Fig. 2. Import of CIC into isolated yeast mitochondria. (A) Mitochondria were isolated from yeast strains lacking the import receptor Tom70 (tom70) or Tom20 (tom20). Wild-type mitochondria (WT) were isolated in parallel. ³⁵S-labeled pCIC was synthesized in reticulocyte lysate and incubated with the isolated mitochondria at 25°C. Aliquots were removed at different time points as indicated and cooled-down to 0°C. The mitochondria were incubated with 250 µg/ml proteinase K for 20 minutes and isolated again by centrifugation. Imported proteins were analyzed by SDS-PAGE and fluorography. The relative amount of imported pCIC was determined using a phosphoimager. The highest value was set to 100% (control). Within import-times of 10 minutes, up to 11% of added pCIC were imported. (B) Import of mCIC, following the same protocol as described in A. (C) Import of Su9-DHFR. (D) Import of AAC.