Specific changes in mitochondrial lipidome alter mitochondrial proteome and increase the geroprotective efficiency of lithocholic acid in chronologically aging yeast

We have previously found that exogenously added lithocholic acid delays yeast chronological aging. We demonstrated that lithocholic acid enters the yeast cell, is sorted to mitochondria, resides in both mitochondrial membranes, changes the relative concentrations of different membrane phospholipids, triggers changes in the concentrations of many mitochondrial proteins, and alters some key aspects of mitochondrial functionality. We hypothesized that the lithocholic acid-driven changes in mitochondrial lipidome may have a causal role in the remodeling of mitochondrial proteome, which may in turn alter the functional state of mitochondria to create a mitochondrial pattern that delays yeast chronological aging. Here, we test this hypothesis by investigating how the ups1?, ups2? and psd1? mutations that eliminate enzymes involved in mitochondrial phospholipid metabolism influence the mitochondrial lipidome. We also assessed how these mutations affect the mitochondrial proteome, influence mitochondrial functionality and impinge on the efficiency of aging delay by lithocholic acid. Our findings provide evidence that 1) lithocholic acid initially creates a distinct pro-longevity pattern of mitochondrial lipidome by proportionally decreasing phosphatidylethanolamine and cardiolipin concentrations to maintain equimolar concentrations of these phospholipids, and by increasing phosphatidic acid concentration; 2) this pattern of mitochondrial lipidome allows to establish a specific, aging-delaying pattern of mitochondrial proteome; and 3) this pattern of mitochondrial proteome plays an essential role in creating a distinctive, geroprotective pattern of mitochondrial functionality.


Supplemental
. Scatter plots comparing the relative concentrations of proteins in mitochondria purified from WT or ups1Δ (short-lived) cells cultured with or without LCA. Mitochondria were purified from WT or ups1Δ cells recovered on day 2, 4 or 7 of cell culturing. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. Scatter plots comparing the relative abundance of mitochondrial proteins between specified datasets were plotted on a log-log scale spanning six orders of magnitude. Data on the relative abundance of mitochondrial proteins are presented as means of 2 independent experiments.
Supplemental Figure S6. The ups1Δ mutation alters the concentrations of many mitochondrial proteins in yeast cultured with or without LCA. (A -F) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in WT, by the ups1Δ mutation in the absence of LCA, and by LCA in ups1Δ cells; WT cells were recovered on day 2, 4 or 7 of culturing. (G and J) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in WT cells recovered on day 2, 4 or 7 of culturing. (H and K) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by the ups1Δ mutation in the absence of LCA; ups1Δ cells were recovered on day 2, 4 or 7 of culturing. (I and L) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in ups1Δ cells recovered on day 2, 4 or 7 of culturing. Figure S7. Scatter plots comparing the relative concentrations of proteins in mitochondria purified from WT or ups2Δ (long-lived) cells cultured with or without LCA. Mitochondria were purified from WT or ups2Δ cells recovered on day 2, 4 or 7 of cell culturing. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. Scatter plots comparing the relative abundance of mitochondrial proteins between specified datasets were plotted on a log-log scale spanning six orders of magnitude. Data on the relative abundance of mitochondrial proteins are presented as means of 2 independent experiments.

Supplemental
Supplemental Figure S8. The ups2Δ mutation alters the concentrations of many mitochondrial proteins in yeast cultured with or without LCA. (A -F) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in WT, by the ups2Δ mutation in the absence of LCA, and by LCA in ups2Δ cells; WT cells were recovered on day 2, 4 or 7 of culturing. (G and J) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in WT cells recovered on day 2, 4 or 7 of culturing. (H and K) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by the ups2Δ mutation in the absence of LCA; ups2Δ cells were recovered on day 2, 4 or 7 of culturing. (I and L) Venn diagrams showing a comparison of the datasets of relative concentrations of mitochondrial proteins that are statistically significantly downregulated or upregulated by LCA in ups2Δ cells recovered on day 2, 4 or 7 of culturing.
Supplemental Table S1. The relative concentrations of proteins in mitochondria purified from WT cells cultured with or without LCA. Mitochondria were purified from WT cells recovered on day 2 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S5. The relative concentrations of proteins in mitochondria purified from WT or ups1Δ cells cultured without LCA. Mitochondria were purified from WT or ups1Δ cells recovered on day 4 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S6. The relative concentrations of proteins in mitochondria purified from WT or ups1Δ cells cultured without LCA. Mitochondria were purified from WT or ups1Δ cells recovered on day 7 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S7. The relative concentrations of proteins in mitochondria purified from ups1Δ cells cultured with or without LCA. Mitochondria were purified from ups1Δ cells recovered on day 2 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S8. The relative concentrations of proteins in mitochondria purified from ups1Δ cells cultured with or without LCA. Mitochondria were purified from ups1Δ cells recovered on day 4 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S9. The relative concentrations of proteins in mitochondria purified from ups1Δ cells cultured with or without LCA. Mitochondria were purified from ups1Δ cells recovered on day 7 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S10. The relative concentrations of proteins in mitochondria purified from WT or ups2Δ cells cultured without LCA. Mitochondria were purified from WT or ups2Δ cells recovered on day 2 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S11. The relative concentrations of proteins in mitochondria purified from WT or ups2Δ cells cultured without LCA. Mitochondria were purified from WT or ups2Δ cells recovered on day 4 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S12. The relative concentrations of proteins in mitochondria purified from WT or ups2Δ cells cultured without LCA. Mitochondria were purified from WT or ups2Δ cells recovered on day 7 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S13. The relative concentrations of proteins in mitochondria purified from ups2Δ cells cultured with or without LCA. Mitochondria were purified from ups2Δ cells recovered on day 2 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S14. The relative concentrations of proteins in mitochondria purified from ups2Δ cells cultured with or without LCA. Mitochondria were purified from ups2Δ cells recovered on day 4 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S15. The relative concentrations of proteins in mitochondria purified from ups2Δ cells cultured with or without LCA. Mitochondria were purified from ups2Δ cells recovered on day 7 of cell culturing with or without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between specified datasets. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S16. Proteins that are downregulated by the ups2Δ mutation (but not by the ups1Δ mutation) in cells cultured without LCA are involved in various biological processes. Mitochondria were purified from cells recovered on day 2, 4 or 7 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between ups2Δ and WT. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S17. Proteins that are upregulated by the ups2Δ mutation (but not by the ups1Δ mutation) in cells cultured without LCA are involved in various biological processes. Mitochondria were purified from cells recovered on day 2, 4 or 7 of cell culturing without LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between ups2Δ and WT. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S18. Proteins that are downregulated by LCA only in ups2Δ cells (but not in WT or ups1Δ cells) are involved in several biological processes. Mitochondria were purified from cells recovered on day 2, 4 or 7 of cell culturing with LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between ups2Δ cells cultured with or without LCA. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments. Supplemental Table S19. Proteins that are upregulated by LCA only in ups2Δ cells (but not in WT or ups1Δ cells) are involved in various biological processes. Mitochondria were purified from cells recovered on day 2, 4 or 7 of cell culturing with LCA. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in Materials and methods. The exponentially modified protein abundance index (emPAI) provided by the ″Proteome Discoverer″ software was used to calculate the relative abundance of mitochondrial proteins between ups2Δ cells cultured with or without LCA. Data on the relative concentrations of mitochondrial proteins are presented as means of 2 independent experiments.

Day Protein
Ratio ups2 + LCA/ ups2 Biological process 2 Aac1 1.51 Metabolite transport (mitochondrial inner membrane ADP/ATP translocator; exchanges cytosolic ADP for mitochondrially synthesized ATP) Aim17 1.52 Unknown Pet9 1.52 Metabolite transport (major ADP/ATP carrier of the mitochondrial inner membrane; exchanges cytosolic ADP for mitochondrially synthesized ATP; also imports heme and ATP) Hem15 1.54 Metabolite synthesis (ferrochelatase; a mitochondrial inner membrane protein, catalyzes insertion of ferrous iron into protoporphyrin IX, the eighth and final step in the heme biosynthetic pathway) Sdh4 1.55 TCA cycle and ETC/respiration (membrane anchor subunit of succinate dehydrogenase; involved in coupling the oxidation of succinate to the transfer of electrons to ubiquinone as part of the TCA cycle and th Cbp3 1.55 Proteostasis and protein synthesis (mitochondrial protein required for assembly of cytochrome bc1 complex; forms a complex with Cbp6p that binds to mt ribosomes near the polypeptide tunnel exit and promo Cbp3p-Cbp6p complex also interacts with newly synthesized cytochrome b (Cobp) and Cbp4p to promote assembly of Cobp into the cytochrome bc1 complex) Tcb1 1.