Fig. 4. Import of pre-proteins into mitoplasts. (A) Mitoplasts were prepared by osmotic shock (swelling, SW) of yeast mitochondria. Reticulocyte lysate containing ³⁵S-labeled Su9-DHFR was incubated for 20 minutes at 25°C with mitochondria (lanes 1 and 2) or mitoplasts (lanes 3 and 4). Valinomycin and oligomycin were added to samples 2 and 4 to dissipate the membrane potential (-). To degrade non-imported protein, 50 µg/ml proteinase K were subsequently added to all samples. The mitochondria were isolated again and analyzed by SDS-PAGE and fluorography. (B) Import of AAC, pCIC and mCIC. The experiment was performed as described in A. (C) Import of AAC, pCIC, and mCIC into intact mitochondria. The ³⁵S-labeled proteins were imported into intact yeast mitochondria for 20 minutes at 25°C. The mitochondria of samples 3 and 4 were subsequently subjected to osmotic shock (+ SW). Following treatment with proteinase K, mitochondria and mitoplasts were isolated again by centrifugation.