Fig. 1. Import of CIC into isolated rat liver mitochondria. (A) Isolated rat liver mitochondria were preincubated with antibodies directed against Tom20 or Tom70, respectively, at increasing concentrations. Reticulocyte lysate containing the ³⁵S-labeled pCIC or mCIC was added and the samples were incubated for 20 minutes at 25°C. The mitochondria were then cooled-down to 0°C and treated with 50 µg/ml proteinase K to remove non-imported protein. Proteolysis was stopped by addition of PMSF, the mitochondria were isolated again by centrifugation, and imported proteins were analyzed by SDS-PAGE and autoradiography. The relative amounts of ³⁵S-labeled protein were determined using a phosphoimager. The amounts of imported protein in the absence of antibodies were set to 100% (control). (B) Import of AAC and Su9-DHFR (a hybrid protein containing the presequence of subunit 9 of the mitochondrial ATP synthase coupled to dihydrofolate reductase), following the same protocol as in the experiments of A. p, precursor protein; m, mature protein.