Characterization of the initial steps of precursor import into rat liver mitoplasts

Mitochondria have two independent protein-import machineries, one in the outer membrane (the Tom system) and the other in the inner membrane (the Tim system). Here, we have characterized the initial steps of precursor import into rat liver mitoplasts. The import reaction was separated into two stages, consisting of precursor binding to the mitoplasts at 0-10 degreesC, and a subsequent chase reaction at 30 degreesC. This assay revealed four distinct precursor-import steps: DeltaPsi-dependent initial binding of the precursor, precursor transfer to the Tim23-Tim17 stage, DeltaPsi-dependent translocation of the presequence across the inner membrane, and the complete translocation of the mature portion of the precursor. Antibodies against the intermembrane space domain of Tim23 inhibited neither the precursor binding nor the subsequent translocation of the presequence across the inner membrane. In contrast, the antibodies inhibited the complete translocation of the mature domain of the precursor across the inner membrane. Immunoprecipitation with anti-Tim23 IgGs revealed that the precursor-Tim23 complex increased with time and temperature after the initial targeting of the precursor to the mitoplasts. These results suggest that the precursor is first targeted to the inner membrane component DeltaPsi-dependently, then transferred to the Tim system consisting of Tim23-Tim17, and finally imported into the matrix.