Figure 1.

Characterization of assembly defects of ATP synthase in Δoxa1 and Δcox18 mutants. (A) Mitochondria from wild-type (WT), Δoxa1, Δcox18, Δoxa1, Δcox18, and Δimp1 strains were solubilized with buffer containing 2% digitonin and subjected to CN-PAGE followed by in-gel ATPase activity assay in the absence or presence of oligomycin (5 μg/ml, + or − oligom, as indicated). The position of the dimeric (V_dim) and monomeric (V_mon) forms of the F₁F_o-ATP synthase are indicated. (B) Mitochondria from WT and Δoxa1 strains were solubilized with either 1 or 2% digitonin as indicated subjected to CN-PAGE, and Western blotted. ATP synthase complexes were detected with an anti-F₁ antiserum. The presence of F₁-containing subcomplexes in the Δoxa1 mitochondria are indicated by an asterisk (*). (C) Mitochondria from rho⁰, WT, Δoxa1, Δcox18, Δimp1, and Δcox18, Δoxa1 strains were subjected to SDS-PAGE and Western blotted using antisera against F₁ subunit β (Atpβ), F_o subunits Atp4, Atp6, Atp9, and the inner membrane protein Tim23. For the analysis of the Atp9 levels, mitochondria were treated with H₂O₂ (see Materials and Methods) to disperse SDS-resistant Atp9 oligomers to monomeric proteins, before the SDS-PAGE analysis.