Figure 7.

Localization of Atp23p in yeast mitochondria. (A) Mitochondria (Mit) and the postmitochondrial supernatant fraction (PMS) consisting mainly of cyotosolic proteins were prepared from the W303ΔATP23/ST13 (ST13), an atp23 mutant with a chromosomally integrated copy of ATP23 fused to a sequence coding for the HA epitope. Samples of mitochondria and PMS containing 40 μg protein were separated on a 12% polyacrylamide gel, transferred to nitrocellulose, and probed with a monoclonal antibody against the HA tag. After a second reaction with anti-mouse IgG coupled to peroxidase, the antibody–antigen complexes were visualized with the SuperSignal chemiluminescent substrate kit (Pierce, Rockford, IL). (B) Mitochondria (Mit) of the transformant W303ΔATP23/ST13 expressing HA-tagged Atp23 was suspended at a protein concentration of 10 mg/ml in 0.6 M sorbitol, 20 mM Tris-HCl, pH 7.5, and 0.5 M EDTA (STE) and was disrupted by sonic irradiation for 3 s with a Branson Sonifier microtip. The suspension was centrifuged at 100,000 × g_av for 20 min. The supernatant (Sup) was collected, and the pellet (SMP) consisting of submitochondrial membrane vesicles was suspended in the starting volume of STE. Mitochondria (40 μg protein) and equivalent volumes of the supernatant and membrane pellet after sonic irradiation were separated on a 12% polyacrylamide gel and processed as in panel A. (C) Submitochondrial membranes (SMP) were suspended in STE at a protein concentration of 10 mg/ml and extracted with the indicated concentrations of potassium deoxycholate (DOC) in the presence of 1 M KCl. After centrifugation at 100,000 × g_av for 15 min, the extracts were collected and the pellets suspended in the starting volume of STE. Samples corresponding to 40 μg of the submitochondrial membranes were separated on a 12% polyacrylamide gel and processed as in A. The positions of mature subunit 6 (Atp6) is indicated by the arrow. (D) Mitochondria were prepared by the method of Glick (1985) from the W303ΔATP23/ST13 the atp23 null mutant with a chromosomally integrated copy of the ATP23 fusion gene expressing the protein with a C-terminal HA tag. The mitochondria were suspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.4, at a protein 8 mg/ml in 0.6 M sorbitol, 20 mM HEPES, pH 7.5 (SH). Equal samples of mitochondria were diluted with 8 volumes of either 0.6 M sorbitol, 20 mM HEPES, pH 7.5, or 20 mM HEPES, pH 7.5, to cause lysis of mitochondria to mitoplasts. Proteinase K (prot K) was added to one-half of each sample to a final concentration of 100 μg/ml. After incubation for 60 min on ice, the reaction was quenched with 2 mM phenylmethylsulfonyl fluoride, and the mitochondria and mitoplasts were recovered by centrifugation at 100,000 × g_av for 10 min. The pellets were suspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.5, and precipitated by addition of 0.1 volume of 50% trichloroacetic acid. The precipitated proteins were dissolved in Laemmli sample buffer and heated for 5 min at 90°C. Mitochondrial (Mt) and mitoplast (Mp) proteins (40 μg) were separated by SDS-PAGE on a 12% polyacrylamide gel, transferred to nitrocellulose, and probed with a mAb against the HA tag and with rabbit polyclonal antibodies against α-ketoglutarate dehydrogenase (α-KGD), Sco1p, and cytochrome b₂ (Cyt b₂). Antibody-antigen complexes were visualized as in panel A after a secondary reaction with either anti-mouse or anti-rabbit IgG.