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

Lack of Mpc1 is accompanied by an increase of Ald enzymatic activities

Since an impairment in the import of pyruvate into mitochondria linked to MPC1 deletion significantly restricted CLS (Fig. 2A) 8, we decided to analyze in more detail the metabolic changes underlying this short-lived phenotype. Initially, in the context of a standard CLS experiment 3, we measured the levels of some metabolites such as pyruvate, ethanol and acetate. These last two compounds are produced during glucose fermentation following decarboxylation of cytosolic pyruvate to acetaldehyde by pyruvate decarboxylase (Pdc) (Fig. 1). Only upon glucose depletion, does the diauxic shift occurs and yeast cells switch to a respiration-based metabolism of the fermentation C2 by-products. Finally, when these carbon/energy sources are fully exhausted, cells enter a quiescent stationary phase. At the diauxic shift, in the mpc1∆ culture the amount of ethanol and acetate was similar to that in the wild type (wt) culture (Fig. S1, 2B and C). Differently, during the post-diauxic phase, in the mutant the consumption of ethanol, which is re-introduced into the metabolism via its oxidation to acetate (Fig. 1), was not affected significantly compared to that in the wt (specific consumption rate, qEtOH, of 1.43 ± 0.04 mmol•g•DW^-1•h^-1 for the mutant and 1.12 ± 0.06 mmol•g•DW^-1•h^-1 for the wt) (Fig. S1 and 2B), while the acetate continued to accumulate in the medium (Fig. 2C). Such a prolonged secretion of acetate throughout the ethanol consumption phase suggests that in the mpc1∆ mutant there is an imbalance between acetate production rate from acetaldehyde and its conversion rate into acetyl-CoA. In fact, the acetate transport relies on an active transport for the dissociated form of the acid (subjected to glucose repression) accompanied by passive/facilitated diffusion of the undissociated acid 24. During the post-diauxic phase the pH of the medium is far below the pKa of acetic acid (4.75) 25 and according to the Henderson-Hasselbalch equation, acetic acid is substantially undissociated: 98.6 % at pH 2.9 (the value we measured at Day 3 after the diauxic shift). Consequently, in a condition where transmembrane diffusion strongly prevails over the active transport, the acetate export/import will take place according to the gradient between the intracellular and extracellular concentrations of acetate.

Concerning intracellular pyruvate, its concentration was higher in the mpc1∆ mutant compared to that in the wt not only in exponential phase, as already observed by 22, but also at/after the diauxic shift (Fig. 2D). This was also associated with an increase in the extracellular pyruvate (Fig. 2E) which reflects an overflow of pyruvate within the cytosol. Similar results were obtained when the experiments were also performed by growing the histidine-prototroph mpc1∆ mutant (mcp1∆::HIS3) in a histidine-supplemented medium as previously carried out for the wt (Fig. S2) indicating that the different composition of amino acids in the medium does not affect the results.

Afterwards, we measured the enzymatic activities of alcohol dehydrogenases (Adhs) catalysing the interconversion of acetaldehyde and ethanol 26 and of acetaldehyde dehydrogenases (Alds) which produce acetate by oxidizing the acetaldehyde generated from pyruvate during fermentation and that obtained during ethanol oxidation (Fig. 1).

No significant difference was found between the wt and the mpc1∆ strain in the Adh activity levels in exponential phase (Fig. 3A), where the Adh1 isoenzyme is chiefly responsible for ethanol formation from acetaldehyde, consistent with the similar amounts of ethanol detected in both cultures (Fig. 2B). Similarly, at/after the diauxic-shift where the cytosolic Adh2 is the major ethanol oxidizer, Adh activities displayed no significant difference (Fig. 3A). On the contrary, during all the growth phases analyzed, Ald activity levels were higher in the mutant compared with the wt (Fig. 3B). In particular, a great increase was observed for Ald6 which is the major cytosolic isoform and is not glucose-repressed 27 compared with that of the mitochondrial counterparts Ald5 and Ald4 (Fig. 3C and D) indicating that the mutant exhibits an increased ability to generate acetate, especially the cytosolic one, which can be used as substrate to produce acetyl-CoA. Accordingly, in the mutant, the nucleocytosolic Acs2-mediated pathway is upregulated during chronological aging 8. Moreover, an increased cytosolic acetate pool can also account for the extracellular acetate detected in the mpc1∆ culture (Fig. 2C) whose prolonged accumulation, however, indicates that the flux towards its formation exceeds its utilization. This takes place despite the upregulation of Acs2 enzymatic activity 8 suggesting that the enzyme and/or the flux downstream is/are working at maximum capacity in line with data which show that increase in Acs activity does not result in enhanced acetate utilization 28,29.

Lack of Mpc1 is accompanied by an increase of malic enzyme activity and a decrease in respiration

Starting from these results, we focused on the mitochondrially localized TCA cycle which can be fed with acetyl-CoA generated either from acetate or following oxidation of mitochondrial pyruvate. We measured the levels of citrate, succinate and malate which are intermediates of this cycle but also metabolic connections with the glyoxylate shunt. This is an anaplerotic device of the TCA cycle which allows the formation of C4 units from C2 units (acetate) by bypassing oxidative decarboxylation (Fig. 4A) 30. At/after the diauxic shift, a clear global decrease was observed for all three intermediates in the mpc1∆ cells compared with the wt counterparts (Fig. 4B-D). This decrease was particularly marked for malate which can be used to generate pyruvate in the mitochondria for biosynthetic purposes. This reaction of oxidative decarboxylation is catalyzed by the mitochondrial malic enzyme encoded by MAE1 31. As shown in Fig. 4E, in the mpc1∆ mutant during the post-diauxic phase, the malic enzyme activity was doubled in comparison with the wt, suggesting that the impairment of pyruvate import into mitochondria linked to Mpc1 loss is compensated by a flux redirection through the Mae1-dependent alternative route. This can explain the severe growth defect observed by 22 when the mpc1∆ allele was combined with MAE1 deletion.

Moreover, when cells switched to a respiration-based metabolism by using ethanol and acetate, the glyoxylate shunt becomes operative and begins replenishing the TCA cycle intermediates. In addition, during growth on C2 compounds, this shunt is the exclusive source of oxaloacetate which is the substrate of phosphoenolpyruvate carboxykinase (Pck1), the key enzyme of gluconeogenesis 32. Measurements of the enzymatic activities of isocitrate lyase (Icl1), which is one of the unique enzymes of the glyoxylate shunt, and Pck1 indicated that these activities were higher in mpc1∆ cells compared with wt ones (Fig. 5A and B). Concomitantly, in mpc1∆ cells cellular respiration decreased (Fig. 5C). Icl1 is localized in the cytosol and from isocitrate it generates succinate and the name-giving metabolite glyoxylate which condenses with acetyl-CoA yielding malate. The last one can return to the mitochondria (Fig. 4A). Similarly, the major fate of cytosolic succinate is assumed to be its transfer into mitochondria 30. Moreover, its transport by the Sfc1 carrier provides cytosolic fumarate for conversion to malate which can be used for gluconeogenesis 33. Thus, taken together, these data indicate that in the mpc1∆ mutant, an increase in the glyoxylate shunt might represent an increase in metabolite feeding from the cytosol to support a mitochondrial impaired TCA cycle. In this context, the cytosol of the mutant can provide the metabolic environment required to fulfill the increased requirement of substrates for the glyoxylate shunt. In fact, the end-product of the Acs2 synthetase, which is increased in the mutant 8, is the nucleocytosolic acetyl-CoA. In the cytosol, this metabolite, following condensation with oxaloacetate, produces citrate which is then isomerized to isocitrate (the substrate of Icl1). In addition, the cytosol of the mutant might also be a suitable environment which can “promote” Pck1 enzymatic activity. In fact, Pck1 is acetylated by Esa1 and this acetylation is required for its enzymatic activity: an increase of Pck1 enzymatic activity is associated with an increase of the acetylated form of the enzyme 34,35. Accumulating evidence indicates that the availability of acetyl-CoA, the donor substrate for acetylation, can be a metabolic input for the acetylation itself 36,37,38, so it is reasonable to hypothesize that changes of acetyl-CoA levels may also influence Esa1 activity.

Carnitine restores chronological longevity of the mpc1∆ mutant

After the diauxic shift, a metabolic change from fermentation to respiration takes place implying that energy metabolism relies on mitochondrial functionality. Since in the mpc1∆ cells we observed a decrease in respiration, we decided to analyze mitochondrial membrane potential and morphology by using the fluorescent dye, 3,3”-dihexyloxacarbocyanine iodide (DiOC₆) 39. In fact, mitochondrial morphology reflects the functional status of mitochondria and is regulated by the orchestrated balance of two opposing events: fission and fusion of mitochondria 40. As shown in Fig. 6A, a typical tubular network was observed for wt cells whereas for the mutant fluorescent punctiform structures appeared at Day 2 after the diauxic shift. These structures are indicative of mitochondrial fragmentation and are linked to an elevated activity of the mitochondrial fission machinery 41. In addition to an altered morphology, the mitochondria of the mutant displayed a time-dependent reduction in membrane potential and, at Day 4, did not accumulate DiOC₆ (Fig.6A). Mitochondrial dysfunctions are intrinsically related to reactive oxygen species (ROS) of which superoxide anion is one of the most potentially harmful. This radical derives mainly from leakage of electrons from the respiratory chain and, among others, can target mitochondria with detrimental effects 42,43. Chronologically aging mpc1∆ cells had a higher ROS content, measured as the superoxide-driven conversion of non-fluorescent dihydroethidium (DHE) into fluorescent ethidium (Eth), compared with that of the wt cells (Fig. 6B). Notably, culturing mpc1∆ cells in a carnitine-supplemented medium was sufficient to avoid this phenomenon (Fig. 6C). Moreover, oxygen consumption measurements indicated that in these cells cellular respiration increased (Fig. 6D). In S.cerevisiae, carnitine is involved in a process referred to as the carnitine shuttle which allows the transport of acetyl-CoA to the mitochondria. This transport system which is non-functional unless carnitine is supplied with the medium 44,45, involves the transfer of the acetyl moiety of acetyl-CoA to carnitine and the subsequent transport of the acetylcarnitine to the mitochondria. Here, a mitochondrial carnitine acetyltransferase catalyses the reverse reaction generating carnitine and acetyl-CoA which enters the TCA cycle 44,46. As shown in Fig. 7A-C, the supplemental carnitine did not significantly affect the levels of citrate, succinate and malate in the wt whilst this was not the case for the mpc1∆ mutant where the levels of all three intermediates increased and were restored to wt-like ones. No effect was observed on the malic enzyme activity which at/after the post diauxic shift in the mpc1∆ cells was still the double of that of the wt (Fig. 7D) suggesting that the presence of carnitine does not abolish the Mae1-dependent flux towards mitochondrial pyruvate generation. Concomitantly, in the mpc1∆ cells the enzymatic activities of Icl1 and Pck1 were reduced to the physiological levels measured in the wt (Fig. 7D and E). Thus, all this suggests that in the mpc1∆ mutant the activation of the carnitine shuttle can properly feed the TCA cycle by supplying acetyl-CoA to the mitochondria. Hence, the compensative metabolite feeding from the cytosol provided by the glyoxylate shunt seems to be no longer required. Moreover, following carnitine supplementation, during the post-diauxic phase no effect was observed on the ethanol consumption in both the wt and the mutant strains (Fig. 8A). Similarly, the acetate utilization in the wt was not affected, while in the mpc1∆ mutant its utilization was promoted (Fig. 8B). This indicates that in the latter the activation of the carnitine shuttle and the consequent acetyl-CoA transport to the mitochondria can result in an enhancement in the flux downstream from the acetate activation allowing acetate utilization. Finally, in the mpc1∆ mutant these metabolic changes matched the almost completely restored chronological longevity (Fig. 8C).

In conclusion, these data collectively indicate that the lack of the Mpc1 transporter brings about a chain of metabolic events which, in order to counteract the decrease of the pyruvate supply in the mitochondria, by influencing the global acetyl-CoA metabolism ultimately restrict cell survival during chronological aging. In particular, after the diauxic shift when cells utilize the earlier produced ethanol/acetate and increase their respiration demand, one of the metabolic traits of the mpc1∆ mutant is a TCA cycle operating in a “branched” fashion with a propensity to shunt intermediates towards pyruvate generation via the malic enzyme. This kind of not-complete cyclic functioning of the TCA cycle by depleting it of intermediates influences not only the respiration, which is reduced in the mutant, but also might reduce mitochondrial acetyl-CoA pool. In fact, a TCA cycle characterized by low levels of intermediates (Fig. 4B-D) generates less succinyl-CoA. This is the substrate for the CoA-transferase's reaction from succinyl-CoA to acetate, catalyzed by Ach1 in cells released from glucose repression 20. In the mitochondria, this reaction allows the production of acetyl-CoA 8. Moreover, the CoA-transferase's reaction is using acetate as acceptor, which implies that Ach1 is also important for acetate detoxification and mitochondrial functionality during chronological aging 47. Consequently, in a condition where acetate-generating activities of Ald4/Ald5 are increased, as it is the case in the mutant, a reduction in CoA-transferase's enzymatic activity could play a causative role in promoting/enhancing the mitochondrial damage observed in the mutant.

Furthermore, since during the utilization of ethanol and acetate, the sole possible route for the net synthesis of C4 dicarboxylic acids for replenishing the TCA cycle of intermediates is the glyoxylate shunt, it follows that in the mpc1∆ cells this anaplerotic shunt is enhanced in order to keep the “branched” TCA cycle functioning. In turn, it follows that the pathway providing the cytosolic acetyl-CoA must be increased to support an enhanced glyoxylate demand. In line with this, the cytosolic Ald6 enzymatic activity is increased (Fig. 3C) and hyperactivation of the Acs2 activity has been detected 8. Interestingly, this synthetase is responsible not only for supplying acetyl-CoA for carbon metabolism, but also for protein acetylation, particularly of histones 19. In addition, it has been shown that during chronological aging, upregulation of Acs2 activity culminates in histone H3 hyperacetylation associated with transcriptional downregulation of several autophagy-essential ATG genes 48. In this context, mpc1∆ cells display an age-dependent loss of autophagy 8; this feature, given the reciprocal cross-talk between autophagy and mitochondria, can negatively affect the removal of the damaged mitochondria of the mutant and consequently contribute to its inability to maintain proper cellular homeostasis during the aging process. Notably, Ald6, which is responsible of generating cytosolic acetate, is degraded preferentially by autophagy 49 and the persistence of its enzymatic activity seems to be disadvantageous for the survival during nitrogen starvation 50. Thus, mpc1∆ cells make up for their impairment in mitochondrial pyruvate with a metabolic rewiring in which the pro-aging outcome prevails.

Yeast strains and growth conditions

The mcp1∆ mutant (mcp1∆::HIS3) was generated by PCR-based methods in a BY4741 background (MATa his3∆-1 leu2∆-0 met15∆-0 ura3∆-0) and the accuracy of gene replacement was verified by PCR with flanking and internal primers. At least two different clones were tested for any experiment. Yeast cells were grown in batches at 30°C in minimal medium (Difco Yeast Nitrogen Base without amino acids, 6.7 g/L) with 2% glucose and the required supplements added in excess to a final concentration of 200 mg/L, except for leucine at 500 mg/L to avoid auxotrophy starvation 51,52. L-carnitine (Sigma) was supplemented to a concentration of 10 mg/L. Strains were inoculated at the same cellular density (culture volume no more than 20% of the flask volume) and growth was monitored by determining cell number using a Coulter Counter-Particle Count and Size Analyser, as described 53. Duplication times (Td) were obtained by linear regression of the cell number increase over time on a semi-logarithmic plot.

CLS determination

Survival experiments in expired medium were performed on cells grown in minimal medium/2% glucose and the required supplements as described above. During growth, cell number and extracellular glucose, ethanol and acetic acid were measured in order to define the growth profile (exponential phase, diauxic shift, post-diauxic phase and stationary phase) of the culture (Fig. S1). Cell survival was monitored by harvesting aliquots of cells starting with 72 h (Day 3, first age-point) after the diauxic shift (Day 0). CLS was measured according to 51 by counting colony-forming units (CFUs) every 2-3 days. The number of CFUs on Day 3 was considered the initial survival (100%).

Metabolite measurements and enzymatic assays

At designated time points, aliquots of the yeast cultures were centrifuged and both pellets (washed twice) and supernatants were frozen at −80°C until used. Rapid sampling for intracellular metabolite measurements was performed according to the leakage-free cold methanol quenching method developed by 54 in which pure methanol at ≤ -40°C and a ratio of cell culture to quenching solvent of 1:5 (final methanol concentration ≥ 83%) were used. Metabolites from the cell pellets were extracted in 5 ml of a solution of 75% (v/v) boiling absolute ethanol containing 0.25 M Hepes, pH 7.5, as described in 55. The concentrations of glucose, ethanol, acetate, pyruvate, citrate, succinate and malate were determined using enzymatic assays (K-HKGLU, K-ETOH, K-ACET, K-PYRUV, K-SUCC, K-CITR and K-LMALR kits from Megazyme). Ethanol specific consumption rate (qEtOH), expressed in mmol•g•DW ^-1•h^-1, was calculated from measured cell dry weights (DWs) and extracellular ethanol concentrations. DW was measured as described 56.

All the enzymatic activities were assayed immediately after preparation of cell-free extracts. Cells were resuspended in 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM MgCl₂ and 1 mM dithiothreitol and broken with acid-washed glass beads by shaking on a vortex for several cycles interspersed with cooling on ice. The activities of cytosolic and mitochondrial aldehyde dehydrogenase (Ald) were measured as described by 57, of alcohol dehydrogenase (Adh) according to 58, of phosphoenolpyruvate carboxykinase (Pck1) and isocitrate lyase (Icl1) as in 34. Malic enzyme activities were determined according to 31 with either 0.4 mM NAD⁺ or NADP⁺ as the redox cofactor. The enzymatic activity was measured in the decarboxylation direction to avoid interference with pyruvate decarboxylase and Adh. Total protein concentration was estimated using the BCA^TM Protein Assay Kit (Pierce).

Oxygen consumption and fluorescence microscopy

The basal oxygen consumption of intact cells was measured at 30°C using a ‘Clark-type’ oxygen electrode in a thermostatically controlled chamber (Oxygraph System, Hansatech Instruments, Norfolk, UK) as previously reported 25. Data were recorded at sampling intervals of 1 s (Oxygraph Plus software, Hansatech Instruments, Norfolk, UK). All assays were conducted in biological triplicate.

ROS were detected with dihydroethidium (DHE, Sigma) according to 59. The mitochondrial membrane potential was assessed by staining with DiOC₆ (Molecular Probes, Invitrogen), according to 39; cells were also counterstained with propidium iodide to discriminate between live and dead cells. A Nikon Eclipse E600 fluorescence microscope equipped with a Leica DC 350F ccd camera was used. Digital images were acquired using FW4000 software (Leica).

Statistical analysis of data

All values are presented as the mean of three independent experiments with the corresponding Standard Deviation (SD). Three technical replicates were analyzed in each independent experiments. Statistical significance was assessed by one-way ANOVA test. P value of ≤ 0.05 was considered statistically significant.

SUPPLEMENTAL MATERIAL

All supplemental data for this article are also available online at http://microbialcell.com/researcharticles/rewiring-yeast-acetate-metabolism-through-mpc1-loss-of-function-leads-to-mitochondrial-damage-and-decreases-chronological-lifespan/.