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. 1992 Feb;11(2):449–456. doi: 10.1002/j.1460-2075.1992.tb05074.x

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

R Lill 1, R A Stuart 1, M E Drygas 1, F E Nargang 1, W Neupert 1
PMCID: PMC556474  PMID: 1371459

Abstract

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

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Selected References

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  1. Baker K. P., Schaniel A., Vestweber D., Schatz G. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature. 1990 Dec 13;348(6302):605–609. doi: 10.1038/348605a0. [DOI] [PubMed] [Google Scholar]
  2. Baker K. P., Schatz G. Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria. Nature. 1991 Jan 17;349(6306):205–208. doi: 10.1038/349205a0. [DOI] [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Drygas M. E., Lambowitz A. M., Nargang F. E. Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase. J Biol Chem. 1989 Oct 25;264(30):17897–17906. [PubMed] [Google Scholar]
  8. Dumont M. E., Ernst J. F., Hampsey D. M., Sherman F. Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae. EMBO J. 1987 Jan;6(1):235–241. doi: 10.1002/j.1460-2075.1987.tb04744.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dumont M. E., Ernst J. F., Sherman F. Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c. J Biol Chem. 1988 Nov 5;263(31):15928–15937. [PubMed] [Google Scholar]
  10. Freitag H., Janes M., Neupert W. Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial membrane of Neurospora crassa. Eur J Biochem. 1982 Aug;126(1):197–202. doi: 10.1111/j.1432-1033.1982.tb06766.x. [DOI] [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., 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]
  14. Hase T., Riezman H., Suda K., Schatz G. Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane. EMBO J. 1983;2(12):2169–2172. doi: 10.1002/j.1460-2075.1983.tb01718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hennig B., Neupert W. Biogenesis of cytochrome c in Neurospora crassa. Methods Enzymol. 1983;97:261–274. doi: 10.1016/0076-6879(83)97138-0. [DOI] [PubMed] [Google Scholar]
  17. Hines V., Brandt A., Griffiths G., Horstmann H., Brütsch H., Schatz G. Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. EMBO J. 1990 Oct;9(10):3191–3200. doi: 10.1002/j.1460-2075.1990.tb07517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. KANAREK L., HILL R. L. THE PREPARATION AND CHARACTERIZATION OF FUMARASE FROM SWINE HEART MUSCLE. J Biol Chem. 1964 Dec;239:4202–4206. [PubMed] [Google Scholar]
  19. 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]
  20. Kiebler M., Pfaller R., Söllner T., Griffiths G., Horstmann H., Pfanner N., Neupert W. Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature. 1990 Dec 13;348(6302):610–616. doi: 10.1038/348610a0. [DOI] [PubMed] [Google Scholar]
  21. Kleene R., Pfanner N., Pfaller R., Link T. A., Sebald W., Neupert W., Tropschug M. Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria. EMBO J. 1987 Sep;6(9):2627–2633. doi: 10.1002/j.1460-2075.1987.tb02553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Nicholson D. W., Hergersberg C., Neupert W. Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria. J Biol Chem. 1988 Dec 15;263(35):19034–19042. [PubMed] [Google Scholar]
  24. Nicholson D. W., Köhler H., Neupert W. Import of cytochrome c into mitochondria. Cytochrome c heme lyase. Eur J Biochem. 1987 Apr 1;164(1):147–157. doi: 10.1111/j.1432-1033.1987.tb11006.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. Pfanner N., Neupert W. The mitochondrial protein import apparatus. Annu Rev Biochem. 1990;59:331–353. doi: 10.1146/annurev.bi.59.070190.001555. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Pfanner N., Söllner T., Neupert W. Mitochondrial import receptors for precursor proteins. Trends Biochem Sci. 1991 Feb;16(2):63–67. doi: 10.1016/0968-0004(91)90026-r. [DOI] [PubMed] [Google Scholar]
  33. Pfanner N., Tropschug M., Neupert W. Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors. Cell. 1987 Jun 19;49(6):815–823. doi: 10.1016/0092-8674(87)90619-2. [DOI] [PubMed] [Google Scholar]
  34. Pollock R. A., Hartl F. U., Cheng M. Y., Ostermann J., Horwich A., Neupert W. The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related. EMBO J. 1988 Nov;7(11):3493–3500. doi: 10.1002/j.1460-2075.1988.tb03225.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. Rietveld A., Berkhout T. A., Roenhorst A., Marsh D., de Kruijff B. Preferential association of apocytochrome c with negatively charged phospholipids in mixed model membranes. Biochim Biophys Acta. 1986 Jun 13;858(1):38–46. doi: 10.1016/0005-2736(86)90289-0. [DOI] [PubMed] [Google Scholar]
  37. Riezman H., Hay R., Witte C., Nelson N., Schatz G. Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins. EMBO J. 1983;2(7):1113–1118. doi: 10.1002/j.1460-2075.1983.tb01554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schleyer M., Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985 Nov;43(1):339–350. doi: 10.1016/0092-8674(85)90039-x. [DOI] [PubMed] [Google Scholar]
  39. Schmidt B., Wachter E., Sebald W., Neupert W. Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9. Eur J Biochem. 1984 Nov 2;144(3):581–588. doi: 10.1111/j.1432-1033.1984.tb08505.x. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Schwaiger M., Herzog V., Neupert W. Characterization of translocation contact sites involved in the import of mitochondrial proteins. J Cell Biol. 1987 Jul;105(1):235–246. doi: 10.1083/jcb.105.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Steger H. F., Söllner T., Kiebler M., Dietmeier K. A., Pfaller R., Trülzsch K. S., Tropschug M., Neupert W., Pfanner N. Import of ADP/ATP carrier into mitochondria: two receptors act in parallel. J Cell Biol. 1990 Dec;111(6 Pt 1):2353–2363. doi: 10.1083/jcb.111.6.2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Stuart R. A., Nicholson D. W., Neupert W. Early steps in mitochondrial protein import: receptor functions can be substituted by the membrane insertion activity of apocytochrome c. Cell. 1990 Jan 12;60(1):31–43. doi: 10.1016/0092-8674(90)90713-o. [DOI] [PubMed] [Google Scholar]
  45. Stueber D., Ibrahimi I., Cutler D., Dobberstein B., Bujard H. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J. 1984 Dec 20;3(13):3143–3148. doi: 10.1002/j.1460-2075.1984.tb02271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Söllner T., Griffiths G., Pfaller R., Pfanner N., Neupert W. MOM19, an import receptor for mitochondrial precursor proteins. Cell. 1989 Dec 22;59(6):1061–1070. doi: 10.1016/0092-8674(89)90762-9. [DOI] [PubMed] [Google Scholar]
  47. Söllner T., Pfaller R., Griffiths G., Pfanner N., Neupert W. A mitochondrial import receptor for the ADP/ATP carrier. Cell. 1990 Jul 13;62(1):107–115. doi: 10.1016/0092-8674(90)90244-9. [DOI] [PubMed] [Google Scholar]
  48. Vestweber D., Brunner J., Baker A., Schatz G. A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature. 1989 Sep 21;341(6239):205–209. doi: 10.1038/341205a0. [DOI] [PubMed] [Google Scholar]
  49. Witte C., Jensen R. E., Yaffe M. P., Schatz G. MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease. EMBO J. 1988 May;7(5):1439–1447. doi: 10.1002/j.1460-2075.1988.tb02961.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zimmermann R., Hennig B., Neupert W. Different transport pathways of individual precursor proteins in mitochondria. Eur J Biochem. 1981 Jun 1;116(3):455–460. doi: 10.1111/j.1432-1033.1981.tb05357.x. [DOI] [PubMed] [Google Scholar]
  51. Zimmermann R., Neupert W. Transport of proteins into mitochondria. Posttranslational transfer of ADP/ATP carrier into mitochondria in vitro. Eur J Biochem. 1980 Aug;109(1):217–229. doi: 10.1111/j.1432-1033.1980.tb04787.x. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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