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. 1995 Mar;139(3):1211–1221. doi: 10.1093/genetics/139.3.1211

Inactivation of Genes Encoding Subunits of the Peripheral and Membrane Arms of Neurospora Mitochondrial Complex I and Effects on Enzyme Assembly

M Duarte 1, R Sousa 1, A Videira 1
PMCID: PMC1206451  PMID: 7768434

Abstract

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.

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Selected References

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  1. Alves P. C., Videira A. Disruption of the gene coding for the 21.3-kDa subunit of the peripheral arm of complex I from Neurospora crassa. J Biol Chem. 1994 Mar 11;269(10):7777–7784. [PubMed] [Google Scholar]
  2. Azevedo J. E., Duarte M., Belo J. A., Werner S., Videira A. Complementary DNA sequences of the 24 kDa and 21 kDa subunits of complex I from Neurospora. Biochim Biophys Acta. 1994 Nov 1;1188(1-2):159–161. doi: 10.1016/0005-2728(94)90034-5. [DOI] [PubMed] [Google Scholar]
  3. Azevedo J. E., Nehls U., Eckerskorn C., Heinrich H., Rothe H., Weiss H., Werner S. Primary structure and mitochondrial import in vitro of the 20.9 kDa subunit of complex I from Neurospora crassa. Biochem J. 1992 Nov 15;288(Pt 1):29–34. doi: 10.1042/bj2880029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fearnley I. M., Walker J. E. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim Biophys Acta. 1992 Dec 7;1140(2):105–134. doi: 10.1016/0005-2728(92)90001-i. [DOI] [PubMed] [Google Scholar]
  5. Fecke W., Sled V. D., Ohnishi T., Weiss H. Disruption of the gene encoding the NADH-binding subunit of NADH: ubiquinone oxidoreductase in Neurospora crassa. Formation of a partially assembled enzyme without FMN and the iron-sulphur cluster N-3. Eur J Biochem. 1994 Mar 1;220(2):551–558. doi: 10.1111/j.1432-1033.1994.tb18655.x. [DOI] [PubMed] [Google Scholar]
  6. Harkness T. A., Metzenberg R. L., Schneider H., Lill R., Neupert W., Nargang F. E. Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP". Genetics. 1994 Jan;136(1):107–118. doi: 10.1093/genetics/136.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hatefi Y. Preparation and properties of NADH: ubiquinone oxidoreductase (complexI), EC 1.6.5.3. Methods Enzymol. 1978;53:11–14. doi: 10.1016/s0076-6879(78)53006-1. [DOI] [PubMed] [Google Scholar]
  8. Hofhaus G., Attardi G. 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. EMBO J. 1993 Aug;12(8):3043–3048. doi: 10.1002/j.1460-2075.1993.tb05973.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hofhaus G., Weiss H., Leonard K. Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I). J Mol Biol. 1991 Oct 5;221(3):1027–1043. doi: 10.1016/0022-2836(91)80190-6. [DOI] [PubMed] [Google Scholar]
  10. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nehls U., Friedrich T., Schmiede A., Ohnishi T., Weiss H. Characterization of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption. J Mol Biol. 1992 Oct 20;227(4):1032–1042. doi: 10.1016/0022-2836(92)90519-p. [DOI] [PubMed] [Google Scholar]
  12. Preis D., Weidner U., Conzen C., Azevedo J. E., Nehls U., Röhlen D., van der Pas J., Sackmann U., Schneider R., Werner S. Primary structures of two subunits of NADH: ubiquinone reductase from Neurospora crassa concerned with NADH-oxidation. Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus. Biochim Biophys Acta. 1991 Aug 27;1090(1):133–138. doi: 10.1016/0167-4781(91)90049-r. [DOI] [PubMed] [Google Scholar]
  13. Prömper C., Schneider R., Weiss H. The role of the proton-pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger. Eur J Biochem. 1993 Aug 15;216(1):223–230. doi: 10.1111/j.1432-1033.1993.tb18136.x. [DOI] [PubMed] [Google Scholar]
  14. Schmidt M., Friedrich T., Wallrath J., Ohnishi T., Weiss H. Accumulation of the pre-assembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa. FEBS Lett. 1992 Nov 16;313(1):8–11. doi: 10.1016/0014-5793(92)81172-i. [DOI] [PubMed] [Google Scholar]
  15. Selker E. U., Garrett P. W. DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6870–6874. doi: 10.1073/pnas.85.18.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Selker E. U. Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet. 1990;24:579–613. doi: 10.1146/annurev.ge.24.120190.003051. [DOI] [PubMed] [Google Scholar]
  17. Smith D. A. A mutant affecting meiosis in Neurospora. Genetics. 1975 May;80(1):125–133. doi: 10.1093/genetics/80.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  19. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tuschen G., Sackmann U., Nehls U., Haiker H., Buse G., Weiss H. Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits. J Mol Biol. 1990 Jun 20;213(4):845–857. doi: 10.1016/S0022-2836(05)80268-2. [DOI] [PubMed] [Google Scholar]
  21. Van der Pas J. C., Röhlen D. A., Weidner U., Weiss H. Primary structure of the nuclear-encoded 29.9 kDa subunit of NADH: ubiquinone reductase from Neurospora crassa mitochondria. Biochim Biophys Acta. 1991 Jul 23;1089(3):389–390. doi: 10.1016/0167-4781(91)90181-k. [DOI] [PubMed] [Google Scholar]
  22. Videira A., Azevedo J. E., Werner S., Cabral P. The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) from Neurospora crassa: cDNA cloning and chromosomal mapping of the gene. Biochem J. 1993 May 1;291(Pt 3):729–732. doi: 10.1042/bj2910729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Videira A., Tropschug M., Werner S. Primary structure and expression of a nuclear-coded subunit of complex I homologous to proteins specified by the chloroplast genome. Biochem Biophys Res Commun. 1990 Sep 28;171(3):1168–1174. doi: 10.1016/0006-291x(90)90807-y. [DOI] [PubMed] [Google Scholar]
  24. Videira A., Tropschug M., Werner S. Primary structure, in vitro expression and import into mitochondria of a 29/21-kDa subunit of complex I from Neurospora crassa. Biochem Biophys Res Commun. 1990 Jan 15;166(1):280–285. doi: 10.1016/0006-291x(90)91942-l. [DOI] [PubMed] [Google Scholar]
  25. Videira A., Tropschüg M., Wachter E., Schneider H., Werner S. Molecular cloning of subunits of complex I from Neurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide. J Biol Chem. 1990 Aug 5;265(22):13060–13065. [PubMed] [Google Scholar]
  26. Videira A., Werner S. Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa. Eur J Biochem. 1989 May 1;181(2):493–502. doi: 10.1111/j.1432-1033.1989.tb14751.x. [DOI] [PubMed] [Google Scholar]
  27. Vollmer S. J., Yanofsky C. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Walker J. E., Arizmendi J. M., Dupuis A., Fearnley I. M., Finel M., Medd S. M., Pilkington S. J., Runswick M. J., Skehel J. M. Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction. J Mol Biol. 1992 Aug 20;226(4):1051–1072. doi: 10.1016/0022-2836(92)91052-q. [DOI] [PubMed] [Google Scholar]
  29. Walker J. E. The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys. 1992 Aug;25(3):253–324. doi: 10.1017/s003358350000425x. [DOI] [PubMed] [Google Scholar]
  30. Wallace D. C. Mitochondrial diseases: genotype versus phenotype. Trends Genet. 1993 Apr;9(4):128–133. doi: 10.1016/0168-9525(93)90207-x. [DOI] [PubMed] [Google Scholar]
  31. Weidner U., Geier S., Ptock A., Friedrich T., Leif H., Weiss H. The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol. 1993 Sep 5;233(1):109–122. doi: 10.1006/jmbi.1993.1488. [DOI] [PubMed] [Google Scholar]
  32. Weiss H., Friedrich T., Hofhaus G., Preis D. The respiratory-chain NADH dehydrogenase (complex I) of mitochondria. Eur J Biochem. 1991 May 8;197(3):563–576. doi: 10.1111/j.1432-1033.1991.tb15945.x. [DOI] [PubMed] [Google Scholar]
  33. Werner S. Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa. Eur J Biochem. 1977 Sep 15;79(1):103–110. doi: 10.1111/j.1432-1033.1977.tb11788.x. [DOI] [PubMed] [Google Scholar]
  34. Werner S., Sebald W. Immunological techniques for studies on the biogenesis of mitochondrial membrane proteins. Methods Biochem Anal. 1981;27:109–170. doi: 10.1002/9780470110478.ch3. [DOI] [PubMed] [Google Scholar]
  35. Zauner R., Christner J., Jung G., Borchart U., Machleidt W., Videira A., Werner S. Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA. Eur J Biochem. 1985 Aug 1;150(3):447–454. doi: 10.1111/j.1432-1033.1985.tb09042.x. [DOI] [PubMed] [Google Scholar]

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