Figure 6.

Mia40 is involved in the assembly pathway of small Tim proteins. (A) Mitochondria from WT and mia40-4 cells grown at 19°C were analyzed by SDS–PAGE and immunodecoration. Mia40, Tim10, Mge1 and Tom40, 15 μg mitochondrial protein (odd-numbered lanes), 45 μg mitochondrial protein (even-numbered lanes); other proteins, 25 μg mitochondrial protein (odd-numbered lanes), 50 μg mitochondrial protein (even-numbered lanes). (B) Energized mitochondria isolated from WT and mia40-4 cells grown at 19°C were incubated with the radiolabeled precursor of Tim9 at 30°C. The mitochondria were solubilized with digitonin and analyzed by BN–PAGE and autoradiography. (C) Tim9 was imported into WT and mia40-4 mitochondria at 30°C, followed by treatment with proteinase K. The mitochondria were reisolated and subjected to SDS–PAGE and autoradiography. (D) Mitochondria (μg of protein) from WT and tim10-2 cells grown at 24°C were analyzed by SDS–PAGE and immunodecoration. (E) Tim9 was imported into WT and tim10-2 mitochondria at 30°C, followed by proteinase K treatment and SDS–PAGE. (F) Tim9 was imported into WT and tim10-2 mitochondria at 30°C and analyzed by BN–PAGE and digital autoradiography. (G) Radiolabeled Cox19 and Cox17 were imported into WT or mia40-4 mitochondria in the presence of 10 μM CuCl₂. After swelling and incubation with antibodies as indicated, the mitoplasts were reisolated and analyzed by BN–PAGE.