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. 1984 Sep;3(9):2137–2143. doi: 10.1002/j.1460-2075.1984.tb02103.x

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

I Sadler, K Suda, G Schatz, F Kaudewitz, A Haid
PMCID: PMC557655  PMID: 6092058

Abstract

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.

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Selected References

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  1. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [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. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  5. Cryer D. R., Eccleshall R., Marmur J. Isolation of yeast DNA. Methods Cell Biol. 1975;12:39–44. doi: 10.1016/s0091-679x(08)60950-4. [DOI] [PubMed] [Google Scholar]
  6. Daum G., Gasser S. M., Schatz G. Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13075–13080. [PubMed] [Google Scholar]
  7. Gasser S. M., Daum G., Schatz G. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J Biol Chem. 1982 Nov 10;257(21):13034–13041. [PubMed] [Google Scholar]
  8. Gasser S. M. Import of polypeptides into isolated yeast mitochondria. Methods Enzymol. 1983;97:329–336. doi: 10.1016/0076-6879(83)97145-8. [DOI] [PubMed] [Google Scholar]
  9. Guarente L., Lalonde B., Gifford P., Alani E. Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell. 1984 Feb;36(2):503–511. doi: 10.1016/0092-8674(84)90243-5. [DOI] [PubMed] [Google Scholar]
  10. Guarente L., Mason T. Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell. 1983 Apr;32(4):1279–1286. doi: 10.1016/0092-8674(83)90309-4. [DOI] [PubMed] [Google Scholar]
  11. Haid A., Suissa M. Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:192–205. doi: 10.1016/s0076-6879(83)96017-2. [DOI] [PubMed] [Google Scholar]
  12. Harvey A., Yep B., Sellin D. Developmental achievement of trainable mentally retarded children. Folia Allergol (Roma) 1966 May-Jun;13(3):100–108. [PubMed] [Google Scholar]
  13. Hay R., Böhni P., Gasser S. How mitochondria import proteins. Biochim Biophys Acta. 1984 Jan 27;779(1):65–87. doi: 10.1016/0304-4157(84)90004-2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kaput J., Goltz S., Blobel G. Nucleotide sequence of the yeast nuclear gene for cytochrome c peroxidase precursor. Functional implications of the pre sequence for protein transport into mitochondria. J Biol Chem. 1982 Dec 25;257(24):15054–15058. [PubMed] [Google Scholar]
  16. Kedes L. H., Chang A. C., Houseman D., Cohen S. N. Isolation of histone genes from unfractionated sea urchin DNA by subculture cloning in E. coli. Nature. 1975 Jun 12;255(5509):533–538. doi: 10.1038/255533a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  19. Ludwig B., Suda K., Cerletti N. Cytochrome c1 from Paracoccus denitrificans. Eur J Biochem. 1983 Dec 15;137(3):597–602. doi: 10.1111/j.1432-1033.1983.tb07867.x. [DOI] [PubMed] [Google Scholar]
  20. Maccecchini M. L., Rudin Y., Blobel G., Schatz G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl Acad Sci U S A. 1979 Jan;76(1):343–347. doi: 10.1073/pnas.76.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. McAda P. C., Douglas M. G. A neutral metallo endoprotease involved in the processing of an F1-ATPase subunit precursor in mitochondria. J Biol Chem. 1982 Mar 25;257(6):3177–3182. [PubMed] [Google Scholar]
  23. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nasmyth K. A., Reed S. I. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Malley K., Pratt P., Robertson J., Lilly M., Douglas M. G. Selection of the nuclear gene for the mitochondrial adenine nucleotide translocator by genetic complementation of the op1 mutation in yeast. J Biol Chem. 1982 Feb 25;257(4):2097–2103. [PubMed] [Google Scholar]
  27. Ohashi A., Gibson J., Gregor I., Schatz G. Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent. J Biol Chem. 1982 Nov 10;257(21):13042–13047. [PubMed] [Google Scholar]
  28. 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]
  29. Riezman H., Hay R., Witte C., Nelson N., Schatz G. Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins. EMBO J. 1983;2(7):1113–1118. doi: 10.1002/j.1460-2075.1983.tb01554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Saltzgaber-Muller J., Kunapuli S. P., Douglas M. G. Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit. J Biol Chem. 1983 Oct 10;258(19):11465–11470. [PubMed] [Google Scholar]
  31. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schatz G., Butow R. A. How are proteins imported into mitochondria? Cell. 1983 Feb;32(2):316–318. doi: 10.1016/0092-8674(83)90450-6. [DOI] [PubMed] [Google Scholar]
  34. Suchanek G., Kreil G. Translation of melittin messenger RNA in vitro yields a product terminating with glutaminylglycine rather than with glutaminamide. Proc Natl Acad Sci U S A. 1977 Mar;74(3):975–978. doi: 10.1073/pnas.74.3.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Teintze M., Slaughter M., Weiss H., Neupert W. Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria. J Biol Chem. 1982 Sep 10;257(17):10364–10371. [PubMed] [Google Scholar]
  36. 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]
  37. Viebrock A., Perz A., Sebald W. The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA. EMBO J. 1982;1(5):565–571. doi: 10.1002/j.1460-2075.1982.tb01209.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wakabayashi S., Matsubara H., Kim C. H., Kawai K., King T. E. The complete amino acid sequence of bovine heart cytochrome C1. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1548–1554. doi: 10.1016/s0006-291x(80)80042-8. [DOI] [PubMed] [Google Scholar]
  39. von Heijne G. Membrane proteins: the amino acid composition of membrane-penetrating segments. Eur J Biochem. 1981 Nov;120(2):275–278. doi: 10.1111/j.1432-1033.1981.tb05700.x. [DOI] [PubMed] [Google Scholar]

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