Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 May;10(5):1873–1881. doi: 10.1128/mcb.10.5.1873

Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix.

S M Glaser 1, B R Miller 1, M G Cumsky 1
PMCID: PMC360532  PMID: 2157966

Abstract

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.

Full text

PDF
1873

Images in this article

1877Image
on p.1877
1878Image
on p.1878
1878Image
on p.1878

Selected References

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

  1. Attardi G., Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289–333. doi: 10.1146/annurev.cb.04.110188.001445. [DOI] [PubMed] [Google Scholar]
  2. Bedwell D. M., Klionsky D. J., Emr S. D. The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information. Mol Cell Biol. 1987 Nov;7(11):4038–4047. doi: 10.1128/mcb.7.11.4038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Böhni P. C., Daum G., Schatz G. Import of proteins into mitochondria. Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides. J Biol Chem. 1983 Apr 25;258(8):4937–4943. [PubMed] [Google Scholar]
  4. Cerletti N., Schatz G. Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer. J Biol Chem. 1979 Aug 25;254(16):7746–7751. [PubMed] [Google Scholar]
  5. Chen W. J., Douglas M. G. Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix. Cell. 1987 Jun 5;49(5):651–658. doi: 10.1016/0092-8674(87)90541-1. [DOI] [PubMed] [Google Scholar]
  6. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  7. Cheng M. Y., Pollock R. A., Hendrick J. P., Horwich A. L. Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4063–4067. doi: 10.1073/pnas.84.12.4063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cumsky M. G., Ko C., Trueblood C. E., Poyton R. O. Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2235–2239. doi: 10.1073/pnas.82.8.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cumsky M. G., Trueblood C. E., Ko C., Poyton R. O. Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V. Mol Cell Biol. 1987 Oct;7(10):3511–3519. doi: 10.1128/mcb.7.10.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  13. Eilers M., Oppliger W., Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987 Apr;6(4):1073–1077. doi: 10.1002/j.1460-2075.1987.tb04860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Glaser S. M., Trueblood C. E., Dircks L. K., Poyton R. O., Cumsky M. G. Functional analysis of mitochondrial protein import in yeast. J Cell Biochem. 1988 Mar;36(3):275–287. doi: 10.1002/jcb.240360308. [DOI] [PubMed] [Google Scholar]
  17. Hartl F. U., Ostermann J., Guiard B., Neupert W. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell. 1987 Dec 24;51(6):1027–1037. doi: 10.1016/0092-8674(87)90589-7. [DOI] [PubMed] [Google Scholar]
  18. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  19. Hartl F. U., Schmidt B., Wachter E., Weiss H., Neupert W. Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. Cell. 1986 Dec 26;47(6):939–951. doi: 10.1016/0092-8674(86)90809-3. [DOI] [PubMed] [Google Scholar]
  20. Hase T., Müller U., Riezman H., Schatz G. A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus. EMBO J. 1984 Dec 20;3(13):3157–3164. doi: 10.1002/j.1460-2075.1984.tb02274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hawlitschek G., Schneider H., Schmidt B., Tropschug M., Hartl F. U., Neupert W. Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell. 1988 Jun 3;53(5):795–806. doi: 10.1016/0092-8674(88)90096-7. [DOI] [PubMed] [Google Scholar]
  22. Horwich A. L., Kalousek F., Fenton W. A., Pollock R. A., Rosenberg L. E. Targeting of pre-ornithine transcarbamylase to mitochondria: definition of critical regions and residues in the leader peptide. Cell. 1986 Feb 14;44(3):451–459. doi: 10.1016/0092-8674(86)90466-6. [DOI] [PubMed] [Google Scholar]
  23. Horwich A. L., Kalousek F., Mellman I., Rosenberg L. E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J. 1985 May;4(5):1129–1135. doi: 10.1002/j.1460-2075.1985.tb03750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hurt E. C., Pesold-Hurt B., Schatz G. The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. FEBS Lett. 1984 Dec 10;178(2):306–310. doi: 10.1016/0014-5793(84)80622-5. [DOI] [PubMed] [Google Scholar]
  25. Hurt E. C., Pesold-Hurt B., Suda K., Oppliger W., Schatz G. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 1985 Aug;4(8):2061–2068. doi: 10.1002/j.1460-2075.1985.tb03892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Isaya G., Fenton W. A., Hendrick J. P., Furtak K., Kalousek F., Rosenberg L. E. Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells. Mol Cell Biol. 1988 Dec;8(12):5150–5158. doi: 10.1128/mcb.8.12.5150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Keng T., Alani E., Guarente L. The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix. Mol Cell Biol. 1986 Feb;6(2):355–364. doi: 10.1128/mcb.6.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. McEwen J. E., Cameron V. L., Poyton R. O. Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. J Bacteriol. 1985 Mar;161(3):831–835. doi: 10.1128/jb.161.3.831-835.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murakami H., Pain D., Blobel G. 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J Cell Biol. 1988 Dec;107(6 Pt 1):2051–2057. doi: 10.1083/jcb.107.6.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nguyen M., Bell A. W., Shore G. C. Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain. J Cell Biol. 1988 May;106(5):1499–1505. doi: 10.1083/jcb.106.5.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ohba M., Schatz G. Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J. 1987 Jul;6(7):2117–2122. doi: 10.1002/j.1460-2075.1987.tb02478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohba M., Schatz G. Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane. EMBO J. 1987 Jul;6(7):2109–2115. doi: 10.1002/j.1460-2075.1987.tb02477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  36. Pfaller R., Neupert W. High-affinity binding sites involved in the import of porin into mitochondria. EMBO J. 1987 Sep;6(9):2635–2642. doi: 10.1002/j.1460-2075.1987.tb02554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pfaller R., Steger H. F., Rassow J., Pfanner N., Neupert W. Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site. J Cell Biol. 1988 Dec;107(6 Pt 2):2483–2490. doi: 10.1083/jcb.107.6.2483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pfanner N., Hartl F. U., Neupert W. Import of proteins into mitochondria: a multi-step process. Eur J Biochem. 1988 Aug 1;175(2):205–212. doi: 10.1111/j.1432-1033.1988.tb14185.x. [DOI] [PubMed] [Google Scholar]
  39. Pfanner N., Neupert W. Distinct steps in the import of ADP/ATP carrier into mitochondria. J Biol Chem. 1987 Jun 5;262(16):7528–7536. [PubMed] [Google Scholar]
  40. Pfanner N., Neupert W. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986 Dec 15;209(2):152–156. doi: 10.1016/0014-5793(86)81101-2. [DOI] [PubMed] [Google Scholar]
  41. Pfanner N., Neupert W. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 1985 Nov;4(11):2819–2825. doi: 10.1002/j.1460-2075.1985.tb04009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Power S. D., Lochrie M. A., Poyton R. O. The nuclear-coded subunits of yeast cytochrome c oxidase. III. Identification of homologous subunits in yeast, bovine heart, and Neurospora crassa cytochrome c oxidases. J Biol Chem. 1984 May 25;259(10):6575–6578. [PubMed] [Google Scholar]
  43. 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]
  44. Poyton R. O. Cooperative interaction between mitochondrial and nuclear genomes: cytochrome c oxidase assembly as a model. Curr Top Cell Regul. 1980;17:231–295. doi: 10.1016/b978-0-12-152817-1.50012-2. [DOI] [PubMed] [Google Scholar]
  45. Sachs M. S., Bertrand H., Metzenberg R. L., RajBhandary U. L. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V. Mol Cell Biol. 1989 Feb;9(2):566–577. doi: 10.1128/mcb.9.2.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schleyer M., Schmidt B., Neupert W. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur J Biochem. 1982 Jun 15;125(1):109–116. doi: 10.1111/j.1432-1033.1982.tb06657.x. [DOI] [PubMed] [Google Scholar]
  47. Trueblood C. E., Poyton R. O. Differential effectiveness of yeast cytochrome c oxidase subunit genes results from differences in expression not function. Mol Cell Biol. 1987 Oct;7(10):3520–3526. doi: 10.1128/mcb.7.10.3520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Verner K., Schatz G. Protein translocation across membranes. Science. 1988 Sep 9;241(4871):1307–1313. doi: 10.1126/science.2842866. [DOI] [PubMed] [Google Scholar]
  49. Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
  50. Zwizinski C., Schleyer M., Neupert W. Proteinaceous receptors for the import of mitochondrial precursor proteins. J Biol Chem. 1984 Jun 25;259(12):7850–7856. [PubMed] [Google Scholar]
  51. van Loon A. P., Brändli A. W., Schatz G. The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting. Cell. 1986 Mar 14;44(5):801–812. doi: 10.1016/0092-8674(86)90846-9. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES