Dataset Information

The affinity purification and characterization of ATP synthase complexes from mitochondria.

ABSTRACT: The mitochondrial F?-ATPase inhibitor protein, IF?, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the ?-helical inhibitory region of the bound IF? occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the ?-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF? with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF? with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

SUBMITTER: Runswick MJ

PROVIDER: S-EPMC3603449 | biostudies-other | 2013 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

The affinity purification and characterization of ATP synthase complexes from mitochondria.

Runswick Michael J MJ Bason John V JV Montgomery Martin G MG Robinson Graham C GC Fearnley Ian M IM Walker John E JE

Open biology 20130213 2

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming ...[more]

PMID: 23407638

Similar Datasets

Project description:The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.

Dataset Information

The affinity purification and characterization of ATP synthase complexes from mitochondria.

Publications

The affinity purification and characterization of ATP synthase complexes from mitochondria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets