title: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.
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 His(6) 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. F(1)F(o) ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.
authors: Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, IL 60064, USA.
authors: Vijayakanth
authors: V
authors: Pagadala
authors: Luke
authors: L
authors: Vistain
authors: Jindrich
authors: J
authors: Symersky
authors: David M
authors: DM
authors: Mueller
mesh_terms: D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D050356:Lipid Metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D025261:Mitochondrial Proton-Translocating ATPases