Abstract
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 this complex and the binding mode of the inhibitor endow IF1 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 IF1 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.
Highlights
The F-ATP synthase, or F1Fo-ATPase, from mitochondria is an abundant multisubunit assembly associated with the inner membranes of the organelle [1,2]
The structure of the membrane extrinsic region of the enzyme was completed by the addition of the structure of the peripheral stalk, which demonstrated its mode of attachment to the F1 domain via an interaction between the oligomycin sensitivity conferral protein (OSCP) with the N-terminal region of one of the three a-subunits [8,9,15]
As described here, we have developed a simple, rapid and mild purification of the F1Fo-ATPase complex from mitochondria that takes advantage of the exquisitely specific inhibition of the mitochondrial F1Fo-ATPase by its natural inhibitor protein, IF1 [17]
Summary
The F-ATP synthase, or F1Fo-ATPase, from mitochondria is an abundant multisubunit assembly associated with the inner membranes of the organelle [1,2]. The high-resolution structural analysis of the F-ATP synthase has been conducted by the detailed analysis of constituent domains, largely by X-ray crystallography [3,4,5,6,7,8,9,10]. These substructures have been assembled into an overall mosaic structure within the constraints of a 32 Aresolution overall structure determined by cryo-electron microscopy of single particle images of the intact enzyme complex [10,11]. One possible approach to filling this lacuna would be to crystallize the intact F1Fo-ATPase complex, and to determine its high-resolution structure by X-ray crystallography
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