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. 1990 Jul;87(13):4996–5000. doi: 10.1073/pnas.87.13.4996

Reversible import of apocytochrome c into mitochondria.

T B Hakvoort 1, J R Sprinkle 1, E Margoliash 1
PMCID: PMC54248  PMID: 2164215

Abstract

35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.

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Selected References

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  1. Dumont M. D., Mathews A. J., Nall B. T., Baim S. B., Eustice D. C., Sherman F. Differential stability of two apo-isocytochromes c in the yeast Saccharomyces cerevisiae. J Biol Chem. 1990 Feb 15;265(5):2733–2739. [PubMed] [Google Scholar]
  2. 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]
  3. Fisher W. R., Taniuchi H., Anfinsen C. B. On the role of heme in the formation of the structure of cytochrome c. J Biol Chem. 1973 May 10;248(9):3188–3195. [PubMed] [Google Scholar]
  4. Greenawalt J. W. The isolation of outer and inner mitochondrial membranes. Methods Enzymol. 1974;31:310–323. doi: 10.1016/0076-6879(74)31033-6. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Hennig B., Koehler H., Neupert W. Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4963–4967. doi: 10.1073/pnas.80.16.4963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hennig B., Neupert W. Assembly of cytochrome c. Apocytochrome c is bound to specific sites on mitochondria before its conversion to holocytochrome c. Eur J Biochem. 1981 Dec;121(1):203–212. doi: 10.1111/j.1432-1033.1981.tb06450.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Klotz I. M. Ligand--receptor interactions: facts and fantasies. Q Rev Biophys. 1985 Aug;18(3):227–259. doi: 10.1017/s0033583500000354. [DOI] [PubMed] [Google Scholar]
  11. Korb H., Neupert W. Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c. Eur J Biochem. 1978 Nov 15;91(2):609–620. doi: 10.1111/j.1432-1033.1978.tb12714.x. [DOI] [PubMed] [Google Scholar]
  12. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  13. Matlib M. A., O'Brien P. J. Properties of rat liver mitochondria with intermembrane Cytochrome c. Arch Biochem Biophys. 1976 Mar;173(1):27–33. doi: 10.1016/0003-9861(76)90230-7. [DOI] [PubMed] [Google Scholar]
  14. Morimoto T., Matsuura S., Arpin M. Biosynthesis of cytochrome c and its posttranslational transfer into mitochondria. Methods Enzymol. 1983;97:408-26, 621-3. doi: 10.1016/0076-6879(83)97152-5. [DOI] [PubMed] [Google Scholar]
  15. Nargang F. E., Drygas M. E., Kwong P. L., Nicholson D. W., Neupert W. A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria. J Biol Chem. 1988 Jul 5;263(19):9388–9394. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Nicholson D. W., Neupert W. Import of cytochrome c into mitochondria: reduction of heme, mediated by NADH and flavin nucleotides, is obligatory for its covalent linkage to apocytochrome c. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4340–4344. doi: 10.1073/pnas.86.12.4340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  20. Swanson M. S., Zieminn S. M., Miller D. D., Garber E. A., Margoliash E. Developmental expression of nuclear genes that encode mitochondrial proteins: insect cytochromes c. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1964–1968. doi: 10.1073/pnas.82.7.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Taniuchi H., Basile G., Taniuchi M., Veloso D. Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase. J Biol Chem. 1983 Sep 25;258(18):10963–10966. [PubMed] [Google Scholar]
  22. Veloso D., Basile G., Taniuchi H. Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase. J Biol Chem. 1981 Aug 25;256(16):8646–8651. [PubMed] [Google Scholar]
  23. Zhou L. X., Jordi W., De Kruijff B. Influence of heme and importance of the N-terminal part of the protein and physical state of model membranes for the apocytochrome c-lipid interaction. Biochim Biophys Acta. 1988 Jul 7;942(1):115–124. doi: 10.1016/0005-2736(88)90280-5. [DOI] [PubMed] [Google Scholar]
  24. 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]

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