Proton pumping complex I increases growth yield in Candida utilis.

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and cellular maintenance processes. A crucial parameter for growth evaluation is its yield, i.e. the efficiency of the transformation processes. The yeast Candida utilis is of peculiar interest since its mitochondria exhibit a complex I that is proposed to pump protons but also an external NADH dehydrogenase that do not pump protons. Here, we show that in C. utilis cells grown on non-fermentable media, growth yield is 30% higher as compared to that of Saccharomyces cerevisiae that do not exhibit a complex I. Moreover, ADP/O determination in C. utilis shows that electrons coming from internal NADH dehydrogenase go through proton pumping complex I, whereas electrons coming from external NADH dehydrogenases do not go through proton pumping complex I. Furthermore, we show that electron competition strictly depends on extra-mitochondrial NADH concentration, i.e. the higher the extra-mitochondrial NADH concentration, the higher the competition process with a right way for electrons coming from external NADH dehydrogenases. Such a complex regulation in C. utilis allows an increase in growth yield when cytosolic NADH is not plentiful but still favors the cytosolic NADH re-oxidation at high NADH, favoring biomass generation metabolic pathways.

The role of mitochondrial biogenesis and ROS in the control of energy supply in proliferating cells.

In yeast, there is a constant growth yield during proliferation on non-fermentable substrate where the ATP generated originates from oxidative phosphorylation. This constant growth yield is due to a tight adjustment between the growth rate and the cellular mitochondrial amount. We showed that this cellular mitochondrial amount is strictly controlled by mitochondrial biogenesis. Moreover, the Ras/cAMP pathway is the cellular signaling pathway involved in the regulation of mitochondrial biogenesis, with a direct relationship between the activity of this pathway and the cellular amount of mitochondria. The cAMP protein kinase Tpk3p is the catalytic subunit specifically involved in the regulation of mitochondrial biogenesis through regulation of the mitochondrial ROS production. An overflow of mitochondrial ROS decreases mitochondrial biogenesis through a decrease in the transcriptional co-activator Hap4p, which can be assimilated to mitochondria quality control. Moreover, the glutathione redox state is shown as being an intermediate in the regulation of mitochondrial biogenesis. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Mitochondrial oxidative phosphorylation is regulated by fructose 1,6-bisphosphate. A possible role in Crabtree effect induction?

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Profound effects of the general anesthetic etomidate on oxidative phosphorylation without effects on their yield.

We investigated the effects of the general anesthetic Etomidate on oxidative phosphorylation in isolated rat liver mitochondria. The study of each electron transfer site shows that there is an inhibition: mainly at complex I but also, to a lesser extent, at complex III. Moreover, with succinate as substrate, the increase in non-phosphorylating respiration is accompanied by a decrease in DeltaPsi. However, this effect is not due to classical uncoupling of oxidative phosphorylation, since ADP addition at high Etomidate concentrations restores the transmembrane difference of electrical potential. Also, in the same range of Etomidate concentration, the ATP/O ratio is not significantly affected. In conclusion, the main effect of Etomidate is to decrease the oxidative phosphorylation rate without changing yield. The H(+) leak which appears under non-phosphorylating conditions becomes negligible in physiological conditions.

Growth yield homeostasis in respiring yeast is due to a strict mitochondrial content adjustment.

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes taking place during net biomass formation and cell property maintenance. A crucial parameter for growth description is its yield, i.e. the efficiency of the transformation from substrate consumption to biomass formation. Using numerous yeast strains growing on different respiratory media, we have shown that the growth yield is identical regardless of the strain, growth phase, and respiratory substrate used. This homeostasis is the consequence of a strict linear relationship between growth and respiratory rates. Moreover, in all conditions tested, the oxygen consumption rate was strictly controlled by the cellular content of respiratory chain compounds in such a way that, in vivo, the steady state of oxidative phosphorylation was kept constant. Thus, the growth yield homeostasis depends on the tight adjustment of the cellular content of respiratory chain compounds to the growth rate. Any process leading to a defect in this adjustment allows an energy waste and consequently an energy yield decrease.

The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth.

During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield. On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae. We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases. In decreased energy demand, the Deltatpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate. Moreover, enzymatic content decreases in the Deltatpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified. Our study points to an important decrease in the cytochrome c content in the Deltatpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase.

Competition of electrons to enter the respiratory chain: a new regulatory mechanism of oxidative metabolism in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are the external NADH dehydrogenases (Nde1p and Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p. Subsequently, glycerol 3-phosphate donates electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). At saturating concentrations of NADH, the activation of external NADH dehydrogenases completely inhibits glycerol 3-phosphate oxidation. Studies on the functionally isolated enzymes demonstrated that neither Nde1p nor Nde2p directly inhibits Gut2p. Thus, the inhibition of glycerol 3-phosphate oxidation may be caused by competition for the entrance of electrons into the respiratory chain. Using single deletion mutants of Nde1p or Nde2p, we have shown that glycerol 3-phosphate oxidation via Gut2p is inhibited fully when NADH is oxidized via Nde1p, whereas only 50% of glycerol 3-phosphate oxidation is inhibited when Nde2p is functioning. By comparing respiratory rates with different respiratory substrates, we show that electrons from Nde1p are favored over electrons coming from Ndip (internal NADH dehydrogenase) and that when electrons come from either Nde1p or Nde2p and succinodehydrogenase, their use by the respiratory chain is shared to a comparable extent. This suggests a very specific competition for electron entrance into the respiratory chain, which may be caused by the supramolecular organization of the respiratory chain. The physiological consequences of such regulation are discussed.

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae.

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.