The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70 (original) (raw)

Abstract

Cytochrome b2 is imported into mitochondria and sorted to the intermembrane space by a bipartite N-terminal presequence, which is a matrix targeting sequenced followed by an intermembrane space sorting signal. The N-terminus of the mature protein forms a folded heme binding domain that depends on the unfoldase function of matrix (mt) Hsp70 for import. We report that the distance between the presequence and the heme binding domain is critical for the ability of mt-Hsp70 to promote import of the domain. Hybrid proteins with 40 or more amino acids between the presequence and the heme binding domain are arrested in the import machinery. The translocation arrest can be overcome by unfolding of the preprotein or by inactivation of the intermembrane space sorting signal. Moreover, the sorting signal prevents backsliding of the precursor polypeptide in the import site in the initial import step, when the signal has not made contact with the matrix. The results indicate that the sorting signal interacts with component(s) of the inner membrane/intermembrane space during the initial import step and promotes an early divergence of b2 preproteins from the general matrix import pathway, precluding an unfolding role for mt-Hsp70 in the translocation of most of the mature portions of a preprotein. We propose a sorting model of cytochrome b2 which explains the apparently divergent previous results by a unifying hypothesis.

6043

Images in this article

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. Chuck S. L., Yao Z., Blackhart B. D., McCarthy B. J., Lingappa V. R. New variation on the translocation of proteins during early biogenesis of apolipoprotein B. Nature. 1990 Jul 26;346(6282):382–385. doi: 10.1038/346382a0. [DOI] [PubMed] [Google Scholar]
  4. Cyr D. M., Stuart R. A., Neupert W. A matrix ATP requirement for presequence translocation across the inner membrane of mitochondria. J Biol Chem. 1993 Nov 15;268(32):23751–23754. [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. Dobberstein B. Protein transport. On the beaten pathway. Nature. 1994 Feb 17;367(6464):599–600. doi: 10.1038/367599a0. [DOI] [PubMed] [Google Scholar]
  7. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  8. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gafvelin G., von Heijne G. Topological "frustration" in multispanning E. coli inner membrane proteins. Cell. 1994 May 6;77(3):401–412. doi: 10.1016/0092-8674(94)90155-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Glick B. S. Can Hsp70 proteins act as force-generating motors? Cell. 1995 Jan 13;80(1):11–14. doi: 10.1016/0092-8674(95)90444-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gruhler A., Ono H., Guiard B., Neupert W., Stuart R. A. A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting. EMBO J. 1995 Apr 3;14(7):1349–1359. doi: 10.1002/j.1460-2075.1995.tb07121.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gärtner F., Voos W., Querol A., Miller B. R., Craig E. A., Cumsky M. G., Pfanner N. Mitochondrial import of subunit Va of cytochrome c oxidase characterized with yeast mutants. J Biol Chem. 1995 Feb 24;270(8):3788–3795. doi: 10.1074/jbc.270.8.3788. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Jungnickel B., Rapoport T. A., Hartmann E. Protein translocation: common themes from bacteria to man. FEBS Lett. 1994 Jun 6;346(1):73–77. doi: 10.1016/0014-5793(94)00367-x. [DOI] [PubMed] [Google Scholar]
  20. Kang P. J., Ostermann J., Shilling J., Neupert W., Craig E. A., Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990 Nov 8;348(6297):137–143. doi: 10.1038/348137a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Kronidou N. G., Oppliger W., Bolliger L., Hannavy K., Glick B. S., Schatz G., Horst M. Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12818–12822. doi: 10.1073/pnas.91.26.12818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Mahlke K., Pfanner N., Martin J., Horwich A. L., Hartl F. U., Neupert W. Sorting pathways of mitochondrial inner membrane proteins. Eur J Biochem. 1990 Sep 11;192(2):551–555. doi: 10.1111/j.1432-1033.1990.tb19260.x. [DOI] [PubMed] [Google Scholar]
  26. Mayer A., Lill R., Neupert W. Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria. J Cell Biol. 1993 Jun;121(6):1233–1243. doi: 10.1083/jcb.121.6.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mayer A., Neupert W., Lill R. Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding. Cell. 1995 Jan 13;80(1):127–137. doi: 10.1016/0092-8674(95)90457-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Ono H., Gruhler A., Stuart R. A., Guiard B., Schwarz E., Neupert W. Sorting of cytochrome b2 to the intermembrane space of mitochondria. Kinetic analysis of intermediates demonstrates passage through the matrix. J Biol Chem. 1995 Jul 14;270(28):16932–16938. doi: 10.1074/jbc.270.28.16932. [DOI] [PubMed] [Google Scholar]
  30. Pace C. N. Conformational stability of globular proteins. Trends Biochem Sci. 1990 Jan;15(1):14–17. doi: 10.1016/0968-0004(90)90124-t. [DOI] [PubMed] [Google Scholar]
  31. Pfanner N., Craig E. A., Meijer M. The protein import machinery of the mitochondrial inner membrane. Trends Biochem Sci. 1994 Sep;19(9):368–372. doi: 10.1016/0968-0004(94)90113-9. [DOI] [PubMed] [Google Scholar]
  32. Pfanner N., Hartl F. U., Guiard B., Neupert W. Mitochondrial precursor proteins are imported through a hydrophilic membrane environment. Eur J Biochem. 1987 Dec 1;169(2):289–293. doi: 10.1111/j.1432-1033.1987.tb13610.x. [DOI] [PubMed] [Google Scholar]
  33. Pfanner N., Meijer M. Protein sorting. Pulling in the proteins. Curr Biol. 1995 Feb 1;5(2):132–135. doi: 10.1016/s0960-9822(95)00033-9. [DOI] [PubMed] [Google Scholar]
  34. Pratje E., Guiard B. One nuclear gene controls the removal of transient pre-sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. EMBO J. 1986 Jun;5(6):1313–1317. doi: 10.1002/j.1460-2075.1986.tb04361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rassow J., Guiard B., Wienhues U., Herzog V., Hartl F. U., Neupert W. Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol. 1989 Oct;109(4 Pt 1):1421–1428. doi: 10.1083/jcb.109.4.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Rassow J., Maarse A. C., Krainer E., Kübrich M., Müller H., Meijer M., Craig E. A., Pfanner N. Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44. J Cell Biol. 1994 Dec;127(6 Pt 1):1547–1556. doi: 10.1083/jcb.127.6.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reid G. A., Yonetani T., Schatz G. Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells. J Biol Chem. 1982 Nov 10;257(21):13068–13074. [PubMed] [Google Scholar]
  39. Rojo E. E., Stuart R. A., Neupert W. Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP. EMBO J. 1995 Jul 17;14(14):3445–3451. doi: 10.1002/j.1460-2075.1995.tb07350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rospert S., Müller S., Schatz G., Glick B. S. Fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial intermembrane space independently of hsp60. J Biol Chem. 1994 Jun 24;269(25):17279–17288. [PubMed] [Google Scholar]
  41. Schekman R. Translocation gets a push. Cell. 1994 Sep 23;78(6):911–913. doi: 10.1016/0092-8674(94)90265-8. [DOI] [PubMed] [Google Scholar]
  42. Schiebel E., Driessen A. J., Hartl F. U., Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. doi: 10.1016/0092-8674(91)90317-r. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. Söllner T., Rassow J., Pfanner N. Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol. 1991;34:345–358. doi: 10.1016/s0091-679x(08)61689-1. [DOI] [PubMed] [Google Scholar]
  48. Ungermann C., Neupert W., Cyr D. M. The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science. 1994 Nov 18;266(5188):1250–1253. doi: 10.1126/science.7973708. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Wachter C., Schatz G., Glick B. S. Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix. Mol Biol Cell. 1994 Apr;5(4):465–474. doi: 10.1091/mbc.5.4.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wachter C., Schatz G., Glick B. S. Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator. EMBO J. 1992 Dec;11(13):4787–4794. doi: 10.1002/j.1460-2075.1992.tb05584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]