in the wild type for each strain. The error bars correspond to standard error. The rse2⁺(rse2^Y300D) strain corresponds to a strain in which an ectopic copy of the mutated rse2^Y300D has been integrated.

Figure 4.—

Structure of the rse2 and rse3 genes. The amino acids mutated in the rse2 gene (top) and the rse3 gene (bottom) are indicated. Exons (E) are shown as solid boxes and introns as solid lines. The first and the last amino acids of each protein are indicated. Sites within exons that contain a motif identified as a possible zinc cluster are indicated by thin lines below the genes.

Figure 5.—

Quantification of fbp, pck, and aox expression. Total RNA was extracted from cultures of the wild-type strain grown under normal conditions (solid) or in the presence of antimycin A (10 μg/ml) (open) and from cultures of the rse2^Y300D (light shading) and rse3^G642V (dark shading) mutants grown under normal conditions. qPCR reactions were performed on cDNA to quantify the level of fbp, pck, aox, and gpd transcripts in each strain. Experiments were performed at least three times. As in Figure 3, in each experiment, the level of fbp, pck, and aox transcripts was normalized using the level of gpd transcripts as a reference. The diagram shows the level of fbp, pck, and aox transcripts in the different strains and culture conditions relative to the level of these transcripts in the wild type. The error bars correspond to standard error.

Figure 6.—

Life-span analyses. For each genotype, at least 18 subcultures (representing the two mating types) were grown on M2 medium at 27° in race tubes. Data were plotted as the cumulative survival in time using Kaplan–Meier estimates. The mean longevity in centimeters ± standard error is in parentheses.