Figure 2 Protein oxidation in the IMS is substrate-specific

FIGURE 2: Dre2 is not in but on mitochondria.

(A) Mitochondria of Dre2-overexpressing cells were sonicated in the presence of 50 µg/ml proteinase K so that only intrinsically protease-stable proteins remain undigested. Proteins were collected by TCA-precipitation and visualized by Western blotting. Cox2 and Mrpl40 are protease-resistant due to their integration into large protein complexes.

(B) Mitochondria of Dre2-overexpressing cells were lysed with 1% Triton X-100. The extract was either directly loaded to the gel (T, total) or after separation of supernatant (S) and pellet (P) fractions by centrifugation for 30 min at 140,000 x g.

(C) Mitochondria were isolated from wild type or GAL-MIA40 cells. The levels of endogenous Dre2 and control proteins were analyzed by Western blotting.

(D) Wild type or GAL-MIA40 cells were transformed with a Dre2-overexpression plasmid and grown in glucose (D) or galactose (Gal) containing medium. Mitochondria were isolated and subjected to Western blotting with antibodies against the depicted proteins.

(E) Mitochondria were isolated from a Dre2-overexpressing strain and incubated with SEH buffer (0.6 M sorbitol, 5 mM EDTA, 20 mM Hepes pH 7.4) in the absence or presence of 0.5 M sodium chloride or urea for 10 min at 30°C. Mitochondria were reisolated by centrifugation. Proteins from the mitochondrial (M) and supernatant (S) fraction were collected by TCA precipitation. The Dre2 signals were quantified and the proportion of Dre2 is indicated that was removed from the mitochondria in the washing step.

(F) Mitochondria of Dre2-overexpressing cells were incubated in SEH buffer for 10 min at 30°C. Mitochondria were reisolated by centrifugation for 7 min at 16,000 x g and dissolved in SEH buffer containing 0.5 M NaCl or urea, respectively. Incubation and centrifugation steps were repeated with increasing concentrations of salt or urea as indicated. Proteins from the supernatant was precipitated by TCA. P, mitochondrial pellet after all washing steps. T, total representing 50 µg mitochondria that were directly loaded.