Figure 8.

The sorting sequence influences the Δψ-dependence of translocation of the matrix-targeting sequence. (A and B) The preproteins of b₂(▵47–65)-DHFR, b₂(K48I, R49C)-DHFR, and b₂(A63P)-DHFR were imported into mitochondria for 2.5 min at 25°C in the presence of the indicated concentrations of CCCP. After import, the samples were split, and one half was treated with proteinase K (+PK; 40 μg/ml) for 15 min on ice, while the other half was left untreated (−PK). After reisolation and separation by SDS-PAGE, the amounts of processed proteins were quantified by phosphorimage analysis. The amount of protein processed in the absence of CCCP was set to 100% (control). Bars indicate the SEs of the means (from four independent experiments). p, precursor; i, intermediate-sized form. (C) Coimmunoprecipitation with antimtHsp70. pb₂(▵47–65)-DHFR was imported into mitochondria for 5 min at 25°C in the presence of the indicated concentrations of CCCP. The mitochondria were reisolated. A portion of each sample was directly analyzed by SDS-PAGE and digital autoradiography (upper panel). The rest of each sample was lysed by nonionic detergent in the presence of EDTA and was subjected to immunoprecipitation with antibodies against mtHsp70 (αHsp70). The bound material was analyzed by SDS-PAGE and digital autoradiography (lower panel). The precursor and processed forms were quantified. The total amount (precursor plus processed form) associated with the mitochondria in the absence of CCCP was set to 100% (control). (D and E) Δψ-dependence of processing of membrane-spanning translocation intermediates. The preproteins of b₂(▵47–65)-DHFR, b₂(K48I, R49C)-DHFR, and b₂(A63P)-DHFR were incubated with isolated mitochondria in the presence of 5 μM MTX and different concentrations of CCCP for 15 min at 25°C. After reisolation and separation by SDS-PAGE, the amounts of processed proteins were quantified by phosphorimage analysis. The amount of each protein processed by the mitochondria in the absence of CCCP was set to 100% (control). Bars indicate the SEs of the means (from four independent experiments).