Fig. 6. Characterization of the mitochondria-targeting signal of rTOM70. (A) Basic amino acids at the C-terminal flanking region of the TMD are critical for targeting rTOM70. COS-7 cells were transfected with the indicated constructs in the expression vectors. After 24 hours, the cells were incubated with Mito Tracker, then the cells were fixed and immunostained with IgG against rabbit polyclonal anti-HA epitope tag antibodies and FITC-conjugated antibodies against rabbit IgG. To monitor the localization of rTOM70S6HA, cells were treated with 5 µg/ml BFA for 1 hour, fixed and immunostained with rabbit polyclonal IgGs against rat Calnexin and FITC-conjugated antibodies against rabbit IgGs. To detect localization of the GFP fusions in COS-7 cells, the cells were co-stained with Mito Tracker, and fluorescence images were obtained using a confocal microscope. (B) Binding and insertion of rTOM70HA, rTOM70S6HA or rTOM70(1-69)GFP into mitochondria in vitro. Reticulocyte-lysate-synthesized ³⁵S-labeled proteins were subjected to mitochondrial import. To measure mitochondria binding of these proteins, the reaction mixtures were centrifuged to isolate mitochondria (P) and the supernatant (S). To verify membrane integration (`Import'), the reaction mixtures were treated with 100 mM sodium carbonate (pH 11.5) at 0°C for 30 minutes and centrifuged to separate the supernatant (S) and membrane (P) fractions. Both fractions were subjected to SDS-PAGE and subsequent fluoroimage analysis. (C) Membrane integration of rTOM70 into trypsin-treated mitochondria. Mitochondrial import assay of rTOM70 was performed using 20 µg/ml trypsin-treated or untreated mitochondria as described in (B). An aliquot of sodium-carbonate-treated mitochondria was centrifuged to separate the membrane (P) and supernatant (S) fractions, and both fractions were subjected to immunoblot analysis using the antibodies against the indicated proteins.