FIGURE 6.

MtHsp70 promotes the assembly of Hsp60 complexes. A, ³⁵S-labeled Hsp60 was imported into WT and ssc1–42 mitochondria at the indicated time points. Where indicated, isolated mitochondria (Mito) were subjected to in vitro heat shock, or the membrane potential (Δψ) was dissipated prior to import. Proteins were analyzed by SDS-PAGE (SDS) or blue native (BN) electrophoresis and autoradiography. B, quantification of the import and assembly (Hsp60₁₄) experiments in A. The longest import time point in WT mitochondria was set to 100% (control). Mean ± S.E. of four independent experiments is depicted. C, ³⁵S-labeled Hsp60 was imported into WT and ssc1–42 mitochondria for a short time (Pulse), followed by further incubation after block of the import (Chase). Proteins were analyzed by SDS-PAGE or blue native electrophoresis and autoradiography. D, ³⁵S-labeled Hsp60 was imported into WT and mtHsp70_His mitochondria for a short time (Pulse), followed by further incubation after block of the import (Chase). Mitochondria were lysed with digitonin, and proteins were purified via Ni²⁺-NTA-agarose. Bound proteins were eluted with ATP and analyzed by SDS-PAGE or blue native electrophoresis and autoradiography.