Fig. 5. Participation of Tim44 and mitochondrial Hsp70 (mtHsp70) in import of pre-proteins. (A) Mitochondria were isolated from the temperature-sensitive yeast strain tim44-8, wild-type mitochondria (WT) were isolated in parallel under the same conditions. To induce the phenotype, the isolated mitochondria were incubated for 15 minutes at 37°C. The mitochondria were then incubated with reticulocyte lysate containing ³⁵S-labeled pCIC at 25°C. Samples were taken after different time points as indicated, and treated with 250 µg/ml proteinase K for 20 min at 0°C. The proteins were subjected to SDS-PAGE and autoradiography. The relative amounts of imported pCIC were determined using a phosphoimager. The highest value was set to 100% (control). (B) Import of Su9-DHFR. The experiment followed the same protocol as described in A, using mitochondria of the same preparation. (C) Import of AAC. (D) Import of pCIC into mitochondria of the temperature-sensitive yeast strain ssc1-3. The gene SSC1 encodes the major 70 kDa heat shock protein of the mitochondrial matrix. (E) Import of Su9-DHFR into mitochondria from the same preparation as in experiment D. (F) Import of AAC.