Macromitophagy is a longevity assurance process that in chronologically aging yeast limited in calorie supply sustains functional mitochondria and maintains cellular lipid homeostasis

Abstract

Macromitophagy controls mitochondrial quality and quantity. It involves the sequestration of dysfunctional or excessive mitochondria within double-membrane autophagosomes, which then fuse with the vacuole/lysosome to deliver these mitochondria for degradation. To investigate a physiological role of macromitophagy in yeast, we examined how theatg32Δ-dependent mutational block of this process influences the chronological lifespan of cells grown in a nutrient-rich medium containing low (0.2%) concentration of glucose. Under these longevity-extending conditions of caloric restriction (CR) yeast cells are not starving. We also assessed a role of macromitophagy in lifespan extension by lithocholic acid (LCA), a bile acid that prolongs yeast longevity under CR conditions. Our findings imply that macromitophagy is a longevity assurance process underlying the synergistic beneficial effects of CR and LCA on yeast lifespan. Our analysis of how the atg32Δ mutation influences mitochondrial morphology, composition and function revealed that macromitophagy is required to maintain a network of healthy mitochondria. Our comparative analysis of the membrane lipidomes of organelles purified from wild-type and atg32Δ cells revealed that macromitophagy is required for maintaining cellular lipid homeostasis. We concluded that macromitophagy defines yeast longevity by modulating vital cellular processes inside and outside of mitochondria.

Figure 1. Under CR conditions, the atg32Δ-dependent mutational block of macromitophagy substantially shor-tens yeast CLS and abolishes the longevity-extending effect of LCA

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose in the presence or absence of 50 μM LCA. Effect of theatg32Δ mutation and LCA on survival (A) and on the mean (B) and maximum (C) lifespans of chronologically aging yeast grown under CR conditions on 0.2% glucose. Data are presented as means ± SEM (n = 6-8; *p < 0.01). Abbreviations: Diauxic (D), logarithmic (L), post-diauxic (PD) or stationary (ST) growth phase.

Figure 2. Under CR conditions, the atg36Δ-dependent mutational block of macropexophagy does not alter yeast CLS and does not compromise the longevity-extending efficacy of LCA

WT and atg36Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose in the presence or absence of 50 μM LCA. Effect of theatg36Δ mutation and LCA on survival (A) and on the mean (B) and maximum (C) lifespans of chronologically aging yeast grown under CR conditions on 0.2% glucose. Data are presented as means ± SEM (n = 5-6; *p < 0.01; ns, not significant). Abbreviations: Diauxic (D), logarithmic (L), post-diauxic (PD) or stationary (ST) growth phase.

Figure 3. Under CR conditions,the atg32Δ-dependent mutational block of macromito-phagy alters the age-related dynamics of changes in mitochondrial size, number, shape, morphology and network appearance

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. (A) Transmission electron micrographs of WT and atg32Δ cells recovered on day 1, 2 or 4 of cell culturing. M, mitochondrion. Bar, 1 μm. (B) Percentage of mitochondria in WT and atg32Δ cells having the indicated relative area of mitochondrion section. The relative area of mitochondrion section was calculated as (area of mitochondrion section/area of cell section) × 100. Data are presented as means ± SEM (at least 100 cells of each strain were used for morphometric analysis at each time-point). (C) Numbers of mitochondria in WT and atg32Δ cells. The data of morphometric analysis are expressed as the number of mitochondria per μm³ of cell section ± SEM (at least 100 cells of each strain were used for morphometric analysis at each time-point). (D) Morphology of mitochondria in WT and atg32Δ cells recovered on day 1, 2 or 4 of cell culturing. Mitochondria were visualized by indirect immunofluorescence microscopy using monoclonal anti-porin primary antibodies and Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibodies. (E) The percentage of cells exhibiting fragmented mitochondria was calculated. At least 800 cells of each strain were used for quantitation at each time-point. Data are presented as mean ± SEM (n = 3).

Figure 4. Under CR conditions, the atg32Δ-dependent mutational block of macromitophagy alters the age-related chronology of changes in vital mitochondrial functions

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. Effect of theatg36Δ mutation on the rate of oxygen consumption by cells (A), electrochemical potential across the inner mitochondrial membrane IMM (ΔΨ) (B), level of cytochrome c in purified mitochondria (M) and the cytosolic fraction (C) (C), and cellular level of ATP (D). Data in A, B and D are presented as means ± SEM (n = 6-9).

Figure 5. Under CR conditions, the atg32Δ mutation reduces the levels of several large protein super-complexes in the IMM, likely due to their dissociation into individual proteins or small protein subcomplexes

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. Mitochondria were purified from WT and atg32Δ cells recovered on day 4 of culturing. (A) Digitonin-solubilized protein complexes from the inner membrane of these mitochondria were separated on a linear 4-13% acrylamide gradient gel for first-dimension blue native PAGE (1-D BN-PAGE). Arrows mark the positions of protein supercomplexes 2, 3, 4 and 5 whose levels were reduced in the IMM of atg32Δ cells. (B and C) Equal quantities (10 μg) of protein from these mitochondria were subjected to first-dimension SDS-PAGE (1-D SDS-PAGE) and analyzed by quantitative immunoblotting with antibodies to porin (loading control), Cyt1p (cytochrome c1, a component of the respiratory complex III), Cox2p (subunit II of cytochrome c oxidase, a component of the respiratory complex IV) or F1α/β (alpha and beta subunits of the mitochondrial F1ATPase, the respiratory complex V). Data in C are presented as means ± SEM (n = 3-4; ns, not significant).

Figure 6. Under CR conditions, the atg32Δ mutation eliminates the non-respiratory protein supercomplex 5 in the IMM and alters compositions of other protein supercomplexes recovered by 1-D BN-PAGE

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. Mitochondria were purified from WT and atg32Δ cells recovered on day 4 of culturing. Digitonin-solubilized protein supercomplexes from the inner membrane of mitochondria purified from WT (A) or atg32Δ (B) cells were separated by first-dimension blue native PAGE (1-D BN-PAGE) and then resolved by second-dimension tricine-SDS-PAGE (2-D SDS-PAGE). Following silver staining, the separated by 2-D SDS-PAGE proteins were identified by mass spectrometry. Arrows in A mark the non-respiratory protein supercomplex 5 lacking in the IMM of atg32Δ cells as well as individual proteins or respiratory protein complexes dissociated from other protein supercomplexes in the IMM of these mutant cells.

Figure 7. Under CR conditions, the atg32Δ mutation reduces capacity of the mitochondrial ETC, lowers the efficacy of coupling between ADP phosphorylation and electron transport, compromises the integrity of the IMM, and disproportionally decreases activities of all OXPHOS enzymes

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. Mitochondria were purified from WT and atg32Δ cells recovered on day 4 of culturing. The rates of state III (A and B), state IV (D and E) and UC (G and H) respiration were measured using NADH (A, D and G) or succinate (B, E and H) as a respiratory substrate. The ratios of state III rate/state IV rate (RCR; J and K), state III rate/UC rate (M and N) and ADP/O (P and Q) were determined using NADH (J, M and P) or succinate (K, N and Q) as a respiratory substrate. Enzymatic activities of the OXPHOS enzymes NADH:decylubiquinone oxidoreductase (NQR; C), succinate:decylubiquinone-2,6-dichlorophenolindo-phenol oxidoreductase (complex II) (F), ubiquinol:cytochrome c oxidoreductase (complex III) (I), cytochrome c oxidase (complex IV) (L) and F₁F₀-ATP synthase (complex V) (O) were assessed. For each of these OXPHOS enzymes, the ratio “activity in mitochondria of atg32Δ strain/activity in mitochondria of WT strain” was calculated (R).

Figure 8. Under CR conditions, the atg32Δ mutation increases the level of mitochondrially produced ROS, elevates the extent of oxidative damage to mitochondrial proteins and membrane lipids, and rises the frequencies of mutations in mtDNA

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. (A) The dynamics of age-dependent changes in the intracellular levels of ROS during chronological aging of yeast. ROS were visualized using dihydrorhodamine 123 (DHR). At least 1,000 cells were used for quantitation of DHR staining for each of 4 independent experiments. Data are presented as mean ± SEM. (B and C) Carbonylated proteins (B) and oxidatively damaged membrane lipids (C) in purified mitochondria were determined as described in Methods. Data are presented as mean ± SEM (n = 2-3). (D) The percentage of respiratory-deficient cells unable to grow in medium containing 3% glycerol because they carried large mtDNA deletions (rho⁻) or lacked mtDNA (rho°). Data are presented as mean ± SEM (n = 5). (E) The frequencies of rib2 and rib3 point mutations in mtDNA caused resistance to erythromycin. Data are presented as mean ± SEM (n = 4).

Figure 9. Under CR conditions, the atg32Δ mutation alters levels of several membrane lipid species in mitochondria, the ER and the PM

WT and atg32Δ strains were cultured in the nutrient-rich YP medium initially containing 0.2% glucose. Mitochondria, the ER and the PM were purified from WT and atg32Δ cells recovered on day 1, 2 or 4 of culturing. Following extraction of membrane lipids from purified mitochondria, ER and PM, various lipid species were identified and quantitated by mass spectrometry as described in Methods. Data are presented as means ± SEM (n = 3; *p < 0.01; ns, not significant).

Figure 10. A working model for a mechanism that in atg32Δ cells underlies the spatiotemporal dynamics of age-related changes in lipid synthesis in the ER and mitochondria as well as in lipid transport via mitochondria-ER (MAM) and PM-ER (PAM) junctions

During logarithmic (A), diauxic (B) and post-diauxic (C) phases of growth under CR conditions, a remodelling of lipid synthesis and transport in atg32Δ cells alters the membrane lipidomes of mitochondria, the ER and the PM. From the data of lipidomic analysis, we inferred an outline of lipid synthesis and transport processes that were activated (red arrows) or inhibited (blue arrows) by the atg32Δ-dependent mutational block of macromitophagy; the thickness of these arrows correlates with the rates of the processes taking place in atg32Δ cells. Arrows next to the names of lipid species denote those of them whose concentrations are elevated (red arrows) or reduced (blue arrows) in atg32Δ cells. See text for details.