Current Genetics

, Volume 20, Issue 1–2, pp 87–90 | Cite as

Over-expression, purification and determination of the proteolytic processing site of the yeast mitochondrial CBS1 protein

  • A. Körte
  • U. Michaelis
  • F. Lottspeich
  • G. Rödel
Original Articles


Yeast transformats harboring the CBS1 gene under the control of the strong ADC1 promoter on a high copy number plasmid express the mitochondrial CBS1 protein at artificially high levels. Over-expressed protein is imported into mitochondria and correctly processed to vield the mature mitochondrial 23.5 kDa form, but differs in its solubility properties from CBS1 in wild-type mitochondria. It forms insoluble protein aggregates, which are refractory to solubilization with 1% Taurodeoxycholate. We exploited this observation to separate CBS1 from the bulk of mitochondrial proteins and to isolate CBS1 after SDS gel electrophoresis. Determination of the amino-terminal amino acids of the purified protein reveals that the mature CBS1 protein starts with Ile30, at the characteristic distance of +2 amino acids from an arginine residue (Arg28). The cleavage site shows a remarkable homology to that of subunit 9 of the F0F1 ATPase from Neurospora crassa.

Key words

CBS1 protein Protein purification Mitochondrial presequence Protein sequencing 


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  1. Ammerer G (1983) Methods Enzymol 101: 192–201Google Scholar
  2. Daum G, Böhni PC, Schatz G (1982) J Biol Chem 257: 13028–13033Google Scholar
  3. Eckerskorn C, Mewes W, Goretzki H, Lottspeich F (1988) Eur J Biochem 176: 509–519Google Scholar
  4. Edman P, Begg G (1967) Eur J Biochem 1: 80–91Google Scholar
  5. Forsbach V, Pillar T, Gottenöf T, Rödel G (1989) Mol Gen Genet 218: 57–63Google Scholar
  6. Gampel A, Nishikimi M, Tzagoloff A (1989) Mol Cell Biol 9: 5424–5433Google Scholar
  7. Hartl F-U, Pfanner N, Nicholson DW, neupert W (1989) Biochim Biophys Acta 988: 1–45Google Scholar
  8. Hawlischek G, Schneider H, Schmidt B, Tropschug M, Hartl F-U, Neupert W (1988) Cell 53: 795–806Google Scholar
  9. Jensen RE, Yaffe MP (1988) EMBO J 7: 3863–3871Google Scholar
  10. Körte A, Forsbach V, Gottenöf T, Rödel G (1989) Mol Gen Genet 217: 162–167Google Scholar
  11. Laemmli UK (1970) Nature 227: 680–685Google Scholar
  12. Marres CAM, Van Loon APGM, Oudshoorn P, Van Steeg H, Grivell LA, Slater EC (1985) Eur J Biochem 14: 153–161Google Scholar
  13. Michaelis U, Rödel G (1990) Mol Gen Genet 223: 394–400Google Scholar
  14. Muroff I, Tzagoloff A (1990) EMBO J 9: 2765–2773Google Scholar
  15. Pollock RA, Hartl F-U, Cheng MY, Ostermann J, Horwich A, Neupert W (1988) EMBO J 7: 3493–3500Google Scholar
  16. Rödel G, Körte A, Kaudewitz F (1985) Curr Genet 9: 641–648Google Scholar
  17. Rödel G, Fox TD (1987) Mol Gen Genet 206: 45–50Google Scholar
  18. Schmidt B, Wachter E, Sebald W, Neupert W (1984) Eur J Biochem 144: 581–588Google Scholar
  19. Schneider A, Behrens M, Scherer P, Pratje E, Michaelis G, Schatz G (1991) EMBO J 10: 247–254Google Scholar
  20. Weber ER, Dieckmann CL (1990) J Biol Chem 265: 1594–1600Google Scholar
  21. Witte C, Jensen RE, Yaffe MP, Schatz G (1988) EMBO J 7: 1439–1447Google Scholar
  22. Yang M, Jensen RE, Yaffe MP, Oppliger W, Schatz G (1988) EMBO J 7: 3857–3862Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • A. Körte
    • 1
  • U. Michaelis
    • 1
  • F. Lottspeich
    • 2
  • G. Rödel
    • 3
  1. 1.Lehrstuhl für GenetikUniversität MünchenMünchenFederal Republic of Germany
  2. 2.Max-Planck-Institut für BiochemicGenzentrumMartinsriedFederal Republic of Germany
  3. 3.Labor für Molekulare Biologie und Allgemeine Pathologie, Institut für PathologieUniversität UlmMünchen 45Federal Republic of Germany

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