Rewiring yeast acetate metabolism through MPC1 loss of function leads to mitochondrial damage and decreases chronological lifespan

Abstract

During growth on fermentable substrates, such as glucose, pyruvate, which is the end-product of glycolysis, can be used to generate acetyl-CoA in the cytosol via acetaldehyde and acetate, or in mitochondria by direct oxidative decarboxylation. In the latter case, the mitochondrial pyruvate carrier (MPC) is responsible for pyruvate transport into mitochondrial matrix space. During chronological aging, yeast cells which lack the major structural subunit Mpc1 display a reduced lifespan accompanied by an age-dependent loss of autophagy. Here, we show that the impairment of pyruvate import into mitochondria linked to Mpc1 loss is compensated by a flux redirection of TCA cycle intermediates through the malic enzyme-dependent alternative route. In such a way, the TCA cycle operates in a “branched” fashion to generate pyruvate and is depleted of intermediates. Mutant cells cope with this depletion by increasing the activity of glyoxylate cycle and of the pathway which provides the nucleocytosolic acetyl-CoA. Moreover, cellular respiration decreases and ROS accumulate in the mitochondria which, in turn, undergo severe damage. These acquired traits in concert with the reduced autophagy restrict cell survival of the mpc1∆ mutant during chronological aging. Conversely, the activation of the carnitine shuttle by supplying acetyl-CoA to the mitochondria is sufficient to abrogate the short-lived phenotype of the mutant.

The role of autophagy in the regulation of yeast life span

“…This is further supported by a 2014 study by Orlandi et al that demonstrated that impairment of the yeast mitochondrial pyruvate carrier (Mpc1) reduced autophagy and shortened CLS. 78 In mpc1 mutant cells, decreased pyruvate import into mitochondria resulted in depletion of tricarboxylic acid (TCA) cycle intermediates, with a concomitant reduction in the generation of nucleocytosolic AcCoA. Reduced AcCoA levels were associated with decreased autophagic flux, possibly through a decrease in SAGA-mediated histone acetylation, as described above.…”

Targeting the Mitochondrial Pyruvate Carrier for Neuroprotection

“…Genetic deletion of all three yeast paralogues is tolerated in rich medium, but even the mpc1 Δ and mpc2 Δ mutants exhibit measurable growth impairment in non-fermentable carbon sources or with amino acid depletion [35]. Moreover, cellular changes and adaptations in mpc1 Δ mutants tend to increase oxidative damage of the mitochondria and restrict cell survival during yeast chronological aging [40]. Genetic ablation of either MPC1 [41] or MPC2 [42] in mouse resulted in embryonic lethality.…”

Altered Expression of Mitochondrial NAD+ Carriers Influences Yeast Chronological Lifespan by Modulating Cytosolic and Mitochondrial Metabolism

“…Cells were grown in batches at 30°C in minimal medium (Difco Yeast Nitrogen Base without amino acids, 6.7 g/L) with 2% w/v glucose. Auxotrophies were compensated for with supplements added in excess (Orlandi et al, 2014). Cell number and cellular volumes were determined using a Coulter Counter-Particle Count and Size Analyser (Vanoni et al, 1983).…”

“…At designated time-points, aliquots of the yeast cultures were centrifuged, and both pellets (washed twice) and supernatants were collected and frozen at −80°C until used. Rapid sampling for intracellular metabolite measurements was performed as previously described (Orlandi et al, 2014). The concentrations of glucose, ethanol, citrate, succinate, and malate were determined using enzymatic assays (K-HKGLU, K-ETOH, K-SUCC, K-CITR, and K-LMALR kits from Megazyme).…”

“…Immediately after preparation of cell-free extracts (Orlandi et al, 2014), the activities of cytosolic and mitochondrial aldehyde dehydrogenase (Ald) were assayed according to Aranda and del Olmo (2003), of phosphoenolpyruvate carboxykinase (Pck1) and isocitrate lyase (Icl1) as described in de Jong-Gubbels et al (1995). Total protein concentration was estimated using the BCA TM Protein Assay Kit (Pierce).…”