FIG. 2.

The precursor of Oxa1 forms a productive translocation intermediate. (A) Radiolabeled Oxa1 precursor was imported for 5 min at 25°C into mitochondria in the presence of FCCP, as indicated. The reaction mixtures were subsequently split in half and either subjected to proteinase K (Prot. K) treatment and SDS-PAGE or left untreated, solubilized in buffer containing 1% (wt/vol) digitonin, and subjected to BN-PAGE analysis. mOxa1, mature Oxa1. (B) Import of ³⁵S-labeled Oxa1 precursor was performed either in the presence of a Δψ, in the presence of valinomycin (Val), or in the presence of 60 μM FCCP for 20 min at 25°C. After reisolation, mitochondria were resuspended in fresh import buffer and subjected to a second incubation at 10°C for the indicated times. During the second incubation, one sample remained in the presence of Δψ, one sample remained without a Δψ (Val), and samples that had previously received FCCP were now incubated in the presence of a reestablished Δψ. Samples were analyzed by BN-PAGE after solubilization or subjected to SDS-PAGE after proteinase K treatment.