The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species

Abstract

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

Figure 1. Proton-pumping activity of the F-ATPase from P. angusta

The purified enzyme was reconstituted into liposomes. (A) Quenching of ACMA. The upper and lower lines correspond to the reconstituted F-ATPase from P. angusta in the presence and absence of oligomycin respectively. (B) ATP synthesis by the F-ATPase from P. angusta reconstituted into liposomes. Filled circles, F-ATPase only; filled squares, F-ATPase in the presence of oligomycin; filled triangles, F-ATPase in the presence of gramicidin. The rate of ATP synthesis was 8.9 s⁻¹.

Figure 2. Subunit compositions of fungal F-ATPases

The subunits of the F-ATPases purified from (A) Y. lipolytica, (B) P. pastoris, (C) P. angusta and (D) S. cerevisiae were separated by SDS/PAGE. They were identified by MS analysis of tryptic peptides derived from the gel bands. (A) and (B) show active enzymes; (C) and (D) show the complex of the enzyme with the inhibitor protein bovI-(1–60)–His₁₀. OSCP, oligomycin-sensitivity-conferring protein.

Figure 3. The thermal stability of purified fungal F-ATPases

The enzymes were heated at a range of temperatures for 10 min. (A) ATP hydrolase activities of the enzymes. (B) Sensitivity of those activities to inhibition by oligomycin. The triangles, squares and filled circles correspond to the bovine enzyme and the enzymes from S. cerevisiae and P. angusta respectively.

Figure 4. Alignment of the sequences of fungal subunits k and l

The N-terminal methionine residues in the k and l subunits from P. angusta and S. cerevisiae are removed post-translationally. In Y. lipolytica and P. pastoris, the protein has not been characterized. There is no subunit l in Y. lipolytica. A predicted transmembrane α-helix is indicated by a black bar.