The accumulation of acetic acid in stationary phase budding yeast cultures is considered to be the primary mechanism of chronological aging in yeast and recent results suggest that the mechanism of acetic acid toxicity in yeast could be related to the induction of growth signaling pathways and oxidative stress (Burhans and Weinberger, 2009
; Burtner et al., 2009
). Recent publications showed that buffering medium could inhibit the age-dependent accumulation of reactive oxygen species preferentially superoxide anions that is produced by dysfunctional mitochondria (Burhans and Weinberger, 2009
; Pan et al., 2011
). The accumulation of acetic acid in stationary phase induces oxidative stress, a factor previously implicated in chronological aging of yeast and aging in other organisms as well. Interestingly, the accumulation of acetic acid in stationary phase cultures inhibits growth arrest of cells in G1 and is preferentially toxic to cells that fail to undergo a G1 arrest (Burhans and Weinberger, 2009
We used a multifactorial experimental design to investigate the impact of acetic acid on cellular growth kinetics in dependence on glucose concentration as well as extracellular pH, covering the whole range of combinations in experimental conditions. Raising the extracellular pH clearly reduced the toxic influence of acetic acid. The accumulation of undissociated acids within the cell is a function of Δ pH and glucose concentration in the medium (Thomas et al., 2002
). By raising the pH to a value higher than the pKa of the acid, the concentration of undissociated acid is reduced for a given amount of total acid, placing less stress on cells. Raising the extracellular glucose concentration results in increased intracellular ATP supporting the activity of ATPases (Thomas et al., 2002
). Both conditions result in a lower waste of energy for maintenance of the pHin in the range optimal for growth. The outcome is a decreased inhibitory effect of acetic acid on yeast growth and metabolism.
The protecting effect of high glucose concentrations observed in our experiments could be explained by several mechanisms. Acetic acid can enter the cells only in its undissociated, uncharged form. The charged acetate anion is generally considered as non-toxic (Piper et al., 2001
). A higher pH on the cytosolic side of the membrane can cause a substantial fraction of this acid to dissociate to the anion, a form which is relatively membrane-impermeable and that therefore will accumulate inside the cell resulting in intracellular acidification (Piper et al., 2001
In budding yeast and many other fungi, intracellular acidification activates highly conserved Ras2 and cAMP-dependent signaling pathways that respond to glucose (Thevelein and de Winde, 1999
). Thus, despite the oxidative stress, inhibition of glycolysis, induction of gluconeogenesis and synthesis of storage carbohydrates, acetic acid treated cells are continuously subjected to growth signals that promote entry into S phase. Constitutive activation of the Ras-cAMP-PKA pathway would also result in a PKA-dependent loss of mitochondrial function (Gourlay and Ayscough, 2006
), which requires HAP4
transcriptional regulation and accumulation of damaged, high ROS producing mitochondria (Leadsham and Gourlay, 2010
). The result is a conflicting situation. On one side, inhibition of glucose uptake and mitochondria function by acetic acids will result in significant ATP depletion and disturbed pH homeostasis. Available nutrients are then redirected toward synthesis of storage carbohydrates causing insufficient synthesis of dNTPs and inefficient DNA replication. On the other side, Ras-cAMP-PKA activation will stimulate growth. Together, intracellular acid accumulation seems to trigger an inappropriate growth signal and replication stress, which leads to cell death. Feeding with high glucose concentrations can, therefore, prevent the energetic collapse in mitochondria impaired cells where the glycolytic flux is reduced because of low pH. This also fits with the observation that neutralizing buffering of yeast media could extend chronological life span in yeast cells, implicating that other mechanism than just simple acidification of the environment are involved in acetic acid-induced metabolic alterations and apoptosis induction (Pan et al., 2011
Reduced sensitivity of the Δhap4 mutant to acetic acid may be explained by impaired expressions of gluconeogenic, glyoxylate, and TCA cycle enzymes, which are regulated by the Hap 2-3-4-5 complex (Figure ). The low gluconeogenic activity in the Δ hap4 mutant will prevent the synthesis of storage carbohydrates, trehalose and glycogen, in conditions where glucose uptake is inhibited by acetic acid. Lin et al. (2002
) showed that aged cells respond to glucose-deprivation by shifting the metabolism away from glycolysis toward gluconeogenesis and energy storage. In acetic acid induced aging, increased gluconeogenesis and trehalose/glycogen synthesis pathways will compete with amino acids synthesis pathways for the same glycolytic and TCA intermediates. In consequence, this will result in a condition of amino acids starvation and ATP depletion. In contrast, in the Δhap4 mutant the limited available glucose in the cells can be fully used for sustaining the ATP pool and pHin homeostasis and significantly decrease acetic acid toxicity.
Our results clearly show the interdependence between an important metabolic by-product of yeast fermentation, acetic acid, and the efficiency of the cellular metabolism as well as aging of cells. In addition to providing some new insight into the role of extracellular conditions and availability of nutrients on glucose metabolism, respiration, cellular proliferation and aging, which play a central role in different diseases, our results also suggest controlled stress conditions as a means to increase fermentation efficiency. Given the urgent need to optimize the production of fuel ethanol from cellulosic biomass as a more environmental-friendly fossil fuel alternative, our results suggest that investigating the role of metabolic by products and other stress conditions on fermentation could still lead to further optimization.