Advertisement

Mitochondria pp 363-377 | Cite as

Methods to Determine the Status of Mitochondrial ATP Synthase Assembly

  • Sharon H. Ackerman
  • Alexander Tzagoloff
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

The adenosine triphosphate (ATP) synthase (F1-F0 complex) of the mitochondrial inner membrane is responsible for making nearly all of the ATP utilized by eukaryotic organisms. The enzyme is an oligomer of more than 20 different subunits, 14 of which are essential for its catalytic activity. The other subunits function in the regulation and structure of the complex. Subunits essential for catalytic activity make up the proton pore, the bulk of the F1 headpiece, and the two stalks that physically and functionally couple the catalytic and proton-translocating activities of the ATP synthase. Saccharomyces cerevisiae provides an excellent model system for studying mutations that affect assembly of the complex because of the ability of this organism to survive on the ATP produced from fermentation in the absence of mitochondrial respiration or oxidative phosphorylation. Studies of such mutants have been instrumental in identifying novel molecular chaperones that act at discrete steps of F1-F0 assembly. Here, we describe some experimental approaches useful in assessing the status of F1-F0 assembly.

Key Words

ATPase assays ATPase mutants F1-F0 assembly isopycnic gradients mitochondrial ATP synthase reconstitution of F1-F0 sedimentation of F1 and F1-F0 

References

  1. 1.
    Boyer, P. D. (1997) The ATP synthase-a splendid molecular machine. Annu. Rev. Biochem. 66, 717–749.CrossRefPubMedGoogle Scholar
  2. 2.
    Ackerman, S. H. and Tzagoloff, A. (2005) Function, structure, and biogenesis of mitochondrial ATP synthase. Prog. Nucl. Acids Res. Mol. Biol. 80, 95–133.CrossRefGoogle Scholar
  3. 3.
    Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schagger, H. (1998) Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J. 17, 7170–7178.CrossRefPubMedGoogle Scholar
  4. 4.
    Nakamoto, R. K., Ketchum, C. J., and al-Shawi, M. K. (1999) Rotational coupling in the F0F1 ATP synthase. Annu. Rev. Biophys. Biomol. Struct. 28, 205–234.CrossRefPubMedGoogle Scholar
  5. 5.
    Faye, G., Kujawa, C., and Fukuhara, H. (1974) Physical and genetic organization of petite and grande yeast mitochondrial DNA. IV. In vivo transcription products of mitochondrial DNA and localization of 23 S ribosomal RNA in petite mutants of Saccharomyces cerevisiae. J. Mol. Biol. 88, 185–203.CrossRefPubMedGoogle Scholar
  6. 6.
    Tzagoloff, A. (1969) Assembly of the mitochondrial membrane system. II. Synthesis of the mitochondrial adenosine triphosphatase, F1. J. Biol. Chem. 244, 5027–5033.PubMedGoogle Scholar
  7. 7.
    King, E. J. (1932) The colorimetic determination of phosphorus, Biochem. J. 26, 292–297.PubMedGoogle Scholar
  8. 8.
    Pullman, M. E., Penefsky, H. S., Datta, A., and Racker, E. (1960) Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphatase. J. Biol. Chem. 235, 3322–3329.PubMedGoogle Scholar
  9. 9.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefPubMedGoogle Scholar
  10. 10.
    Ackerman, S. H. and Tzagoloff, A. (1990) ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex. J. Biol. Chem. 265, 9952–9959.PubMedGoogle Scholar
  11. 11.
    Velours, J., Arselin de Chateaubodeau, G., Galante, M., and Guerin, B. (1987) Subunit 4 of ATP synthase (F0F1) from yeast mitochondria. Purification, aminoacid composition and partial N-terminal sequence. Eur. J. Biochem. 164, 579–584.CrossRefPubMedGoogle Scholar
  12. 12.
    Ackerman, S. H. and Tzagoloff, A. (1990) Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase. Proc. Natl. Acad. Sci. U. S. A. 87, 4986–4990.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Sharon H. Ackerman
    • 1
  • Alexander Tzagoloff
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyWayne State University School of MedicineDetroit
  2. 2.Department of Biological SciencesColumbia UniversityNew York

Personalised recommendations