Fig. 6. Inhibition of protein import by increasing concentrations of KCl. (A) AAC and Su9-DHFR were synthesized in reticulocyte lysate in the presence of ³⁵S-labeled methionine. The reticulocyte lysates were diluted with BSA-buffer pH 7.2, containing different concentrations of KCl as indicated. Wild-type yeast mitochondria were added and the mixtures were incubated for 10 minutes at 25°C. The samples were then cooled-down to 0°C and proteinase K was added to digest non-imported protein. The mitochondria were isolated again by centrifugation and analyzed by SDS-PAGE and autoradiography. The relative amounts of imported protein were determined using a phosphoimager. The amounts of AAC or Su9-DHFR that had been imported in the presence of 80 mM KCl were set to 100% (control). (B) Import of pCIC and mCIC, following the same procedure as in A. (C) Import of pCIC into wild-type mitochondria (WT), and into mitochondria lacking the import receptor Tom70 (tom70) or Tom20 (tom20), respectively. The import experiment essentially followed the scheme described in A, except that the samples contained either 80 mM KCl (-) or 400 mM KCl (+). (D) Import of mCIC, following the same protocol as described in C. (E) Import of pCIC into wild-type mitochondria. The mitochondria were pretreated with 100 µg/ml trypsin for 15 minutes at 0°C (+) or left without trypsin (-). The mitochondria were incubated with pCIC for 10 minutes at 25°C. The samples were subsequently treated with 250 µg/ml proteinase K (+ PK) or left without protease (- PK). The amounts of imported pCIC were determined as described in (A). (F) Relative amounts of imported pCIC vs bound pCIC after import in the presence of different KCl concentrations. The experiment was carried out as described in B, using parallel samples +/- treatment with proteinase K.