Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Δklnde1 and Δklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum.     

The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis

The phosphoglucose isomerase mutant of the respiratory yeast Kluyveromyces lactis (rag2) is forced to metabolize glucose through the oxidative pentose phosphate pathway and shows an increased respiratory chain activity and reactive oxygen species production. We have proved that the K. lactis rag2 mutant is more resistant to oxidative stress (OS) than the wild type, and higher activities of glutathione reductase (GLR) and catalase contribute to this phenotype. Resistance to OS of the rag2 mutant is reduced when the gene encoding GLR is deleted. The reduction is higher when, in addition, catalase activity is inhibited. In K. lactis, catalase activity is induced by peroxide-mediated OS but GLR is not. We have found that the increase of GLR activity is correlated with that of glucose-6-phosphate dehydrogenase (G6PDH) activity that produces NADPH. G6PDH is positively regulated by an active respiratory chain and GLR plays a role in the reoxidation of the NADPH from the pentose phosphate pathway in these conditions. Cytosolic NADPH is also used by mitochondrial external alternative dehydrogenases. Neither GLR overexpression nor induction of the OS response restores growth on glucose of the rag2 mutant when the mitochondrial reoxidation of cytosolic NADPH is blocked.     

In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.     

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

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.     

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

The external calcium-dependent NADPH dehydrogenase from Neurospora crassa mitochondria

We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an N-terminal transmembrane domain.     

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.     

Sources of NADPH in yeast vary with carbon source

Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984-13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1Delta ald6Delta mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1Delta idp2Delta mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1Delta ald6Delta mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP+ rise dramatically in the zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.     

Pyridine nucleotide transhydrogenations in yeast

Though previously described as very low or absent in yeast, we find significant pyridine nucleotide transhydrogenation (NADPH + acetyl pyridine-NAD+----NADP+ + acetyl pyridine-NADH) activity in yeast extracts when assayed at pH 8-9, and describe here the subcellular distribution and separation of the various molecular forms contributing to the total activity in two yeast species. Gentle subcellular fractionation reveals transhydrogenase activity only in the cytosolic fraction of both Saccharomyces cerevisiae and Candida utilis while intact mitochondria and microsomes are without activity. On sucrose gradient centrifugation, this soluble cytosolic activity proves to be primarily in a high-molecular-weight (greater than 10(6)) band which has salmon-colored fluorescence on uv illumination. Sonication of the particulate subcellular fractions solubilizes substantial transhydrogenase activity from mitochondria of C. utilis (but not from S. cerevisiae) which on sucrose gradients consists of both high (greater than 10(6))- and low-molecular-weight active fractions, each with yellow-green fluorescence. Ammonium sulfate fractionation and sucrose gradient centrifugation of protein solubilized from whole yeast of both species by vigorous homogenization with glass beads confirms the presence and fluorescence of these various molecular weight forms. The relationship of these activities to other enzymatic activities (especially the mitochondrial external NADH dehydrogenase) is discussed.     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.     

MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria

In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature- sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.