Figure 4.

Mmp37p is a matrix-localized protein. (A) Proteinase K (Prot. K) treatment was performed on intact mitochondria (Mitoch.) or mitochondria which had been subjected to hypotonic swelling (Swelling) or Triton X-100 detergent lysis (T X-100) on ice, as indicated. Samples were subjected to SDS-PAGE and Western blotting. (B) Isolated wild-type mitochondria (0.1 mg/ml) were extracted by alkaline treatment using 0.1 M NaCO₃ buffer at the indicated pH. Control mitochondria (pH 7.5) were resuspended in 0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.5, buffer. Samples were sedimented by centrifugation and the membrane pellet and supernatant (after TCA precipitation) were subjected to SDS-PAGE and Western blotting. Note, the Ccpo protein displays an inherent protease stability and hence is not degraded by the proteinase K treatment in the presence of Triton X-100. (C) Wild-type mitochondria were subjected to a sonication treatment as described in Materials and Methods. T, total input; P, membrane vesicle pellet; S, soluble matrix proteins.