Mitochondrial DNA of two independent oligomycin-resistant Chinese hamster ovary cell lines contains a single nucleotide change in the ATPase 6 gene

We have determined the nucleotide sequence of the URF A6L and ATPase 6 genes of the mitochondrial DNA of wild-type Chinese hamster ovary (CHO) cells and of two independently isolated, cytoplasmically inherited CHO mutant cell lines that are resistant to oligomycin, an inhibitor of the mitochondrial ATP synthase (ATPase) complex. Comparison of the nucleotide sequences of the mutants with that of their parental cell line revealed a single nucleotide difference, a G-to-A transition at nucleotide 433 of the ATPase 6 gene. This single base pair change predicts a nonconservative amino acid change, with a glutamic acid residue being replaced by a lysine residue at amino acid 145 of the ATPase 6 gene product in the mutants. This glutamic acid residue and several others in the surrounding amino acid sequence are conserved among all species examined to date. Analyses of several of the biochemical properties of the oligomycin-resistant CHO mutants indicate that the glutamic acid residue at position 145 of subunit 6 of the mitochondrial ATP synthase complex is important for the binding of oligomycin to the enzyme complex, but is not essential for proton translocation.     

An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit

Unlike most organisms, the mitochondrial DNA (mtDNA) of Chlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F(0)F(1)-ATP synthase. We hypothesized that C. reinhardtii ATPase 6 is nucleus encoded and identified cDNAs and a single-copy nuclear gene specifying this subunit (CrATP6, with eight exons, four of which encode a mitochondrial targeting signal). Although the algal and human ATP6 genes are in different subcellular compartments and the encoded polypeptides are highly diverged, their secondary structures are remarkably similar. When CrATP6 was expressed in human cells, a significant amount of the precursor polypeptide was targeted to mitochondria, the mitochondrial targeting signal was cleaved within the organelle, and the mature polypeptide was assembled into human ATP synthase. In spite of the evolutionary distance between algae and mammals, C. reinhardtii ATPase 6 functioned in human cells, because deficiencies in both cell viability and ATP synthesis in transmitochondrial cell lines harboring a pathogenic mutation in the human mtDNA-encoded ATP6 gene were overcome by expression of CrATP6. The ability to express a nucleus-encoded version of a mammalian mtDNA-encoded protein may provide a way to import other highly hydrophobic proteins into mitochondria and could serve as the basis for a gene therapy approach to treat human mitochondrial diseases.     

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.     

Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse

Genetic alterations of FKHR (FOXO1), AF6q21 (FOXO2), and AFX (FOXO4), closely related members of the forkhead family of DNA binding proteins, in human cancers has suggested they play a role in the regulation of cellular differentiation or proliferation. In order to elucidate the function of this gene subfamily during mammalian development, we have identified and characterized three novel mouse genes; Fkhr1 (Foxo1), Fkhr2 (Foxo3), and Afxh (Foxo4), which are closely related to the human FKHR (Foxo1), AF6q21 (FOXO2), and AFX (FOXO4) genes, respectively. The genes are each expressed both during development and in the adult with distinct patterns ranging from ubiquitous [Fkhr2 (Foxo3)] to tissue-specific [Afxh (Foxo4)]. Selection of high-affinity DNA-binding sites from a pool of degenerate oligonucleotides demonstrated that the proteins encoded by these genes recognize a core sequence [(T/A) (A/T) A A C A] similar to that recognized by other forkhead domain-containing proteins. We have also identified additional FKHR-related genes expressed during development in both the chick and zebrafish. Further characterization will provide insight into the roles of members of the FKHR subfamily of forkhead-related genes during both normal and neoplastic development.     

The solution structure of the forkhead box‐O DNA binding domain of Brugia malayi DAF‐16a

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF‐16a, a putative ortholog of Caenorhabditis elegans DAF‐16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF‐16 is involved in the insulin/IGF‐I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF‐16a is a member of the FOXO family of forkhead proteins. Proteins 2014; 82:3490–3496. © 2014 Wiley Periodicals, Inc.     

Biochemical characteristics of Escherichia coli ATP synthase with insulin peptide a fused to the globular part of the γ-subunit

The codon 5383-5385 (CCG) in the atpC gene of the unc operon of Escherichia coli cells was replaced with the sequence encoding peptide A of human insulin. The foreign protein fused to the middle part of the gamma-subunit of ATP synthase affects neither biosynthesis of the chimeric polypeptide nor the integration of the EF(0) x F(1) enzyme into the membranes of the E. coli cells. The inserted peptide A does not inhibit the process of oxidative phosphorylation. The ATPase activity of the mutant EF(0) x F(1) enzyme was equal to that of the wild-type enzyme and was regulated by modifiers in the similar way, suggesting that the space in the stalk area of F(0)/F(1) interaction is enough for the introduction of an additional oligopeptide without changing catalytic properties of the ATP synthase.     

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation

Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".     

Cloning of the cDNA for the Human ATP Synthase OSCP Subunit (ATP50) by Exon Trapping and Mapping to Chromosome 21q22.1-q22.2

Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP50 gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. (Nature 359:380, 1992) YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP50 subunit is a key structural component of the stalk of the mitochondrial respiratory chain F₁F₀-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21).     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

Sequence analysis of the wheat mitochondrial atp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins

The nucleotide sequence of the wheat mitochondrial gene for subunit 6 (atp6) of the F1F0 ATPase complex has been determined. Unlike bacterial, chloroplast or animal/fungal mitochondrial atp6 counterparts, which encode proteins of about 230-270 amino acids, the wheat mitochondrial atp6 homologue comprises the latter part of an open reading frame (ORF) of 386 codons. The ATP6 protein may therefore by synthesized with a long N-terminal presequence. This is supported by the finding that the ORF is preceded by a conserved sequence block closely related to ones preceding several other actively transcribed wheat mitochondrial protein-coding genes. The fused upstream ORF is similar in length, but unrelated in sequence, to those preceding the maize and tobacco mitochondrial atp6 genes. In wheat, the atp6 gene is located on a recombinationally active repeated DNA element, whose length of 1.4 kb corresponds approximately to that of the atp6 mRNA. A comparison of the wheat and maize ATP6 sequences reveals unexpectedly high divergence in the region corresponding to the mature N-terminal domain and may reflect mitochondrial DNA rearrangements during atp6 gene evolution in monocotyledonous plants.     

NAMPT pathway is involved in the FOXO3a-mediated regulation of GADD45A expression

Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD(+). Despite the pivotal role of NAD(+) in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD(+) level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD(+) levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD(+)-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.     

Calcium-binding ATPase inhibitor protein of bovine heart mitochondria. Role in ATP synthesis and effect of Ca2+

Submitochondrial particles (A particles) and phosphorylating electron-transport particles (ETPH) were prepared from bovine heart mitochondria. The A particles either were supplemented with or were depleted of the mitochondrial calcium-binding ATPase inhibitor protein (CaBI). The CaBI-depleted A particles still retained the Pullman-Monroy ATPase inhibitor protein (PMI), and the other particles all contained both CaBI and PMI. ATP synthase and ATPase activities of the particles were measured in similar reaction mixtures by luminescence of firefly luciferin-luciferase. Succinate was the respiratory substrate, and the adenylate kinase inhibitor P1, P5-di(adenosine-5') pentaphosphate was obligatory. The ATP synthase activity of CaBI-depleted A particles was 30-40% of that of the A and ETPH particles, and its ATPase activity was 7-8 times greater. Reconstitution of the CaBI-depleted A particles with CaBI restored the original ATP synthase and ATPase activities. ATP synthase activity rose about 1.7-fold when A particles were supplemented with additional CaBI and ATPase activity dropped to 9% of the original. Varying Ca2+ levels had little or no effect on the ATP synthase and ATPase activities of the CaBI-depleted A particles. In contrast, ATP synthase activity of the other particles was decreased by as much as 70% at the optimal Ca2+ concentration of 1 microM, and the ATPase activity of the A and EPTH particles rose concomitantly by 7-8-fold. The ATP synthase and ATPase activities of all the particles in microM Ca2+ became like those of the CaBI-depleted A particles. These changes were reversible; normal activities were restored as Ca2+ concentrations were raised above 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)