Skip to main content
Genetics logoLink to Genetics
. 2000 Oct;156(2):607–615. doi: 10.1093/genetics/156.2.607

Respiratory chain complex I is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly.

M Duarte 1, A Videira 1
PMCID: PMC1461281  PMID: 11014810

Abstract

We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21. 3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.

Full Text

The Full Text of this article is available as a PDF (275.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almeida T., Duarte M., Melo A. M., Videira A. The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity. Eur J Biochem. 1999 Oct 1;265(1):86–93. doi: 10.1046/j.1432-1327.1999.00668.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Chevallet M., Dupuis A., Lunardi J., van Belzen R., Albracht S. P., Issartel J. P. The NuoI subunit of the Rhodobacter capsulatus respiratory Complex I (equivalent to the bovine TYKY subunit) is required for proper assembly of the membraneous and peripheral domains of the enzyme. Eur J Biochem. 1997 Dec 1;250(2):451–458. doi: 10.1111/j.1432-1033.1997.0451a.x. [DOI] [PubMed] [Google Scholar]
  6. Duarte M., Finel M., Videira A. Primary structure of a ferredoxin-like iron-sulfur subunit of complex I from Neurospora crassa. Biochim Biophys Acta. 1996 Jul 31;1275(3):151–153. doi: 10.1016/0005-2728(96)00033-3. [DOI] [PubMed] [Google Scholar]
  7. Duarte M., Mota N., Pinto L., Videira A. Inactivation of the gene coding for the 30.4-kDa subunit of respiratory chain NADH dehydrogenase: is the enzyme essential for Neurospora? Mol Gen Genet. 1998 Feb;257(3):368–375. doi: 10.1007/s004380050659. [DOI] [PubMed] [Google Scholar]
  8. Duarte M., Schulte U., Videira A. Identification of the TYKY homologous subunit of complex I from Neurospora crassa. Biochim Biophys Acta. 1997 Dec 15;1322(2-3):237–241. doi: 10.1016/s0005-2728(97)00084-4. [DOI] [PubMed] [Google Scholar]
  9. Dupuis A., Chevallet M., Darrouzet E., Duborjal H., Lunardi J., Issartel J. P. The complex I from Rhodobacter capsulatus. Biochim Biophys Acta. 1998 May 6;1364(2):147–165. doi: 10.1016/s0005-2728(98)00025-5. [DOI] [PubMed] [Google Scholar]
  10. Dupuis A., Skehel J. M., Walker J. E. A homologue of a nuclear-coded iron-sulfur protein subunit of bovine mitochondrial complex I is encoded in chloroplast genomes. Biochemistry. 1991 Mar 19;30(11):2954–2960. doi: 10.1021/bi00225a032. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Ferreira A. V., An Z., Metzenberg R. L., Glass N. L. Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa. Genetics. 1998 Mar;148(3):1069–1079. doi: 10.1093/genetics/148.3.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferreirinha F., Duarte M., Melo A. M., Videira A. Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa. Biochem J. 1999 Sep 15;342(Pt 3):551–554. [PMC free article] [PubMed] [Google Scholar]
  14. Finel M. Genetic inactivation of the H(+)-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli. FEBS Lett. 1996 Sep 9;393(1):81–85. doi: 10.1016/0014-5793(96)00831-9. [DOI] [PubMed] [Google Scholar]
  15. Finel M., Majander A. S., Tyynelä J., De Jong A. M., Albracht S. P., Wikström M. Isolation and characterisation of subcomplexes of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Eur J Biochem. 1994 Nov 15;226(1):237–242. doi: 10.1111/j.1432-1033.1994.tb20046.x. [DOI] [PubMed] [Google Scholar]
  16. Fontaine E., Eriksson O., Ichas F., Bernardi P. Regulation of the permeability transition pore in skeletal muscle mitochondria. Modulation By electron flow through the respiratory chain complex i. J Biol Chem. 1998 May 15;273(20):12662–12668. doi: 10.1074/jbc.273.20.12662. [DOI] [PubMed] [Google Scholar]
  17. Friedrich T. The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. Biochim Biophys Acta. 1998 May 6;1364(2):134–146. doi: 10.1016/s0005-2728(98)00024-3. [DOI] [PubMed] [Google Scholar]
  18. Grohmann L., Rasmusson A. G., Heiser V., Thieck O., Brennicke A. The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria. Plant J. 1996 Nov;10(5):793–803. doi: 10.1046/j.1365-313x.1996.10050793.x. [DOI] [PubMed] [Google Scholar]
  19. Gutierres S., Sabar M., Lelandais C., Chetrit P., Diolez P., Degand H., Boutry M., Vedel F., de Kouchkovsky Y., De Paepe R. Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3436–3441. doi: 10.1073/pnas.94.7.3436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harkness T. A., Rothery R. A., Weiner J. H., Werner S., Azevedo J. E., Videira A., Nargang F. E. Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I in Neurospora crassa by repeat induced point mutation (RIP). Curr Genet. 1995 Mar;27(4):339–350. doi: 10.1007/BF00352103. [DOI] [PubMed] [Google Scholar]
  21. Hegde R. The 24-kDa subunit of the bovine mitochondrial NADH:ubiquinone oxidoreductase is a G protein. Biochem Biophys Res Commun. 1998 Mar 27;244(3):620–629. doi: 10.1006/bbrc.1998.8304. [DOI] [PubMed] [Google Scholar]
  22. Heiser V., Brennicke A., Grohmann L. The plant mitochondrial 22 kDa (PSST) subunit of respiratory chain complex I is encoded by a nuclear gene with enhanced transcript levels in flowers. Plant Mol Biol. 1996 Sep;31(6):1195–1204. doi: 10.1007/BF00040836. [DOI] [PubMed] [Google Scholar]
  23. Higuchi M., Proske R. J., Yeh E. T. Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis. Oncogene. 1998 Nov 12;17(19):2515–2524. doi: 10.1038/sj.onc.1202485. [DOI] [PubMed] [Google Scholar]
  24. Landschütze V., Willmitzer L., Müller-Röber B. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers. EMBO J. 1995 Feb 15;14(4):660–666. doi: 10.1002/j.1460-2075.1995.tb07044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Laval-Favre K., Letouvet-Pawlak B., Friedrich T., Alexandre J., Guespin-Michel J. F. A gene involved in both protein secretion during growth and starvation-induced development encodes a subunit of the NADH:ubiquinone oxidoreductase in Myxococcus xanthus. Mol Microbiol. 1997 Mar;23(5):1043–1052. doi: 10.1046/j.1365-2958.1997.2901655.x. [DOI] [PubMed] [Google Scholar]
  26. Laval-Favre K., Letouvet-Pawlak B., Friedrich T., Alexandre J., Guespin-Michel J. F. A gene involved in both protein secretion during growth and starvation-induced development encodes a subunit of the NADH:ubiquinone oxidoreductase in Myxococcus xanthus. Mol Microbiol. 1997 Mar;23(5):1043–1052. doi: 10.1046/j.1365-2958.1997.2901655.x. [DOI] [PubMed] [Google Scholar]
  27. Leif H., Sled V. D., Ohnishi T., Weiss H., Friedrich T. Isolation and characterization of the proton-translocating NADH: ubiquinone oxidoreductase from Escherichia coli. Eur J Biochem. 1995 Jun 1;230(2):538–548. doi: 10.1111/j.1432-1033.1995.tb20594.x. [DOI] [PubMed] [Google Scholar]
  28. Loeffen J., Smeitink J., Triepels R., Smeets R., Schuelke M., Sengers R., Trijbels F., Hamel B., Mullaart R., van den Heuvel L. The first nuclear-encoded complex I mutation in a patient with Leigh syndrome. Am J Hum Genet. 1998 Dec;63(6):1598–1608. doi: 10.1086/302154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marienfeld J. R., Newton K. J. The maize NCS2 abnormal growth mutant has a chimeric nad4-nad7 mitochondrial gene and is associated with reduced complex I function. Genetics. 1994 Nov;138(3):855–863. doi: 10.1093/genetics/138.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Nelson M. A., Kang S., Braun E. L., Crawford M. E., Dolan P. L., Leonard P. M., Mitchell J., Armijo A. M., Bean L., Blueyes E. Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa. Fungal Genet Biol. 1997 Jun;21(3):348–363. doi: 10.1006/fgbi.1997.0986. [DOI] [PubMed] [Google Scholar]
  32. Nowrousian M., Masloff S., Pöggeler S., Kück U. Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity. Mol Cell Biol. 1999 Jan;19(1):450–460. doi: 10.1128/mcb.19.1.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Papa S., Sardanelli A. M., Cocco T., Speranza F., Scacco S. C., Technikova-Dobrova Z. The nuclear-encoded 18 kDa (IP) AQDQ subunit of bovine heart complex I is phosphorylated by the mitochondrial cAMP-dependent protein kinase. FEBS Lett. 1996 Feb 5;379(3):299–301. doi: 10.1016/0014-5793(95)01532-9. [DOI] [PubMed] [Google Scholar]
  34. Pla M., Mathieu C., De Paepe R., Chétrit P., Vedel F. Deletion of the last two exons of the mitochondrial nad7 gene results in lack of the NAD7 polypeptide in a Nicotiana sylvestris CMS mutant. Mol Gen Genet. 1995 Jul 22;248(1):79–88. doi: 10.1007/BF02456616. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Procaccio V., Depetris D., Soularue P., Mattei M. G., Lunardi J., Issartel J. P. cDNA sequence and chromosomal localization of the NDUFS8 human gene coding for the 23 kDa subunit of the mitochondrial complex I. Biochim Biophys Acta. 1997 Mar 20;1351(1-2):37–41. doi: 10.1016/s0167-4781(97)00020-1. [DOI] [PubMed] [Google Scholar]
  37. Schneider R., Massow M., Lisowsky T., Weiss H. Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein. Curr Genet. 1995 Dec;29(1):10–17. doi: 10.1007/BF00313188. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. 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]
  44. 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]
  45. 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]
  46. Wallace D. C. Diseases of the mitochondrial DNA. Annu Rev Biochem. 1992;61:1175–1212. doi: 10.1146/annurev.bi.61.070192.005523. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Weiss H., von Jagow G., Klingenberg M., Bücher T. Characterization of Neurospora crassa mitochondria prepared with a grind-mill. Eur J Biochem. 1970 May 1;14(1):75–82. doi: 10.1111/j.1432-1033.1970.tb00263.x. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Xu X., Matsuno-Yagi A., Yagi T. DNA sequencing of the seven remaining structural genes of the gene cluster encoding the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans. Biochemistry. 1993 Jan 26;32(3):968–981. doi: 10.1021/bi00054a030. [DOI] [PubMed] [Google Scholar]
  51. Yagi T., Yano T., Di Bernardo S., Matsuno-Yagi A. Procaryotic complex I (NDH-1), an overview. Biochim Biophys Acta. 1998 May 6;1364(2):125–133. doi: 10.1016/s0005-2728(98)00023-1. [DOI] [PubMed] [Google Scholar]
  52. Yu J., Vassiliev I. R., Jung Y. S., Golbeck J. H., McIntosh L. Strains of synechocystis sp. PCC 6803 with altered PsaC. I. Mutations incorporated in the cysteine ligands of the two [4Fe-4S] clusters FA and FB of photosystem I. J Biol Chem. 1997 Mar 21;272(12):8032–8039. doi: 10.1074/jbc.272.12.8032. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. da Silva M. V., Alves P. C., Duarte M., Mota N., Lobo-da-Cunha A., Harkness T. A., Nargang F. E., Videira A. Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH: ubiquinone reductase of Neurospora mitochondria. Mol Gen Genet. 1996 Aug 27;252(1-2):177–183. doi: 10.1007/BF02173218. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES