Figure 1.

Phenotype of atp23 mutants. (A) Spectra of mitochondrial cytochromes in wild type, atp23 mutants, and revertants. Mitochondria of the wild-type strain W303-1A, the atp23 mutants aE884/UL1 and W303ΔATP23 (ΔATP23), and two independent revertants of W303ΔATP23 (R1, R2) were extracted with potassium deoxycholate at a protein concentration of 5 mg/ml as described previously (Tzagoloff et al., 1975). Difference spectra of the extracts oxidized with potassium ferricyanide and reduced with sodium dithionite were recorded at room temperature. The α-absorption bands corresponding to cytochromes a, a₃, and b and cytochromes c and c₁ are indicated. The percentages of ρ^o/− mutants were 85% for W303ΔATP23, and 20% for the revertants. (B) Sedimentation of mitochondrial ATPase. Mitochondrial of the wild-type strain W303-1A, of the atp23 null mutant aW303ΔATP23, and of revertant W303ΔATP23/R1 were suspended at a protein concentration of 10 mg/ml in 2 mM ATP, 1 mM EDTA, and 20 mM Tris-Cl, pH 7.5. The suspension was adjusted to a final concentration of 0.4% with a 10% solution of Triton X-100 (Tzagoloff and Meagher, 1971) and centrifuged at 250,000 × g_av for 15 min. The clarified extracts (0.5 ml) were mixed with 45 μg of β-galactosidase and applied on top of 5 ml of 7–20% linear sucrose gradients containing 2 mM ATP, 20 mM Tris-Cl, pH 7.5, 1 mM EDTA, and 0.1% Triton X-100. After centrifugation at 65,000 rpm in a Beckman SW65 rotor for 3.5 h, 16 equal fractions were collected and assayed for ATPase and β-galactosidase (Wallenfels, 1962). (C) Western analysis of mitochondrial F_O and F₁ subunits. Mitochondria (40 μg protein) from the same strains as in panel A as well as the atp23 point mutant harboring the wild-type ATP23 gene (aE884/UL1/T1) were separated by SDS-PAGE on a 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with polyclonal antibodies to the α subunit of F₁ and subunits 4 and 6 of F_O. After a second reaction with peroxidase-conjugated anti-rabbit IgG, the antibody-antigen complexes were visualized with the SuperSignal chemiluminescent substrate kit (Pierce, Rockford, IL).