Fig. 6

Impact of mitophagy inhibition on chronological lifespan. A Mitophagy is induced during the stationary phase in low glucose conditions. Mitophagy was monitored by the cleavage of Sdh2-GFP detected by immunoblotting with an anti-GFP antibody. A wild-type strain (972) was grown in rich media containing 3% glucose (3% Glu) or 1% glucose (1% Glu). Samples correspond to TCA extracts of cell cultures after logarithmic growth (day 0) and days 1, 2, and 3 of the stationary state. Sty1 levels were used as a loading control. B Deletion of Atg8 inhibits processing of Sdh2-GFP during the stationary phase. Mitophagy induction in 972 (WT) and atg8Δ strains was monitored by immunoblotting as in A. Samples correspond to TCA extracts from logarithmic growth (day 0) and days 1, 2, and 3 of the stationary state. Sty1 levels were used as a loading control. C Colocalization of the mitochondrial marker Sdh2-GFP and the vacuolar marker Cpy1-mCherry. Wild-type cells grown in rich media with 3% glucose (3% Glu) or 1% glucose (1% Glu) were visualized during logarithmic growth (Log) and day 3 of the stationary phase. Insets show a magnified region containing vacuoles labeled with GFP and Cpy1-mCherry. Scale bar, 5 μm. D atg43-1 mutant blocks mitophagy during the stationary phase at low glucose conditions. Mitophagy in 972 (WT) and atg43-1 strains was monitored by the cleavage of Sdh2-GFP detected by immunoblotting with an anti-GFP antibody. Samples correspond to TCA extracts of cell cultures grown in media with 1% glucose and collected after logarithmic growth (day 0) and days 1, 2, and 3 of the stationary state. Ponceau was used as the loading control. Bar plot represents the mean and SD of the GFP/Sdh2-GFP ratio from three independent experiments. Quantification was performed using Image Lab software. Significant differences between the atg43-1 strain and wild type were determined by a two-sided t-test (***p < 0.001). E Inhibition of mitophagy reduces cell longevity under low glucose conditions (1% Glu). Lifespan of 972 (WT), pka1Δ, sty1Δ, and atg43-1 strains was measured by propidium iodide staining and FACS. Line plot represents the local regression curves for the average survival of each strain (n > 10) at different time points. Each survival curve also displays a 95% confidence interval band. Bar plot depicts the average area under the curve of each strain, and error bars represent SD. Significant differences between deletion strains and wild type were determined by a two-sided t-test (*p < 0.05, **p < 0.01, ***p < 0.001)