Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product.

In most eukaryotic cells, the respiratory chain NADH dehydrogenase (Complex I) is a multimeric enzyme under dual (nuclear and mitochondrial) genetic control. Several genes encoding subunits of this enzyme have been identified in the mitochondrial genome from various organisms, but the functions of these subunits are in most part unknown. We describe here a human cell line in which the enzyme lacks the mtDNA-encoded subunit ND4 due to a frameshift mutation in the gene. In this cell line, the other mtDNA-encoded subunits fail to assemble, while at least some of the nuclear-encoded subunits involved in the redox reactions appear to be assembled normally. In fact, while there is a complete loss of NADH:Q1 oxidoreductase activity, the NADH:Fe(CN)6 oxidoreductase activity is normal. These observations provide the first clear evidence that the ND4 gene product is essential for Complex I activity and give some insights into the function and the structural relationship of this polypeptide to the rest of the enzyme. They are also significant for understanding the pathogenetic mechanism of the ND4 gene mutation associated with Leber's hereditary optic neuropathy.     

The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme.

Seven of the approximately 40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near-homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA-encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA-less (rho0) mouse LL/2-m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the rho0 cell transformants revealed that the mutation causes a loss of assembly of the mtDNA-encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate-dependent respiration in digitonin-permeabilized cells by approximately 90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by approximately 99%. Furthermore, the ND6(-) cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease-causing ND6 gene mutations.     

Efficient selection and characterization of mutants of a human cell line which are defective in mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase.

The mitochondrial NADH dehydrogenase (complex I) in mammalian cells is a multimeric enzyme consisting of approximately 40 subunits, 7 of which are encoded in mitochondrial DNA (mtDNA). Very little is known about the function of these mtDNA-encoded subunits. In this paper, we describe the efficient isolation from a human cell line of mutants affected in any of these subunits. In the course of analysis of eight mutants of the human cell line VA2B selected for their resistance to high concentrations of the complex I inhibitor rotenone, seven were found to be respiration deficient, and among these, six exhibited a specific defect of complex I. Transfer of mitochondria from these six mutants into human mtDNA-less cells revealed, surprisingly, in all cases a cotransfer of the complex I defect but not of the rotenone resistance. This result indicated that the rotenone resistance resulted from a nuclear mutation, while the respiration defect was produced by an mtDNA mutation. A detailed molecular analysis of the six complex I-deficient mutants revealed that two of them exhibited a frameshift mutation in the ND4 gene, in homoplasmic or in heteroplasmic form, resulting in the complete or partial loss, respectively, of the ND4 subunit; two other mutants exhibited a frameshift mutation in the ND5 gene, in near-homoplasmic or heteroplasmic form, resulting in the ND5 subunit being undetectable or strongly decreased, respectively. It was previously reported (G. Hofhaus and G. Attardi, EMBO J. 12:3043-3048, 1993) that the mutant completely lacking the ND4 subunit exhibited a total loss of NADH:Q1 oxidoreductase activity and a lack of assembly of the mtDNA-encoded subunits of complex I. By contrast, in the mutant characterized in this study in which the ND5 subunit was not detectable and which was nearly totally deficient in complex I activity, the capacity to assemble the mtDNA-encoded subunits of the enzyme was preserved, although with a decreased efficiency or a reduced stability of the assembled complex. The two remaining complex I-deficient mutants exhibited a normal rate of synthesis and assembly of the mtDNA-encoded subunits of the enzyme, and the mtDNA mutation(s) responsible for their NADH dehydrogenase defect remains to be identified. The selection scheme used in this work has proven to be very valuable for the isolation of mutants from the VA2B cell line which are affected in different mtDNA-encoded subunits of complex I and may be applicable to other cell lines.     

Analysis of mitochondrial ND4 gene DNA sequence in Finnish families with Leber hereditary optic neuroretinopathy

A mutation in the mitochondrial DNA at nt 11,778 has recently been found in Leber hereditary optic neuroretinopathy (LHON), a maternally inherited ocular disease. The mutation is located in the ND4 gene encoding subunit 4 of the respiratory chain enzyme NADH dehydrogenase. The mutation was subsequently not found in 9 of the 20 known Finnish families with LHON, implying that there are at least two different mutations associated with the disease. Using direct sequencing of PCR-amplified mtDNA, we have now sequenced the entire ND4 region in the families without the nt 11,778 mutation to find the other mutations. No new mutations in the ND4 region were found, suggesting that the putative mtDNA mutation in these families may be in the coding regions for other subunits of NADH dehydrogenase enzyme. The sequence of ND4 gene as found to be highly homogeneous.     

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Inhibition of mitochondrial function in HL60 cells is associated with an increased apoptosis and expression of CD14.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.     

ND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed.     

Cloning and sequence analysis of a mitochondrial gene cluster encoding two NADH dehydrogenase subunits and seven tRNA molecules from Trichoderma reesei

A mitochondrial gene cluster, encoding proteins homologous to NADH dehydrogenase subunits II and III (ND2 and ND3) and seven tRNAs, from Trichoderma reesei QM9414 was cloned and sequenced. These genes are clustered tandemly on the mitochondrial genome of QM9414. Phylogenetic analysis showed that ND2 and ND3 were most closely related to the mitochondrial ND subunits II (71% identity) and III (70% identity) from Podospora anserine. Northern dot blot analysis showed that the nd2 and nd3 genes are actively transcribed in the T. reesei mitochondria.     

Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes

Summary The gene organization of starfish mitochondrial DNA is identical with that of the sea urchin counterpart except for a reported inversion of an approximately 4.6-kb segment containing two structural genes for NADH dehydrogenase subunits 1 and 2 (ND 1 and ND 2). When the codon usage of each structural gene in starfish, sea urchin, and vertebrate mitochondrial DNAs is examined, it is striking that codons ending in T and G are preferentially used more for heavy strand-encoded genes, including starfish ND 1 and ND 2, than for light strand-encoded genes, including sea urchin ND 1 and ND 2. On the contrary, codons ending in A and Care preferentially used for the light strand-encoded genes rather than for the heavy strand-encoded ones. Moreover, G-U base pairs are more frequently found in the possible secondary structures of heavy strandencoded tRNAs than in those of light strand-encoded tRNAs. These observations suggest the existence of a certain constraint operating on mitochondrial genomes from various animal phyla, which results in the accumulation of G and T on one strand, and A and C on the other.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

The decrease of mitochondrial NADH dehydrogenease and drug induced apoptosis in doxorubicin resistant A431 cells

Doxorubicin (DOX) resistant A10A cells derived from human squamous carcinoma A431 cells were found to exhibit a smaller degree of apoptosis after DOX treatment as compared to their parent cells. Induction of reactive oxygen species (ROS) formation and mitochondrial depolarization by DOX were more pronounced in the parent cells than in the A10A cells. The fact that catalase suppressed the DOX effect on ROS induction, mitochondrial depolarization and apoptosis in both cell lines suggests an involvement of ROS in the DOX-induced apoptosis. To investigate the underlying mechanisms for DOX resistance in A10A cells, RT-PCR based differential display was used. One of the clones, which was down-regulated in the A10A cells, had sequence homology with part of the mitochondrial NADH dehydrogenase III (ND3) gene. NADH dehydrogenase plays an important role in generating ROS during DOX treatment. The results indicate that down-regulation of ND3 may at least in part contribute to the mechanism for A10A cells resistant to DOX-induced apoptosis.     

NADH dehydrogenase in Neurospora crassa contains myristic acid covalently linked to the ND5 subunit peptide

The mitochondrial, proton-pumping NADH:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex I catalyzes the first steps of mitochondrial electron transfer and proton translocation. Radiolabel from [(3)H]myristic acid was incorporated by Neurospora crassa into the mitochondrial-encoded, approximately 70 kDa ND5 subunit of NADH dehydrogenase, as shown by immunoprecipitation. This myristate apparently was linked to the peptide through an amide linkage at an invariant lysine residue (Lys546), based upon analyses of proteolysis products. The myristoylated lysine residue occurs in the predicted transmembrane helix 17 (residues 539-563) of ND5. A consensus amino acid sequence around this conserved residue exists in homologous subunits of NADH dehydrogenase. Cytochrome c oxidase subunit 1, in all prokaryotes and eukaryotes, contains this same consensus sequence surrounding the lysine which is myristoylated in N. crassa.     

Ginkgo biloba Extracts EGb 761 and Bilobalide Increase NADH Dehydrogenase mRNA Level and Mitochondrial Respiratory Control Ratio in PC12 Cells

In the present study, we investigated the effect of Ginkgo biloba extract, EGb 761, and one of its components, bilobalide, on gene expression of subunit 1 of mitochondrial NADH dehydrogenase (ND1) in PC12 cells. By Northern blot analysis we found a 2-fold significant increase in ND1 mRNA level, after 48 and 72 h exposure to 100 g/ml EGb 761 and to 10 g/ml bilobalide. We also evaluated, by oxygraphy measurements, mitochondrial respiration during state 3 and state 4. In cells treated with EGb 761 and bilobalide for 48 and 72 h, state 4 respiration was significantly decreased, and the respiratory control ratio was increased. These results provide evidence that EGb 761 and bilobalide exert their protective effects by up-regulating mitochondrial ND1 gene expression and by decreasing state 4 respiration, whose increase is thought to be responsible for oxidative damage.     

Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties.

Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2.