Internal NADH Dehydrogenase Research Articles

Expression of alternative NADH dehydrogenases (NDH-2) in the phytopathogenic fungus Ustilago maydis.

Type 2 alternative NADH dehydrogenases (NDH‐2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH‐2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH‐2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

The role of flavin-containing enzymes in mitochondrial membrane hyperpolarization and ROS production in respiring Saccharomyces cerevisiae cells under heat-shock conditions

Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.

Changes in the OXPHOS system in leaf and root mitochondria of Arabidopsis thaliana subjected to long-term sulphur deficiency

Long-term sulphur (S) deficiency in Arabidopsis thaliana affects the functioning of the mitochondrial oxidative phosphorylation system (OXPHOS) via alteration of the multisubunit NADH-ubiquinone oxidoreductase (Complex I; EC 1.6.5.3), which contains several iron–sulphur clusters. Densitometric analysis of bands of respiratory chain complexes after one-dimensional blue-native polyacrylamide gel electrophoresis (BN-PAGE) showed that levels and in-gel capacities of Complex I in leaf and root mitochondria were lower than those of the control. Two-dimensional BN/SDS-PAGE showed lower abundance of all Complex I subunits, but the qualitative structural composition (subunit expression and mobility) did not change. In mitochondria of S-deficient A. thaliana, impairment of Complex I could be compensated to some extent by additional type II NADH dehydrogenases that do not contain iron–sulphur clusters. The level and capacity of external NADH dehydrogenases in leaf and root mitochondria was higher under S deficiency, but that of internal NADH dehydrogenases did not differ from the control. The amount of COXII (mitochondrial-encoded subunit of cytochrome c oxidase in Complex IV; EC 1.9.3.1) and the capacity of Complex IV were lower under S deficiency, but levels of alternative oxidase, a bypass to Complex IV, did not change. We discuss S deficiency in A. thaliana in relation to the assembly and stability of Complex I and to a bypass of Complex I by external type II NADH dehydrogenases.

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.

Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases.

Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed.

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1.

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved.

Respiratory activity, energy and redox status in sulphur-deficient bean plants

Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic processes. S-deficient bean plants ( Phaseolus vulgaris L. cv. Złota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower dry matter production. Phenotypic effects of S deficiency appeared as depressed shoot growth, paling and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show that S deficiency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex I. Increased activities of internal NADH dehydrogenases (ND in) may at least partially compensate for Complex I impairment. External NADH dehydrogenases (ND ex) activities, as well as protein level and capacity of alternative oxidase (AOX), did not change in S-deficient bean plants. Total ATP concentration severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-deficient bean plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our findings indicate that flexible function of plant mitochondrial respiratory chain could be an important target during adaptations to S deficiency.

The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance

Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally, we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases, respectively, that have a function in FOH resistance. When exposed to FOH, the ΔaifA and ΔndeA strains have increased ROS production while ΔndeB, ΔndeA ΔndeB, and ΔndiA mutant strains showed the same ROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH.

Respiratory chain alternative enzymes as tools to better understand and counteract respiratory chain deficiencies in human cells and animals

Mitochondrial respiratory chain defects are now recognized to underlie a large number of human diseases with a spectacular variety in their phenotypic presentations. Despite progress made in the elucidation of their molecular basis, these diseases remain essentially untreatable. To date, most strategies to counteract these diseases, either in vitro or in vivo have proven unsuccessful. In humans, the respiratory chain lacks several redox active proteins long known in many micro-organisms, as well as in plants, and, as found recently, even in some metazoans. These alternative enzymes, e.g. the cyanide-insensitive alternative oxidase and the internal rotenone-insensitive NADH dehydrogenase, confer a significant flexibility to the respiratory chain, allowing it to overcome potential constraints exerted by the cell phosphorylation potential or by environmental xenobiotics. In plants, these alternative enzymes, activated by a subset of keto-acids, including pyruvate, are essentially engaged under highly reducing conditions. Because these are conditions observed in patients with respiratory chain dysfunction, we made the hypothesis that expression of these proteins might be of benefit in such situations. The observation that a functional alternative oxidase from Ciona intestinalis could be expressed in mammalian cells without obvious detrimental effect has provided a basis to develop a research programme to test the hypothesis, within an ad hoc international consortium, that this paper aims to describe. Combining research on human cells, flies and mice, the project aims, firstly, to verify that expressing these alternative enzymes is physiologically benign, useful as a tool to delineate the mechanisms of respiratory chain dysfunction and, finally, test their potential therapeutic benefit.

The iron–sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

ATP-sensitive Potassium Channel in Mitochondria of the Eukaryotic Microorganism Acanthamoeba castellanii

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.

Substrate‐inducible versions of internal alternative NADH : ubiquinone oxidoreductase from Yarrowia lipolytica

In standard laboratory strains of the obligate aerobic yeast Yarrowia lipolytica, respiratory chain complex I (proton-translocating NADH : ubiquinone oxidoreductase) is an essential enzyme, since alternative NADH dehydrogenase activity is located exclusively at the external face of the mitochondrial inner membrane. Deletions and other loss-of-function mutations in genes for nuclear coded subunits of complex I can be obtained only when an internal version of the latter enzyme, termed NDH2i, is introduced. In contrast to recent findings with Neurospora crassa, external alternative NADH dehydrogenase activity is dispensable in complex I deletion strains of Y. lipolytica. We used regulable promoters to create strains which express internal alternative NADH dehydrogenase in a substrate-dependent manner. The ability to switch between complex I-dependent and -independent mode of growth simply by changing the carbon source is an important prerequisite for screens for both loss-of-function and inhibitor resistance mutation. The isocitrate lyase promoter (pICL1), in combination with a NDH2i allele that results in reduced expression and activity, was most promising. In the presence of complex I inhibitors, this construct allowed growth on acetate, but not on glucose minimal media. A somewhat higher background was observed with the acyl-CoA oxidase 2 (pPOX2) promoter on glucose minimal media.

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.

Yeast AMID Homologue Ndi1p Displays Respiration-restricted Apoptotic Activity and Is Involved in Chronological Aging

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosis-like cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed.

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.

Modelling NADH turnover in plant mitochondria.

NADH is central to the functioning of mitochondrial respiration. It is produced by enzymes in, or associated with, the tricarboxylic acid cycle in the matrix, and it is oxidized by two respiratory chain enzymes in the inner membrane, the rotenone-sensitive complex I and the rotenone-insensitive internal NADH dehydrogenase (ND(in)). A simplified kinetic model for NADH turnover in the matrix of plant mitochondria is presented. Only the two main NADH-producing enzymes, NAD-malate dehydrogenase [EC 1.1.1.37] (MDH) and NAD-malic enzyme [EC 1.1.1.39] (ME), are considered. This model reproduces the complex behaviour of malate oxidation by isolated mitochondria in response to additions of ADP (state 3/state 4), NAD(+) and/or rotenone, as well as to changes in pH. It is found that MDH always operates at or close to equilibrium. Changes in the activity of complex I, ND(in), or ME are predicted to cause clear changes in the pattern of malate oxidation. In general, the model predicts high sensitivity to changes in the ME activity. In contrast, MDH activity can be reduced 100-fold without detectable changes in malate oxidation. It is demonstrated that it is not the high activity, but the equilibrium properties of MDH that are important for the redox-buffering function of MDH in the mitochondrial matrix. Binding of NAD(+) and NADH in the matrix reduces the concentrations of free NAD(+) and NADH, depending on the concentration of binding sites and the binding strength. On the basis of the modelling results it is estimated that a significant proportion of the mitochondrial NAD is bound.

Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic Nicotiana sylvestris.

The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD(P)H dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity was instead 10-fold decreased, indicating that the corresponding N. sylvestris gene had been suppressed. Activities of external and internal rotenone-insensitive NADH dehydrogenases were unchanged in the transgenic lines. The results demonstrate that the St-ndb1 encodes an external dehydrogenase specific for NADPH and dependent on calcium for activity. Transgenic lines overexpressing St-ndb1 had specifically increased protein levels for alternative oxidase and uncoupling protein, as compared to the WT and one co-suppressing line. This indicates cross-talk in the expressional control, or metabolic conditions influencing it, for the different categories of energy-dissipating proteins that bypass oxidative phosphorylation. The potential effects of external NADPH oxidation on other cellular processes are discussed.

Antimycin A treatment decreases respiratory internal rotenone-insensitive NADH oxidation capacity in potato leaves

BackgroundThe plant respiratory chain contains several energy-dissipating enzymes, these being type II NAD(P)H dehydrogenases and the alternative oxidase, not present in mammals. The physiological functions of type II NAD(P)H dehydrogenases are largely unclear and little is known about their responses to stress. In this investigation, potato plants (Solanum tuberosum L., cv. Desiree) were sprayed with antimycin A, an inhibitor of the cytochrome pathway. Enzyme capacities of NAD(P)H dehydrogenases (EC 1.6.5.3) and the alternative oxidase were then analysed in isolated leaf mitochondria.ResultsWe report a specific decrease in internal rotenone-insensitive NADH dehydrogenase capacity in mitochondria from antimycin A-treated leaves. External NADPH dehydrogenase and alternative oxidase capacities remained unaffected by the treatment. Western blotting revealed no change in protein abundance for two characterised NAD(P)H dehydrogenase homologues, NDA1 and NDB1, nor for two subunits of complex I. The alternative oxidase was at most only slightly increased. Transcript levels of nda1, as well as an expressed sequence tag derived from a previously uninvestigated closely related potato homologue, remained unchanged by the treatment. As compared to the daily rhythm-regulated nda1, the novel homologue displayed steady transcript levels over the time investigated.ConclusionsThe internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves. However, the decrease is not due to changes in expression of known nda genes. One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted.

The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria.

An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination.