FIGURE 6:

Effect of oxidative stress on protein import and ATPase activity of mHsp70. (A) Mitochondria isolated from wild type or mutants were either pretreated with 20 μM antimycin A for 20 min or left on ice before import. Mitochondria were treated with valinomycin (5 μm) for 10 min on ice. Reticulocyte lysate–synthesized, ³⁵S-labeled mitochondrial precursor proteins (CoxIV and Su9-DHFR) were imported with or without antimycin A into pretreated wild-type Mge1 and Mge1-M155L mutant mitochondria for different time points. The samples were incubated with (right) and without (left) 25 μg/ml trypsin and analyzed by SDS–PAGE, followed by digital autoradiography as mentioned in Materials and Methods. This is a representative figure of two independent experiments. (B) Purified Mge1 and Mge1-M155L mutant were treated with or without 1 mM H₂O₂ for 20 min in 20 mM HEPES/KOH, pH 7.2, buffer. Then the proteins were incubated with recombinant Hsp70 in ATPase assay buffer, and we analyzed the release of labeled P_i at different time points as indicated in Materials and Methods.