Figure 5.

Destabilization of the HBD in the IMS promotes the retrograde translocation of the kinetically arrested translocation intermediates of the pb₂(260)-HBD fusion proteins. (A) pb₂(260)-HBD fusion proteins. (B) Radiolabeled pb₂(260)^WT-HBD (WT), pb₂(260)^dc1-HBD (dc1), and pb₂(260)^dc2-HBD (dc2) were incubated with isolated yeast mitochondria in import buffer for 10 min at 25°C. The mitochondria were reisolated by centrifugation and were incubated at 25°C for indicated times. The mitochondria were divided into halves and were incubated with or without 100 μg/ml proteinase K for 30 min on ice. The mitochondria without proteinase K treatment were reisolated by centrifugation, and proteins recovered with the mitochondria (pellet) and those in the supernatant (sup) were analyzed by SDS/PAGE and radioimaging. Amounts of the forms processed by Imp1p (the mature-size forms in “pellet” and “sup”) are plotted against incubation times in the left panel. Amounts of the imported proteins (the protease-protected mature-size forms) are plotted against incubation times in the center panel; the amounts at time 0 are set to 0%. Amounts of the retrotranslocated proteins (the mature-size forms in “sup”) are plotted against incubation times in the right panel; the amounts at time 0 are set to 0%. The amounts of the kinetically arrested fusion proteins associated with mitochondria after the first incubation are set to 100%. Squares, wild type (WT); triangles, dc1; circles, dc2.