The affinity purification and characterization of ATP synthase complexes from mitochondria

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.

Figure 1.

Purification of the F₁F_o-ATPase inhibitor complexes from bovine, ovine, porcine and yeast mitochondria by nickel affinity chromatography. The inhibitor was (a,b) I1–60GFPHis and (c) I1–60His. (a) Elution profile of the inhibited bovine F₁F_o-ATPase-I1–60GFPHis complex monitored at 280 nm. (b) SDS–PAGE analysis of fraction 43 (1 ml) from (a). The inhibitor protein is found between the γ- and b-subunits (compare with (c)). (c) Tracks 1–4 are purified inhibited F₁F_o-ATPase-I1–60His from cows, sheep, pigs and S. cerevisiae, respectively. Subunits were identified by mass spectrometric analysis of peptides derived from stained bands.

Figure 2.

Purification of active bovine F₁F_o-ATPase by GST affinity chromatography. (a) SDS–PAGE analysis of fractions 4–13 (each 1 ml) recovered from the column after reversal of inhibition with EDTA. (b) Analysis of pooled fractions of purified bovine F₁F_o-ATPase. Subunits were identified by mass mapping of tryptic peptides derived from stained bands.

Figure 3.

A comparison of the degradation of uncoupled and coupled samples of bovine F₁F_o-ATPase by mild proteolysis. The uncoupled and coupled samples correspond to samples of the enzyme purified in the absence of phospholipids and in the presence of synthetic phospholipids, respectively (table 1). Samples were removed after various times (given in minutes across the top of the gel), and the products were analysed by SDS–PAGE. The migration positions of the subunits of the enzyme complex are indicated on the right-hand side of the gel. BPTI is the bovine pancreatic trypisin inhibitor protein used to terminate proteolysis.

Figure 4.

A comparison of the susceptibility to trypsinolysis of the peripheral stalk and F_o-subunits of bovine F₁F_o-ATPase in samples of the uncoupled and coupled enzymes. Samples were analysed by liquid chromatography linked online to mass spectrometry (LC–MS) at various times during a 2 h period of proteolysis. The histograms show the times at which the various subunits, shown on the abscissa, were last observed. Uncoupled and coupled samples of F₁F_o-ATPase are shown in black and light grey, respectively.

Figure 5.

Proton-pumping activity of affinity-purified F₁F_o-ATPase reconstituted into liposomes. The purified enzyme had an ATP hydrolase activity of 24.8 µmol min⁻¹ mg⁻¹, which was 91% sensitive to oligomycin. The formation of a proton-motive force dependent on ATP hydrolysis was monitored from the change of fluorescence of ACMA. The enzyme was inhibited with (a) oligomycin and (b) the ATPase inhibitor protein, I1–60GFP.

Figure 6.

The synthesis of ATP in proteoliposomes containing bovine F₁F_o-ATPase and bacteriorhodopsin. The enzyme was purified in the presence of phospholipids (POPC : POPG : POPE, 3 : 1 : 1, by wt) and had a specific activity of 19.3 µmol min⁻¹ mg⁻¹ that was 92% sensitive to oligomycin. The assay was carried out in the presence of I1–60GFP to inhibit any uncoupled enzyme. Open circles, in the presence of white light: open triangles, in the presence of red light; open squares, in the presence of white light plus FCCP; filled circles, control in the absence of white light and I1–60GFP.