FIG. 2.

Association of the small Tom proteins with the 400K GIP complex. (A) Stability of the small Tom proteins in the 400K complex following urea treatment. Mitochondria (50 μg of protein) were incubated with the radiolabeled preproteins of the small Tom proteins in import buffer for 10 min at 25°C. After lysis in solubilization buffer containing 1% digitonin and the indicated concentrations of urea, the samples were subjected to BN-PAGE and digital autoradiography. (B) Quantitation of the urea-resistant Tom proteins in the 400K complex (as described for Fig. 1A and panel A). The total amount of each Tom protein in the 400K complex in the absence of urea was set to 100% (control). (C) Carbonate resistance of the small Tom proteins in the 400K complex. Radiolabeled preproteins of the small Tom proteins were imported into mitochondria (50 μg of protein) for 10 min at 25°C. Mitochondrial pellets were resuspended in 100 mM Na₂CO₃ (pH 11.5) and incubated on ice for 30 min. After centrifugation at 100,000 × g for 30 min, the carbonate-resistant pellets were solubilized in 1% digitonin and separated by BN-PAGE (lanes 4 to 6). As a control, mitochondria containing imported Tom proteins were directly lysed and separated by BN-PAGE (lanes 1 to 3). Tom40 was detected by immunodecoration in a control sample and a carbonate-resistant pellet (lanes 7 and 8, respectively). The amount of carbonate-resistant Tom protein was quantified and compared to the total amount of the respective Tom protein in the 400K complex (control) (columns 9 to 12). For comparison, radiolabeled small Tom proteins were imported into mitochondria (lane 13), separated into carbonate-resistant pellet (lane 14) and carbonate-extractable supernatant (Sn.; TCA precipitated; lane 15), and subjected to SDS-PAGE.