Figure 4|. Effect of TbTim54 KD on the mitochondrial import of MCPs.

(A & B) Radiolabeled MCP3, MCP5, and MCP11 served as substrates in an in vitro mitochondrial protein import assay using mitochondria isolated from TbTim54-KD and TbTim17-KD cells grown in the absence (Uninduced) and presence (Induced) of doxycycline for 96 h. Mitochondria were re-isolated at different time points during the assay. The proteins were analyzed by SDS-PAGE and autoradiography. Input lanes represent 10% of the radiolabeled substrates used for each reaction. One set of mitochondria was pretreated with CCCP to disrupt the mitochondrial membrane potential (ΔΨ). These pretreated mitochondria were used to show that import of MCPs was ΔΨ-dependent. (C–H) Intensities of protein bands protected from PK digestion were quantitated by densitometry using Image J. Band intensities of the substrates (MCP3, MCP5, and MCP11) in the uninduced control cells at the 10-min time point were set as 100% (control), and the band intensities at other time points for the uninduced and induced samples were calculated as percentages of these control values. Each experiment was repeated at least three times and standard errors are shown. ** P < 0.01, * < 0.05, t-test.