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. 2022 Mar 2;11:e75658. doi: 10.7554/eLife.75658

Figure 5. Age-related reduction of Tom70 is associated with mitochondrial dysfunctions.

(A) Representative images of Tom70-GFP and mito-RFP in young and aged cells. Both young and old cells were from YPD culture and stained with calcofluor white to visualize the bud scars. (B) Quantification of different GFP-tagged TOM proteins and mitoRFP in young and aged cells. Both young and old cells were from YPD culture and the GFP signal was normalized to corresponding young cells of the same strain. (C, D) Representative images (C) and quantification (D) of mitochondrial membrane potential in young and aged cells from different strains. Different TOM proteins were overexpressed from pGAL promoter. Both young and old cells were from YEP-galactose medium. Mitochondrial membrane potential was indicated by TMRM staining. (E, F) Representative images (E) and quantification (F) of mitochondrial proteins undergo age-associated loss of expression, which can be suppressed by overexpressing TOM70. WT, wild type. Both young and old cells were from YEP-galactose medium. The significant increase of mitochondrial proteins in old TOM70 OE cells (F) is likely due to the continuous induction of mitochondrial proteins by TOM70 OE throughout the entire lifespan. Scale bar for all images: 5 μm. Mitochondrial proteins were visualized by endogenous C-terminal GFP tagging and expressed from their own promoters. Both young and old cells in each experiment went through the same culture medium, experimental procedures, and purification. Images are representative of at least two independent experiments. Bar graphs are the mean and s.e.m. that normalized to young cells for each genotype. Sample sizes of (B, D, F) are given in Supplementary file 4. Data were analyzed with unpaired two-tailed t test: ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Figure 5.

Figure 5—figure supplement 1. Overexpressing Tom70 from a different promoter can rescue age-associated mitochondrial defects.

Figure 5—figure supplement 1.

(A, B) Quantification of mitochondrial proteins abundance (A) and membrane potential (B) in different cells. The age-associated loss of mitochondrial membrane potential and protein expression can be suppressed by overexpressing TOM70 from pZ3EV promoter in the glucose medium. Both wild type (WT) and pZ3EV-TOM70 strains were cultured in YPD supplemented with 100nM β-estradiol before and after biotinylating mother cell wall and culturing for 2 days to age the mother cells. Bar graphs are the mean and s.e.m. that normalized to young cells for each genotype. Data were analyzed with unpaired two-tailed t test: ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant.Sample sizes are given in Supplementary file 4.