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. 1985 Jan;4(1):179–184. doi: 10.1002/j.1460-2075.1985.tb02334.x

The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

W Dowhan, C R Bibus, G Schatz
PMCID: PMC554168  PMID: 2990892

Abstract

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.

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Selected References

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

  1. Andreadis A., Hsu Y. P., Hermodson M., Kohlhaw G., Schimmel P. Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product. J Biol Chem. 1984 Jul 10;259(13):8059–8062. [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  4. Casey R. P., Thelen M., Azzi A. Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity. J Biol Chem. 1980 May 10;255(9):3994–4000. [PubMed] [Google Scholar]
  5. Cumsky M. G., McEwen J. E., Ko C., Poyton R. O. Nuclear genes for mitochondrial proteins. Identification and isolation of a structural gene for subunit V of yeast cytochrome c oxidase. J Biol Chem. 1983 Nov 25;258(22):13418–13421. [PubMed] [Google Scholar]
  6. Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028–13033. [PubMed] [Google Scholar]
  7. Douglas M. G., Butow R. A. Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1083–1086. doi: 10.1073/pnas.73.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  9. Ebner E., Mason T. L., Schatz G. Mitochondrial assembly in respiration-deficient mutants of Saccharomyces cerevisiae. II. Effect of nuclear and extrachromosomal mutations on the formation of cytochrome c oxidase. J Biol Chem. 1973 Aug 10;248(15):5369–5378. [PubMed] [Google Scholar]
  10. Fee J. A., Choc M. G., Findling K. L., Lorence R., Yoshida T. Properties of a copper-containing cytochrome c1aa3 complex: a terminal oxidase of the extreme thermophile Thermus thermophilus HB8. Proc Natl Acad Sci U S A. 1980 Jan;77(1):147–151. doi: 10.1073/pnas.77.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kadenbach B., Hartmann R., Glanville R., Buse G. Tissue-specific genes code for polypeptide VIa of bovine liver and heart cytochrome c oxidase. FEBS Lett. 1982 Feb 22;138(2):236–238. doi: 10.1016/0014-5793(82)80450-x. [DOI] [PubMed] [Google Scholar]
  13. Kadenbach B., Jarausch J., Hartmann R., Merle P. Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure. Anal Biochem. 1983 Mar;129(2):517–521. doi: 10.1016/0003-2697(83)90586-9. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Ludwig B., Schatz G. A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans. Proc Natl Acad Sci U S A. 1980 Jan;77(1):196–200. doi: 10.1073/pnas.77.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lustig A., Padmanaban G., Rabinowitz M. Regulation of the nuclear-coded peptides of yeast cytochrome c oxidase. Biochemistry. 1982 Jan 19;21(2):309–316. doi: 10.1021/bi00531a017. [DOI] [PubMed] [Google Scholar]
  17. Maarse A. C., Van Loon A. P., Riezman H., Gregor I., Schatz G., Grivell L. A. Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein. EMBO J. 1984 Dec 1;3(12):2831–2837. doi: 10.1002/j.1460-2075.1984.tb02216.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  19. Mason T. L., Poyton R. O., Wharton D. C., Schatz G. Cytochrome c oxidase from bakers' yeast. I. Isolation and properties. J Biol Chem. 1973 Feb 25;248(4):1346–1354. [PubMed] [Google Scholar]
  20. Merle P., Kadenbach B. The subunit composition of mammalian cytochrome c oxidase. Eur J Biochem. 1980 Apr;105(3):499–507. doi: 10.1111/j.1432-1033.1980.tb04525.x. [DOI] [PubMed] [Google Scholar]
  21. Navarrete R., Serrano R. Solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents. Biochim Biophys Acta. 1983 Mar 9;728(3):403–408. doi: 10.1016/0005-2736(83)90512-6. [DOI] [PubMed] [Google Scholar]
  22. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Power S. D., Lochrie M. A., Patterson T. E., Poyton R. O. The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane. J Biol Chem. 1984 May 25;259(10):6571–6574. [PubMed] [Google Scholar]
  24. Power S. D., Lochrie M. A., Sevarino K. A., Patterson T. E., Poyton R. O. The nuclear-coded subunits of yeast cytochrome c oxidase. I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reversed phase high performance liquid chromatography. J Biol Chem. 1984 May 25;259(10):6564–6570. [PubMed] [Google Scholar]
  25. Poyton R. O., Schatz G. Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity. J Biol Chem. 1975 Jan 25;250(2):762–766. [PubMed] [Google Scholar]
  26. Riezman H., Hase T., van Loon A. P., Grivell L. A., Suda K., Schatz G. Import of proteins into mitochondria: a 70 kilodalton outer membrane protein with a large carboxy-terminal deletion is still transported to the outer membrane. EMBO J. 1983;2(12):2161–2168. doi: 10.1002/j.1460-2075.1983.tb01717.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Riezman H., Hay R., Gasser S., Daum G., Schneider G., Witte C., Schatz G. The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. EMBO J. 1983;2(7):1105–1111. doi: 10.1002/j.1460-2075.1983.tb01553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Steffens G. C., Buse G., Oppliger W., Ludwig B. Sequence homology of bacterial and mitochondrial cytochrome c oxidases. Partial sequence data of cytochrome c oxidase from Paracoccus denitrificans. Biochem Biophys Res Commun. 1983 Oct 14;116(1):335–340. doi: 10.1016/0006-291x(83)90419-9. [DOI] [PubMed] [Google Scholar]
  31. Steffens G. J., Buse G. Studies on cytochrome c oxidase, IV[1--3]. Primary structure and function of subunit II. Hoppe Seylers Z Physiol Chem. 1979 Apr;360(4):613–619. [PubMed] [Google Scholar]
  32. Suissa M. Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983 Sep;133(2):511–514. doi: 10.1016/0003-2697(83)90117-3. [DOI] [PubMed] [Google Scholar]
  33. Tzagoloff A., Akai A., Needleman R. B. Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes. J Biol Chem. 1975 Oct 25;250(20):8228–8235. [PubMed] [Google Scholar]
  34. Winter D. B., Bruyninckx W. J., Foulke F. G., Grinich N. P., Mason H. S. Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase. J Biol Chem. 1980 Dec 10;255(23):11408–11414. [PubMed] [Google Scholar]

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