Advertisement

Mitochondria pp 325-342 | Cite as

Investigation of Iron-Sulfur Protein Maturation in Eukaryotes

  • Oliver Stehling
  • Paul M. Smith
  • Annette Biederbick
  • Janneke Balk
  • Roland Lill
  • Ulrich Mühlenhoff
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

Iron-sulfur (Fe-S) clusters are cofactors of many proteins that are involved in central biochemical pathways, such as oxidative phosphorylation, photosynthesis, and amino acid biosynthesis. The assembly of these cofactors and the maturation of Fe-S proteins require complex cellular machineries in all kingdoms of life. In eukaryotes, Fe-S protein biogenesis is an essential process, and mitochondria perform a primary role in synthesis. Defects in Fe-S protein maturation in yeast result in respiratory deficiency and auxotrophies for certain amino acids and vitamins that require Fe-S proteins for their biosynthesis. Frequently, heme biosynthesis is also affected. The present compendium describes assays for the analysis of de novo Fe-S cluster and heme formation, cellular iron homeostasis, and the activity of Fe-S cluster- and heme-containing enzymes. These approaches are crucial to elucidate the mechanisms underlying the maturation of Fe-S proteins and may aid in the identification of new members of this evolutionary ancient process.

Key Words

Biosynthesis iron-sulfur proteins heme iron homeostasis mammalian cell culture Saccharomyces cerevisiae 

References

  1. 1.
    Beinert, H., Holm, R. H., and Munck, E. (1997) Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 277, 653–659.PubMedCrossRefGoogle Scholar
  2. 2.
    Johnson, D. C., Dean, D. R., Smith, A. D., and Johnson, M. K. (2004) Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281.CrossRefGoogle Scholar
  3. 3.
    Balk, J. and Lill, R. (2004) The cell’s cookbook for iron-sulfur clusters: recipes for fool’s gold? Chembiochem. 5, 1044–1049.PubMedCrossRefGoogle Scholar
  4. 4.
    Lill, R. and Muhlenhoff, U. (2005) Iron-sulfur-protein biogenesis in eukaryotes. Trends Biochem. Sci. 30, 133–141.PubMedCrossRefGoogle Scholar
  5. 5.
    Kispal, G., Csere, P., Prohl, C., and Lill, R. (1999) The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. EMBO J. 18, 3981–3989.PubMedCrossRefGoogle Scholar
  6. 6.
    Jensen, L. T. and Culotta, V. C. (2000) Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis. Mol. Cell. Biol. 20, 3918–3927.PubMedCrossRefGoogle Scholar
  7. 7.
    Kispal, G., Sipos, K., Lange, H., et al. (2005) Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria. EMBO J. 24, 589–598.PubMedCrossRefGoogle Scholar
  8. 8.
    Yarunin, A., Panse, V. G., Petfalski, E., Dez, C., Tollervey, D., and Hurt, E. C. (2005) Functional link between ribosome formation and biogenesis of iron-sulfur proteins. EMBO J. 24, 580–588.PubMedCrossRefGoogle Scholar
  9. 9.
    Lange, H., Muhlenhoff, U., Denzel, M., Kispal, G., and Lill, R. (2004) The heme synthesis defect of mutants impaired in mitochondrial iron-sulfur protein biogenesis is caused by reversible inhibition of ferrochelatase. J. Biol. Chem. 279, 29,101–29,108.PubMedCrossRefGoogle Scholar
  10. 10.
    Kispal, G., Csere, P., Guiard, B., and Lill, R. (1997) The ABC transporter Atm1p is required for mitochondrial iron homeostasis. FEBS Lett. 418, 346–350.PubMedCrossRefGoogle Scholar
  11. 11.
    Rutherford, J. C., Ojeda, L., Balk, J., Muhlenhoff, U., Lill, R., and Winge, D. R. (2005) Activation of the iron regulon by the yeast Aft1/Aft2 transcription factors depends on mitochondrial but not cytosolic iron-sulfur protein biogenesis. J. Biol. Chem. 280, 10,135–10,140.PubMedCrossRefGoogle Scholar
  12. 12.
    Eisenstein, R. S. (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu. Rev. Nutr. 20, 627–662.PubMedCrossRefGoogle Scholar
  13. 13.
    Muhlenhoff, U., Richhardt, N., Gerber, J., and Lill, R. (2002) Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP, NADH, and reduced iron. J. Biol. Chem. 277, 29,810–29,816.PubMedCrossRefGoogle Scholar
  14. 14.
    Lutz, T., Westermann, B., Neupert, W., and Herrmann, J. M. (2001) The mitochondrial proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria. J. Mol. Biol. 307, 815–825.PubMedCrossRefGoogle Scholar
  15. 15.
    Mullner, E. W., Neupert, B., and Kuhn, L. C. (1989) A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell 58, 373–382.PubMedCrossRefGoogle Scholar
  16. 16.
    Guthrie, C. and Fink, G. R. (1991) Guide to yeast genetics and molecular biology. Meth. Enzymol. 194, 1–863.Google Scholar
  17. 17.
    Diekert, K., de Kroon, A. I., Kispal, G., and Lill, R. (2001) Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 65, 37–51.PubMedCrossRefGoogle Scholar
  18. 18.
    Muhlenhoff, U., Richhardt, N., Ristow, M., Kispal, G., and Lill, R. (2002) The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins. Hum. Mol. Genet. 11, 2025–2036.PubMedCrossRefGoogle Scholar
  19. 19.
    Meyer, J., Moulis, J. M., and Lutz, M. (1986) High-yield chemical assembly of [2Fe-2X] (X = S, Se) clusters into spinach apoferredoxin: product characterisation by Raman spectroscopy. Biochim. Biophys. Acta 871, 243–249.CrossRefGoogle Scholar
  20. 20.
    Lange, H., Kispal, G., and Lill, R. (1999) Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria. J. Biol. Chem. 274, 18,989–18,996.PubMedCrossRefGoogle Scholar
  21. 21.
    Li, J., Kogan, M., Knight, S. A., Pain, D., and Dancis, A. (1999) Yeast mitochondrial protein, Nfs1p, coordinately regulates iron-sulfur cluster proteins, cellular iron uptake, and iron distribution. J. Biol. Chem. 274, 33,025–33,034.PubMedCrossRefGoogle Scholar
  22. 22.
    Makino, T., Kiyonaga, M., and Kina, K. (1988) A sensitive, direct colorimetric assay of serum iron using the chromogen, nitro-PAPS. Clin. Chim. Acta 171, 19–27.PubMedCrossRefGoogle Scholar
  23. 23.
    O’Connell, I. A. R. a. E. L. (1967) Mechanism of aconitase action. I. The hydrogen transfer reaction. J. Biol. Chem. 242, 1870–1879.PubMedGoogle Scholar
  24. 24.
    Drapier, J. C. and Hibbs, J. B., Jr. (1996) Aconitases: a class of metalloproteins highly sensitive to nitric oxide synthesis. Meth. Enzymol. 269, 26–36.PubMedCrossRefGoogle Scholar
  25. 25.
    Hausladen, A. and Fridovich, I. (1996) Measuring nitric oxide and superoxide: rate constants for aconitase reactivity. Meth. Enzymol. 269, 37–41.PubMedCrossRefGoogle Scholar
  26. 26.
    Hatefi, Y. and Stiggall, D. L. (1978) Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Meth. Enzymol. 53, 21–27.PubMedCrossRefGoogle Scholar
  27. 27.
    Birch-Machin, M. A. and Turnbull, D. M. (2001) Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 65, 97–117.PubMedCrossRefGoogle Scholar
  28. 28.
    Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.PubMedCrossRefGoogle Scholar
  29. 29.
    Siegel, L. and Englard, S. (1962) Beef-heart malic dehydrogenases. III. Comparative studies of some properties of M-malic dehydrogenase and S-malic dehydrogenase. Biochim. Biophys. Acta 64, 101–110.PubMedCrossRefGoogle Scholar
  30. 30.
    Leibold, E. A., and Munro, H. N. (1988) Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′-untranslated region of ferritin heavy-and light-subunit mRNAs. Proc. Natl. Acad. Sci. USA. 85, 2171–2175.PubMedCrossRefGoogle Scholar
  31. 31.
    Takahashi, Y., Mitsui, A., and Matsubara, H. (1991) Formation of the Fe-S cluster of ferredoxin in lysed spinach chloroplasts. Plant Physiol. 95, 97–103.PubMedCrossRefGoogle Scholar
  32. 32.
    Suzuki, S., Izumihara, K., and Hase, T. (1991) Plastid import and iron-sulphur cluster assembly of photosynthetic and nonphotosynthetic ferredoxin isoproteins in maize. Plant Physiol. 97, 375–80.PubMedCrossRefGoogle Scholar
  33. 33.
    Stehling, O., Elsasser, H. P., Bruckel, B., Muhlenhoff, U., and Lill, R. (2004) Iron-sulfur protein maturation in human cells: evidence for a function of frataxin. Hum. Mol. Genet. 13, 3007–15. Epub October 27, 2004.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Oliver Stehling
    • 1
  • Paul M. Smith
    • 1
  • Annette Biederbick
    • 1
  • Janneke Balk
    • 1
    • 2
  • Roland Lill
    • 1
  • Ulrich Mühlenhoff
    • 1
  1. 1.Institut für Zytobiologie und ZytopathologiePhilipps-Universität MarburgMarburgGermany
  2. 2.Department of Plant SciencesCambridge UniversityCambridgeUK

Personalised recommendations