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. 2015;14(11):1643-56.
doi: 10.1080/15384101.2015.1026493.

Lithocholic Bile Acid Accumulated in Yeast Mitochondria Orchestrates a Development of an Anti-Aging Cellular Pattern by Causing Age-Related Changes in Cellular Proteome

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Lithocholic Bile Acid Accumulated in Yeast Mitochondria Orchestrates a Development of an Anti-Aging Cellular Pattern by Causing Age-Related Changes in Cellular Proteome

Adam Beach et al. Cell Cycle. .
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Abstract

We have previously revealed that exogenously added lithocholic bile acid (LCA) extends the chronological lifespan of the yeast Saccharomyces cerevisiae, accumulates in mitochondria and alters mitochondrial membrane lipidome. Here, we use quantitative mass spectrometry to show that LCA alters the age-related dynamics of changes in levels of many mitochondrial proteins, as well as numerous proteins in cellular locations outside of mitochondria. These proteins belong to 2 regulons, each modulated by a different mitochondrial dysfunction; we call them a partial mitochondrial dysfunction regulon and an oxidative stress regulon. We found that proteins constituting these regulons (1) can be divided into several "clusters", each of which denotes a distinct type of partial mitochondrial dysfunction that elicits a different signaling pathway mediated by a discrete set of transcription factors; (2) exhibit 3 different patterns of the age-related dynamics of changes in their cellular levels; and (3) are encoded by genes whose expression is regulated by the transcription factors Rtg1p/Rtg2p/Rtg3p, Sfp1p, Aft1p, Yap1p, Msn2p/Msn4p, Skn7p and Hog1p, each of which is essential for longevity extension by LCA. Our findings suggest that LCA-driven changes in mitochondrial lipidome alter mitochondrial proteome and functionality, thereby enabling mitochondria to operate as signaling organelles that orchestrate an establishment of an anti-aging transcriptional program for many longevity-defining nuclear genes. Based on these findings, we propose a model for how such LCA-driven changes early and late in life of chronologically aging yeast cause a stepwise development of an anti-aging cellular pattern and its maintenance throughout lifespan.

Keywords: D, diauxic growth phase; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; ETC, electron transport chain; ISC, iron-sulfur clusters; LCA, lithocholic acid; MAM, mitochondria-associated membrane; OS, oxidative stress; PD, post-diauxic growth phase; PMD, partial mitochondrial dysfunction; ROS, reactive oxygen species; ST, stationary growth phase; TCA, tricarboxylic acid; WT, wild type; anti-aging compounds; cell metabolism; cellular aging; lithocholic bile acid; longevity; mitochondria; mitochondrial proteome; mitochondrial signaling; signal transduction; yeast.

Figures

Figure 1.
Figure 1.
LCA alters the age-related dynamics of changes in levels of numerous mitochondrial proteins implicated in many essential mitochondrial functions. Wild-type cells were cultured in the nutrient-rich YP medium initially containing 0.2% glucose, with 50 50 μM mu;M LCA or without it. Mitochondria were purified from cells recovered on days 2, 5 and 9 of culturing (D, PD and ST growth phases, respectively) as described in “Materials and Methods”. Mass spectrometry-based identification and quantitation of proteins recovered in purified mitochondria were performed as described in “Materials and Methods”. Relative levels of proteins in mitochondria of cells cultured in the presence of LCA (fold difference relative to those in the absence of LCA) are shown. Abbreviations: D, diauxic growth phase; ETC, the mitochondrial electron transport chain; mtDNA, mitochondrial DNA; PD, post-diauxic growth phase; ROS, reactive oxygen species; ST, stationary growth phase; TCA, the tricarboxylic acid cycle in mitochondria.
Figure 2.
Figure 2.
The mitochondrial proteins whose levels are altered in an age-related fashion in yeast cells cultured with LCA can be divided into 2 regulons called a partial mitochondrial dysfunction (PMD) regulon and an oxidative stress (OS) regulon. Each regulon is modulated by a different kind of mitochondrial dysfunction. Based on expression profiles of the genes encoding mitochondrial proteins composing the PMD and OS regulons and considering published data on regulation of such proteins in response to certain mitochondrial dysfunctions, each regulon can be divided into several “clusters”. Each cluster denotes a distinct type of partial mitochondrial dysfunction that elicits a different signaling pathway mediated by a distinct set of transcription factors. Because mitochondrial proteins constituting the PMD and OS regulons exhibit 3 different patterns of the age-related dynamics of changes in their cellular levels, each of these regulons is separated into regulons “type 1”, “type 2” and “type 3”. The names of proteins that belong to more than one PMD or OS regulon are italicized; the names of proteins that are members of both a PMD regulon and an OS regulon are underlined. Abbreviations: D, diauxic growth phase; PD, post-diauxic growth phase; ST, stationary growth phase.
Figure 3.
Figure 3.
LCA alters the age-related dynamics of changes in levels of many mitochondrial proteins and numerous proteins located outside of mitochondria; these mitochondrial and non-mitochondrial proteins have been implicated in various cellular processes. Wild-type cells were cultured in the nutrient-rich YP medium initially containing 0.2% glucose, with 50 50 μM mu;M LCA or without it. Cells were recovered on days 2, 5 and 9 of culturing (D, PD and ST growth phases, respectively). Mass spectrometry-based identification and quantitation of proteins recovered in total lysates of yeast cells were performed as described in “Materials and Methods”. Relative levels of proteins in cells cultured in the presence of LCA (fold difference relative to those in the absence of LCA) are shown. Abbreviations: C, cytosol; D, diauxic growth phase; EE, ergosteryl esters; ETC, the mitochondrial electron transport chain; M, mitochondria; mtDNA, mitochondrial DNA; PD, post-diauxic growth phase; ROS, reactive oxygen species; ST, stationary growth phase; TAG, triacylglycerols; TCA, the tricarboxylic acid cycle in mitochondria.
Figure 4.
Figure 4.
Each of the cellular proteins whose level is changed in yeast cultured with LCA belongs to the following 2 multi-clustered regulons, each modulated by a different kind of mitochondrial dysfunction: 1) the partial mitochondrial dysfunction (PMD) regulon, which consisted of the rho0 (Rtg2p governed) cluster, S1 cluster, general TCA cycle dysfunction cluster, kgd1Δ, kgd2Δ or lpd1Δ cluster, yme1Δmdl1Δ cluster, and afo1Δ (Sfp1p governed) cluster; and 2) the oxidative stress (OS) regulon, which included the Yap1p governed cluster, Msn2p/Msn4p governed cluster, Skn7p governed cluster and Hog1p governed cluster. Each cluster denotes a distinct type of partial mitochondrial dysfunction that elicits a different signaling pathway governed by a distinct set of transcription factors. Because cellular proteins that belong to the PMD and OS regulons display 3 different patterns of age-related changes in their levels, each of these regulons is separated into regulons “type 1”, “type 2” and “type 3”. The names of cellular proteins that belong to more than one PMD or OS regulon are italicized; the names of cellular proteins that are members of both a PMD regulon and an OS regulon are underlined. Abbreviations: D, diauxic growth phase; PD, post-diauxic growth phase; ST, stationary growth phase.
Figure 5.
Figure 5.
Gene-deletion mutations eliminating the Rtg1p, Rtg2p, Rtg3p, Sfp1p, Aft1p, Yap1p, Msn2p/Msn4p, Skn7p or Hog1p transcription factor significantly alter the extent to which LCA extends yeast longevity. Wild-type and mutant cells lacking one (or 2, as in case of msn2/4Δ mutant cells) of the above transcription factors were cultured in the nutrient-rich YP medium initially containing 0.2% glucose, with 50 50 μM mu;M LCA or without it. The chronological lifespans were measured as described in “Materials and Methods”. Data are presented as means ± SEM (n = 4–6; *p < 0.01).
Figure 6.
Figure 6.
A model for how LCA-driven changes in mitochondrial lipidome alter mitochondrial proteome and functionality, thereby enabling mitochondria to function as signaling organelles modulating transcription of many longevity-defining nuclear genes. See text for details. Abbreviations: Ac-CoA, acetyl-CoA; ETC, the mitochondrial electron transport chain; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; ST, stationary growth phase; TCA, the tricarboxylic acid cycle in mitochondria.
Figure 7.
Figure 7.
For figure legend, see page 1654. Figure 7 (See previous page). A model for how LCA-driven changes in mitochondrial proteome and functionality early and late in life of chronologically aging yeast orchestrate a stepwise development of an anti-aging cellular pattern and its maintenance throughout lifespan. From the data of proteomic analysis (Figs. 14) and based on the data of biochemical and cell biological analyses, we inferred an outline of metabolic pathways and processes that were activated (red arrows) or inhibited (green arrows) in cells cultured with exogenous LCA. Arrows next to the names of metabolites, proteins or processes denote those of them whose concentrations or efficiencies were elevated (red arrows) or reduced (green arrows) in cells cultured with exogenous LCA. Abbreviations: Ac-CoA, acetyl-CoA; D, diauxic growth phase; EE, ethyl esters; ER, endoplasmic reticulum; EtOH, ethanol; ETC, the mitochondrial electron transport chain; FFA, free fatty acids; IMM, inner mitochondrial membrane; LD, lipid droplets; mtDNA, mitochondrial DNA; OMM, outer mitochondrial membrane; PD, post-diauxic growth phase; ST, stationary growth phase; TAG, triacylglycerols; TCA, the tricarboxylic acid cycle in mitochondria. See text for details.

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