Figure 5.

The precursors of small Tim proteins associate with Mia40. (A) The radiolabeled precursor of Tim9 was incubated with energized WT mitochondria for 45 min at the indicated temperatures. The mitochondria were reisolated, solubilized by digitonin and subjected to BN–PAGE and autoradiography. Mia40^*, transport intermediate bound to Mia40. (B) Tim9 was imported into WT mitochondria for 10 min at 30°C. The mitochondria were converted to mitoplasts and incubated with antibodies for 30 min on ice. After reisolation and solubilization by digitonin, the samples were analyzed by BN–PAGE and autoradiography. (C) Tim9 was imported into WT mitochondria for 45 min at 30°C in the presence of zinc chloride as indicated. The mitochondria were reisolated, solubilized and subjected to BN–PAGE and autoradiography. Arrowhead, unspecific band. (D) Tim9 was imported into WT mitochondria for 15 min at 15 or 30°C. The mitochondria were lysed in digitonin buffer in the presence or absence of β-mercaptoethanol (β-ME) and analyzed by BN–PAGE. (E) Tim9 was imported into WT mitochondria for 10 min at 30°C where indicated. The mitochondria were subjected to swelling and incubated with antibodies. After reisolation, the mitochondria were mixed as indicated, solubilized with digitonin and analyzed by BN–PAGE and autoradiography. (F) Tim9 was imported into WT mitochondria for 10 min at 30°C. The mitochondria were reisolated, incubated at 30°C as indicated and analyzed by BN–PAGE. (G) Mitochondria isolated from WT and mia40-3 cells grown at 19°C were incubated with the radiolabeled precursors of Tim9 and Tim10 at 30°C. After solubilization with digitonin, the samples were analyzed by BN–PAGE and autoradiography. For comparison, WT mitochondria were analyzed by BN–PAGE and immunodecoration (samples 7 and 8). (H) Tim8 was imported into WT and mia40-3 mitochondria as described in the legend to G.