Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Mar;3(3):651–657. doi: 10.1002/j.1460-2075.1984.tb01862.x

A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria.

S Ohta, G Schatz
PMCID: PMC557402  PMID: 6232136

Abstract

The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.

Full text

PDF
651

Images in this article

652Fig. 1.
on p.652
652Fig. 2.
on p.652
652Fig. 3.
on p.652
653Fig. 4.
on p.653
653Fig. 5.
on p.653
653Fig. 6.
on p.653
654Fig. 7.
on p.654
654Fig. 8.
on p.654
655Fig. 9.
on p.655

Selected References

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

  1. Argan C., Lusty C. J., Shore G. C. Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria. J Biol Chem. 1983 Jun 10;258(11):6667–6670. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Douglas M. G., Koh Y., Dockter M. E., Schatz G. Aurovertin binds to the beta subunit of yeast mitochondrial ATPase. J Biol Chem. 1977 Dec 10;252(23):8333–8335. [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. 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]
  9. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Maccecchini M. L., Rudin Y., Schatz G. Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase. J Biol Chem. 1979 Aug 25;254(16):7468–7471. [PubMed] [Google Scholar]
  12. Miura S., Mori M., Tatibana M. Transport of ornithine carbamoyltransferase precursor into mitochondria. Stimulation by potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s). J Biol Chem. 1983 Jun 10;258(11):6671–6674. [PubMed] [Google Scholar]
  13. 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]
  14. Ohashi A., Schatz G. Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides. J Biol Chem. 1980 Aug 25;255(16):7740–7745. [PubMed] [Google Scholar]
  15. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  16. Reid G. A., Schatz G. Import of proteins into mitochondria. Extramitochondrial pools and post-translational import of mitochondrial protein precursors in vivo. J Biol Chem. 1982 Nov 10;257(21):13062–13067. [PubMed] [Google Scholar]
  17. Reid G. A., Schatz G. Import of proteins into mitochondria. Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides. J Biol Chem. 1982 Nov 10;257(21):13056–13061. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Stähli C., Staehelin T., Miggiano V., Schmidt J., Häring P. High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis. J Immunol Methods. 1980;32(3):297–304. doi: 10.1016/0022-1759(80)90194-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Walter P., Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature. 1982 Oct 21;299(5885):691–698. doi: 10.1038/299691a0. [DOI] [PubMed] [Google Scholar]
  23. Wickerham L. J. A Critical Evaluation of the Nitrogen Assimilation Tests Commonly Used in the Classification of Yeasts. J Bacteriol. 1946 Sep;52(3):293–301. [PMC free article] [PubMed] [Google Scholar]
  24. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  25. Zimmermann R., Paluch U., Neupert W. Cell-free synthesis of cytochrome c. FEBS Lett. 1979 Dec 1;108(1):141–146. doi: 10.1016/0014-5793(79)81196-5. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES