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Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

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Abstract

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.

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References

  • Alves PC, Videira A (1994) Disruption of the gene coding for the 21.3-kDa subunit of the peripheral arm of complex I fromNeurospora crassa. J Biol Chem 269:7777–7784

    Google Scholar 

  • Ausubel FA, Brent R, Kingston RE, Moore DD, Seidmann JG, Smith JA, Struhl K (1992) Current protocols in molecular biology. Greene and Wiley Interscience, New York

    Google Scholar 

  • Azevedo JE, Videira A (1994) Characterization of a membrane fragment of respiratory chain complex I fromNeurospora crassa. Insights on the topology of the ubiquinone-binding site. Int J Biochem 26:505–510

    Google Scholar 

  • Azevedo JE, Nehls U, Eckerskorn C, Heinrich H, Rothe H, Weiss H, Werner S (1992) Primary structure and mitochondrial import in vitro of the 20.9 kDa subunit of complex I fromNeurospora crassa. Biochem J 288:29–34

    Google Scholar 

  • Bertrand H, Pittenger TH (1969) Cytoplasmic mutants selected from continuously growing cultures ofNeurospora crassa. Genetics 61:643–659

    Google Scholar 

  • Bertrand H, Nargang FE, Collins RA, Zagozeski C (1977) Nuclear cytochrome-deficient mutants ofNeurospora crassa: isolation, characterization and genetic mapping. Mol Gen Genet 153:247–257

    Google Scholar 

  • Blake MS, Johnston KH, Russel-Jones GJ, Gotschlich EC (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 136:175–179

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques forNeurospora crassa. Methods Enzymol 17A:79–143

    Google Scholar 

  • Duarte M, Sousa R, Videira A (1995) Inactivation of genes encoding subunits of the peripheral and membrane arms of Neurospora mitochondrial complex I and effects on enzyme assembly. Genetics 139:1211–1221

    Google Scholar 

  • Dupuis A, Skehel JM, Walker JE (1991) NADH:ubiquinone oxidoreductase from bovine mitochondria: cDNA sequence of a 19 kDa cysteine-rich subunit. Biochem J 277:11–15

    Google Scholar 

  • Fearnley IM, Walker JE (1992) Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim Biophys Acta 1140:105–134

    Google Scholar 

  • Filser M, Werner S (1988) Pethidine analogs, a novel class of potent inhibitors of mitochondrial NADH:ubiquinone reductase. Biochem Pharmacol 37:2551–2558

    Google Scholar 

  • Finel M, Majander AS, Tyynela J, de Jong AM, Albracht SPJ, Wikstroem M (1994) Isolation and characterisation of subcomplexes of the mitochondrial NADH:ubiquinone oxidoreductase (Complex I). Eur J Biochem 226:237–242

    Google Scholar 

  • Harkness TAA, Rothery RA, Weiner JH, Werner S, Azevedo JE, Videira A, Nargang FE (1995) Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I inNeurospora crassa by repeat induced point mutation (RIP). Curr Genet 27:339–350

    Google Scholar 

  • Hatefi Y (1978) Preparation and properties of NADH:ubiquinone oxidoreductase (Complex I, EC 1.6.5.3). Methods Enzymol 53:11–14

    Google Scholar 

  • Hatefi Y (1985) The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 59:1015–1069

    Google Scholar 

  • Lee SB, Milgroom MG, Taylor JW (1988) A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet Newslett 35:23–24

    Google Scholar 

  • Majander A, Finel M, Wikstroem M (1994) Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (complex I). J Biol Chem 269:21037–21042

    Google Scholar 

  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056

    Google Scholar 

  • Metzenberg RL, Grotelueschen JS (1992) Disruption of essential genes inNeurospora by RIP. Fungal Genet Newslett 39:37–49

    Google Scholar 

  • Metzenberg RL, Grotelueschen JS (1995) Restriction polymorphism maps ofNeurospora crassa: update. Fungal Genet Newslett 42:82–90

    Google Scholar 

  • Metzenberg RL, Stevens JN, Selker EU, Morzycka-Wroblewska E (1984) A method for finding the genetic map position of cloned DNA fragments. Fungal Genet Newslett 31:35–39

    Google Scholar 

  • Nehls U, Friedrich T, Schmiede A, Ohnishi T, Weiss H (1992) Characterization of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated inNeurospora mitochondria by gene disruption. J Mol Biol 227:1032–1042

    Google Scholar 

  • Osumi M, Miwa N, Teranishi Y, Tanaka A, Fukui S (1974) Ultrastructure ofCandida yeast grown onn-alkanes. Appearance of microbodies and its relationship to high catalase activity. Arch Microbiol 99:181–201

    Google Scholar 

  • Perkins DD (1986) Hints and precautions for the care, feeding and breeding ofNeurospora. Fungal Genet Newslett 35:35–41

    Google Scholar 

  • Preis D, van der Pas JC, Nehls U, Roehlen D, Sackmann U, Jahnke U, Weiss H (1990) The 49 kDa subunit of NADH:ubiquinone reductase fromNeurospora crassa mitochondria: primary structure of the gene and the protein. Curr Genetics 18:59–64

    Google Scholar 

  • Roberts TH, Fredlund KM, Moller IM (1995) Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria. FEBS Lett 373:307–309

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schulte U, Fecke W, Kruell C, Nehls U, Schmiede A, Schneider R, Ohnishi T, Weiss H (1994) In vivo dissection of the mitochondrial respiratory NADH:ubiquinone reductase (complex I). Biochim Biophys Acta 1187:121–124

    Google Scholar 

  • Selker EU (1990) Premeiotic instability of repeated sequences inNeurospora crassa. Annu Rev Genet 24:579–613

    Google Scholar 

  • Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation inNeurospora crassa. Proc Natl Acad Sci 85:6870–6874

    Google Scholar 

  • Staben C, Jensen B, Singer M, Pollock J, Schechtman M, Kinsey J, Selker EU (1989) Use of a bacterial hygromycin B resistance gene as a dominant marker inNeurospora crassa transformation. Fungal Genet Newslett 36:79–81

    Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci 76:4350–4354

    Google Scholar 

  • Tuschen G, Sackmann U, Nehls U, Haiker H, Buse G, Weiss H (1990) Assembly of NADH:ubiquinone reductase (complex I) inNeurospora mitochondria: Independent pathways of nuclear-encoded and mitochondrially encoded subunits. J Mol Biol 213:845–857

    Google Scholar 

  • van der Pas JC, Roehlen DA, Weidner U, Weiss H (1991) Primary structure of the nuclear-encoded 29.9 kDa subunit of NADH: ubiquinone reductase fromNeurospora crassa mitochondria. Biochim Biophys Acta 1089:389–390

    Google Scholar 

  • Videira A, Werner S (1989) Assembly kinetics and identification of precursor proteins of complex I fromNeurospora crassa. Eur J Biochem 181:493–502

    Google Scholar 

  • Videira A, Tropschug M, Wachter E, Schneider H, Werner S (1990a) Molecular cloning of subunits of complex I fromNeurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide. J Biol Chem 265:13060–13065

    Google Scholar 

  • Videira A, Tropschug M, Werner S (1990b) Primary structure and expression of a nuclear-encoded subunit of complex I homologous to proteins specified by the chloroplast genome. Biochem Biophys Res Commun 171:1168–1174

    Google Scholar 

  • Videira A, Azevedo JE, Werner S, Cabral P (1993) The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) fromNeurospora crassa: cDNA cloning and chromosomal mapping of the gene. Biochem J 291:729–732

    Google Scholar 

  • Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes ofNeurospora crassa. Proc Natl Acad Sci 83:4869–4873

    Google Scholar 

  • Walker JE (1992) The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys 25:253–324

    Google Scholar 

  • Weidner U, Geier S, Ptock A, Friedrich T, Leif H, Weiss H (1993) The gene locus of the proton-translocating NADH:ubiquinone oxidoreductase inEscherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol 233:109–122

    Google Scholar 

  • Weiss H, von Jagow G, Klingenberg M, Buecher T (1970) Characterisation ofNeurospora crassa mitochondria prepared with a grind-mill. Eur J Biochem 14:75–82

    Google Scholar 

  • Weiss H, Friedrich T, Hofhaus G, Preis D (1991) The respiratory-chain NADH dehydrogenase (complex I) of mitochondria. Eur J Biochem 197:563–576

    Google Scholar 

  • Werner S (1977) Preparation of polypeptide subunits of cytochrome oxidase fromNeurospora crassa. Eur J Biochem 79:103–110

    Google Scholar 

  • Zauner R, Christner J, Jung G, Borchart U, Machleidt W, Videira A, Werner S (1985) Identification of the polypeptide encoded by theURF-1 gene ofNeurospora crassa mtDNA. Eur J Biochem 150:447–454

    Google Scholar 

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Authors and Affiliations

  1. Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Lg. Prof. Abel Salazar 2, 4050, Porto, Portugal

    M. V. da Silva, P. C. Alves, M. Duarte, N. Mota, A. Lobo-da-Cunha & A. Videira

  2. Unidade Multidisciplinar de Investigação Biomédica, Universidade do Porto, 4050, Porto, Portugal

    P. C. Alves & M. Duarte

  3. Instituto de Biologia Molecular e Celular, Universidade do Porto, 4050, Porto, Portugal

    A. Videira

  4. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, Canada

    T. A. A. Harkness & F. E. Nargang

Authors
  1. M. V. da Silva
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  2. P. C. Alves
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  3. M. Duarte
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  4. N. Mota
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  5. A. Lobo-da-Cunha
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  6. A. Videira
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  7. T. A. A. Harkness
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  8. F. E. Nargang
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Communicated by E. Cerdá-Olmedo

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da Silva, M.V., Alves, P.C., Duarte, M. et al. Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria. Molec. Gen. Genet. 252, 177–183 (1996). https://doi.org/10.1007/BF02173218

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  • DOI: https://doi.org/10.1007/BF02173218

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