Figure 3.

Mitochondria lacking Mdm12/Mmm1 are impaired in assembly of β-barrel proteins. (A, B) Assembly defect of Tom40. ³⁵S-labeled precursor of Tom40 was imported into mitochondria isolated from wild-type (WT), mdm12Δ and mmm1Δ yeast strains as described in Materials and methods. The incubation time for the import reaction is indicated (min). Assembly into complexes was analyzed by digitonin lysis, blue native electrophoresis and digital autoradiography. (C, D) Import of radiolabeled Tom40 precursor into mitochondria from WT, mmm1Δ or mdm12Δ yeast strains. Mutant mitochondria were adjusted to the same amount of TOM complexes and import was performed at 20°C. After treatment with proteinase K (10 μg/ml), the mitochondria were reisolated and analyzed by SDS–PAGE and digital autoradiography. (E, F) Assembly defect of porin. ³⁵S-labeled precursor of porin was imported into isolated yeast mitochondria. Assembly into complexes was analyzed as described for panels (A, B). (G) Assembly of radiolabeled Sam50 precursor in WT and mdm12Δ mitochondria was performed as described for panel A. (H) Assembly of radiolabeled Mdm10 in WT and mdm12Δ mitochondria was performed as described for panel (A).