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The deleterious effects of H202 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by beta-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to Qo site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Qo) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.

In yeast, Ca(2+) and long chain alkylguanidines interact with mitochondria modulating the opening of the yeast mitochondrial unspecific channel. Mammalians possess a similar structure, the mitochondrial permeability transition pore. The composition of these pores is under debate. Among other components, the voltage-dependent anion channel has been proposed as a component of either pore. In yeast from an industrial strain, octylguanidine and calcium closed the yeast mitochondrial unspecific channel. Here, the effects of the cations Ca(2+) or octylguanidine and the voltage-dependent anion channel effector decavanadate were evaluated in yeast mitochondria from either a wild type or a voltage-dependent anion channel deletion laboratory strain. It was observed that in the absence of voltage-dependent anion channel, the yeast mitochondrial unspecific channel was desensitized to Ca(2+), octylguanidine or decavanadate but remained sensitive to phosphate. It is thus suggested that in yeast mitochondria, the voltage-dependent anion channel has a cation binding site where Ca(2+) and octylguanidine interact, conferring cation sensitivity to the yeast mitochondrial unspecific channel.

In mammals, biotin, well known for its role as the cofactor of carboxylases, also controls the expression not only of proteins involved in this function, but also of a large number and variety of other different proteins. As a first step towards looking for a rationale for these phenomena, we intend to compare these regulatory functions of biotin between the rat and the much less evolutionized eukaryote, Saccharomyces cerevisiae. Thus far, we have measured growth in yeast cultured on different concentrations of biotin to choose the experimental conditions to be used (2, 200 and 2000 microM) and have found that a band corresponding to the biotinylated S. cerevisiae Arc1p protein appears at streptavidin Western blots at a biotin concentration above 2000 muM, its density increasing with higher biotin amounts. We will now study changes in yeast transcriptome with these varying concentrations and compare them with changes observed in the rat.

The K+ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K+-mediated mitochondrial swelling observed in the absence of phosphate (PO4) and in the presence of a respiratory substrate has led to propose that large K+ movements occur in yeast mitochondria. Thus, the uptake of K+ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K+ transport; these were mitochondrial swelling and the uptake of the radioactive K+ analog 86Rb+. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO4 concentrations. The high protein concentrations used to measure 86Rb+ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO4 but not at 0 or 4 mM PO4. At 4 mM PO4 swelling was inhibited while, in contrast, 86Rb+ uptake was still observed. The results suggest that an energy-dependent K+ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K+ uptake system, the Mg2+ and quinine sensitivity of both swelling and 86Rb+ uptake were evaluated. Under the conditions where the unspecific pore was closed, K+ transport sensitivity to Mg2+ and quinine increased. In addition, when Zn2+ was added as an antiport inhibitor, uptake of 86Rb+ increased. It is suggested that in yeast mitochondria, the K+ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms., (Copyright 2001 Academic Press.)

In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP. The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added. It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.

We have cloned and sequenced QCR10, the nuclear gene encoding an 8.5-kDa protein proposed to be a subunit of the cytochrome bc1 complex of Saccharomyces cerevisiae. QCR10 includes a 231-base pair open reading frame capable of encoding a protein of 77 amino acids with a predicted molecular mass of 8492 Da. The codons for the amino-terminal methionine and alanine are separated from the remainder of the open reading frame by a 63-base pair intron that contains 5'-donor, 3'-acceptor, and TACTAAC sequences known to be necessary for splicing. The deduced amino acid sequence of the 8.5-kDa yeast protein is 28% identical to that of the 6.4-kDa subunit 11 from the bovine heart cytochrome bc1 complex, and the predicted secondary structures of the two proteins are very similar. Deletion of the chromosomal copy of QCR10 did not affect the growth of yeast on nonfermentable carbon sources. However, deletion of QCR10 synergistically contributes to the temperature-dependent phenotype resulting from deletion of QCR6, the gene for subunit 6 of the cytochrome bc1 complex. In addition, ubiquinol-cytochrome c oxidoreductase activity was reduced 40% in mitochondrial membranes from the QCR10 deletion strain, and the Rieske iron-sulfur protein was lost when the cytochrome bc1 complex lacking the 8.5-kDa protein was purified. We conclude that the 8.5-kDa protein encoded by QCR10 is a subunit of the yeast cytochrome bc1 complex and that the presence of this subunit during assembly is required for stable association of the iron-sulfur protein with the complex.

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.

In mitochondria from Saccharomyces cerevisiae and in the presence of ethanol or NADH, K+ or Na+ increased the rate of O2 uptake in states 3 and uncoupled as well as in sonicated mitochondria. The respiratory control, the ADP:O ratio and the synthesis of ATP also increased. ATP hydrolysis by sonicated mitochondria increased depending on the cation added as follows: K+ = NH4+ = Rb+ Na+ Li+. This correlated with the ionic radii of the cations. Monovalent cations increased the activity of: 1) F1F0ATPase which was sensitive to cation size and 2) complex I of the respiratory chain, which seemed to regulate the rate of oxidative phosphorylation, but did not discriminate between K+ or Na+.

Little information on the effects of cyclohexane at the cellular or subcellular level is available. In Saccharomyces cerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes. In mitochondria isolated from S. cerevisiae, oxygen uptake and ATP synthesis were inhibited, although ATPase activity was not affected. Cyclohexane effects were similar to those reported for beta-pinene and limonene, suggesting that the cyclohexane ring in these monoterpenes may be a determinant for their biological activities.

The effects of beta-pinene on yeast cells were studied. This terpene inhibited respiration with glucose or ethanol as the substrate. The inhibition depended on the ratio of the terpene to the amount of yeast cells; for a fixed concentration of pinene, inhibition decreased as the amount of yeast cells increased. Pinene also inhibited the pumping of protons and K+ transport, but this inhibition was more marked with with ethanol than with glucose as the substrate, indicating the mitochondrial localization of the inhibition. The studies on isolated mitochondria showed a series of effects, starting with the disappearance of the respiratory control and deenergization of the organelles and followed by an inhibition of respiration at higher concentrations of the terpene. The effect on respiration could be localized to the cytochrome b region of the electron transport chain. No effect could be detected on the activity of ATPase. The effects can be ascribed to a localization of pinene on membranes which was also accompanied by a decrease in the fluorescence polarization of diphenyl hexatriene, probably meaning an increase in the fluidity of the membrane, localized preferentially to the mitochondria.