Journal of Bioenergetics and Biomembranes

, 43:333

Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae

  • Vijayakanth Pagadala
  • Luke Vistain
  • Jindrich Symersky
  • David M. Mueller
Article

DOI: 10.1007/s10863-011-9364-5

Cite this article as:
Pagadala, V., Vistain, L., Symersky, J. et al. J Bioenerg Biomembr (2011) 43: 333. doi:10.1007/s10863-011-9364-5

Abstract

The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His6 tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F1Fo ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.

Keywords

ATP synthaseF1Fo ATPaseMitochondrionOxidative phosphorylation

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Vijayakanth Pagadala
    • 1
  • Luke Vistain
    • 1
  • Jindrich Symersky
    • 1
  • David M. Mueller
    • 1
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyRosalind Franklin University of Medicine and Science, The Chicago Medical SchoolNorth ChicagoUSA
  2. 2.North ChicagoUSA