Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Apr 3;14(7):1349–1359. doi: 10.1002/j.1460-2075.1995.tb07121.x

A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.

A Gruhler 1, H Ono 1, B Guiard 1, W Neupert 1, R A Stuart 1
PMCID: PMC398220  PMID: 7729413

Abstract

Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.

Full text

PDF
1350

Images in this article

1350Image
on p.1350
1351Image
on p.1351
1352Image
on p.1352
1353Image
on p.1353
1354Image
on p.1354
1355Image
on p.1355
1356Image
on p.1356

Selected References

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

  1. Beasley E. M., Müller S., Schatz G. The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 1993 Jun;12(6):2303–2311. doi: 10.1002/j.1460-2075.1993.tb05884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blom J., Kübrich M., Rassow J., Voos W., Dekker P. J., Maarse A. C., Meijer M., Pfanner N. The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane. Mol Cell Biol. 1993 Dec;13(12):7364–7371. doi: 10.1128/mcb.13.12.7364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Gambill B. D., Voos W., Kang P. J., Miao B., Langer T., Craig E. A., Pfanner N. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J Cell Biol. 1993 Oct;123(1):109–117. doi: 10.1083/jcb.123.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glick B. S., Beasley E. M., Schatz G. Protein sorting in mitochondria. Trends Biochem Sci. 1992 Nov;17(11):453–459. doi: 10.1016/0968-0004(92)90487-t. [DOI] [PubMed] [Google Scholar]
  8. Glick B. S., Brandt A., Cunningham K., Müller S., Hallberg R. L., Schatz G. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell. 1992 May 29;69(5):809–822. doi: 10.1016/0092-8674(92)90292-k. [DOI] [PubMed] [Google Scholar]
  9. Glick B. S., Wachter C., Reid G. A., Schatz G. Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP. Protein Sci. 1993 Nov;2(11):1901–1917. doi: 10.1002/pro.5560021112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guiard B. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2). EMBO J. 1985 Dec 1;4(12):3265–3272. doi: 10.1002/j.1460-2075.1985.tb04076.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hartl F. U., Neupert W. Protein sorting to mitochondria: evolutionary conservations of folding and assembly. Science. 1990 Feb 23;247(4945):930–938. doi: 10.1126/science.2406905. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Herrmann J. M., Stuart R. A., Craig E. A., Neupert W. Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA. J Cell Biol. 1994 Nov;127(4):893–902. doi: 10.1083/jcb.127.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horst M., Jenö P., Kronidou N. G., Bolliger L., Oppliger W., Scherer P., Manning-Krieg U., Jascur T., Schatz G. Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product. EMBO J. 1993 Aug;12(8):3035–3041. doi: 10.1002/j.1460-2075.1993.tb05972.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jensen R. E., Schmidt S., Mark R. J. Mutations in a 19-amino-acid hydrophobic region of the yeast cytochrome c1 presequence prevent sorting to the mitochondrial intermembrane space. Mol Cell Biol. 1992 Oct;12(10):4677–4686. doi: 10.1128/mcb.12.10.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Koll H., Guiard B., Rassow J., Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell. 1992 Mar 20;68(6):1163–1175. doi: 10.1016/0092-8674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lill R., Stuart R. A., Drygas M. E., Nargang F. E., Neupert W. Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space. EMBO J. 1992 Feb;11(2):449–456. doi: 10.1002/j.1460-2075.1992.tb05074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maarse A. C., Blom J., Grivell L. A., Meijer M. MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 1992 Oct;11(10):3619–3628. doi: 10.1002/j.1460-2075.1992.tb05446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nunnari J., Fox T. D., Walter P. A mitochondrial protease with two catalytic subunits of nonoverlapping specificities. Science. 1993 Dec 24;262(5142):1997–2004. doi: 10.1126/science.8266095. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Rassow J., Hartl F. U., Guiard B., Pfanner N., Neupert W. Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett. 1990 Nov 26;275(1-2):190–194. doi: 10.1016/0014-5793(90)81469-5. [DOI] [PubMed] [Google Scholar]
  25. Schneider A., Behrens M., Scherer P., Pratje E., Michaelis G., Schatz G. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO J. 1991 Feb;10(2):247–254. doi: 10.1002/j.1460-2075.1991.tb07944.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schneider H. C., Berthold J., Bauer M. F., Dietmeier K., Guiard B., Brunner M., Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature. 1994 Oct 27;371(6500):768–774. doi: 10.1038/371768a0. [DOI] [PubMed] [Google Scholar]
  27. Schwarz E., Seytter T., Guiard B., Neupert W. Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal. EMBO J. 1993 Jun;12(6):2295–2302. doi: 10.1002/j.1460-2075.1993.tb05883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stuart R. A., Cyr D. M., Craig E. A., Neupert W. Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem Sci. 1994 Feb;19(2):87–92. doi: 10.1016/0968-0004(94)90041-8. [DOI] [PubMed] [Google Scholar]
  29. Stuart R. A., Gruhler A., van der Klei I., Guiard B., Koll H., Neupert W. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. Eur J Biochem. 1994 Feb 15;220(1):9–18. doi: 10.1111/j.1432-1033.1994.tb18593.x. [DOI] [PubMed] [Google Scholar]
  30. Stuart R. A., Neupert W. Apocytochrome c: an exceptional mitochondrial precursor protein using an exceptional import pathway. Biochimie. 1990 Feb-Mar;72(2-3):115–121. doi: 10.1016/0300-9084(90)90136-5. [DOI] [PubMed] [Google Scholar]
  31. Voos W., Gambill B. D., Guiard B., Pfanner N., Craig E. A. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J Cell Biol. 1993 Oct;123(1):119–126. doi: 10.1083/jcb.123.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Loon A. P., Brändli A. W., Pesold-Hurt B., Blank D., Schatz G. Transport of proteins to the mitochondrial intermembrane space: the 'matrix-targeting' and the 'sorting' domains in the cytochrome c1 presequence. EMBO J. 1987 Aug;6(8):2433–2439. doi: 10.1002/j.1460-2075.1987.tb02522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. van Loon A. P., Schatz G. Transport of proteins to the mitochondrial intermembrane space: the 'sorting' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane. EMBO J. 1987 Aug;6(8):2441–2448. doi: 10.1002/j.1460-2075.1987.tb02523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES