Fig. 3. Accessibility of DIC to proteases after import into mitochondria. ³⁵S-labelled wild-type DIC and the mutant versions CS1, CS2 and CS1/2 were synthesized in reticulocyte lysate and incubated with isolated yeast mitochondria for 5 minutes at 25°C. The mitochondria were then cooled to 0°C and re-isolated by centrifugation. The mitochondria were then resuspended either in 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2 to allow swelling (+SW) of the mitochondria and rupture of the outer membrane, or in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2 to keep the mitochondria intact (–SW). All samples were treated with 250 µg/ml proteinase K for 20 minutes at 0°C. Mitochondria and mitoplasts were re-isolated, the proteins were separated by SDS-PAGE, blotted on nitrocellulose and the radiolabelled proteins were visualized using a PhosphorImager (upper panel). For quantification of each construct, the amount of ³⁵S-labelled protein in the –SW sample was set to 100%. The nitrocellulose was eventually immuno-decorated with polyclonal antibodies directed against the hydrophilic N-terminus of the inner-membrane protein Tim23. PK, proteinase K. Bars represent standard deviation.