Figure 6.

Purified Tim9/Tim10 complex can bind AAC precursor. (A) Purified Tim9/Tim10 complex added to an import reaction containing intact mitochondria inhibits the import of AAC precursor. Mitochondria from wild-type and from a Tom70 null strain were either incubated with purified Tim9/Tim10 complex or mock treated for 2 min at 0°C (+/- Tim9/Tim10). They were then added to import buffer containing radiolabeled AAC, and as a control, pSu9-DHFR. After incubation for 5 or 20 min, mitochondria were reisolated, resuspended in SEM buffer, and divided into two halves. Both halves were kept on ice without (-PK) or with addition of proteinase K (+PK). Imported proteins were analyzed by SDS-PAGE. p, precursor; m, mature form. (B) The indicated amounts of purified Tim9/Tim10 complex were incubated for 10 min at 0°C with radiolabeled AAC and as a control, pSu9-DHFR. These mixtures were then added to import buffer containing 40 μg of wild-type mitochondria. After incubation for 10 min at 25°C proteinase K was added. Imported proteins were analyzed by SDS-PAGE. The amount of import in the absence of added Tim9/Tim10 complex was taken as 100%. (C) Purified Tim9/Tim10 complex was incubated with a mixture of porin and AAC for 30 min at 4°C. The mixture was then split into three aliquots that were added to protein A-coupled Sepharose beads containing prebound antibodies from preimmune serum or antibodies against either Tim9 or Fis1. After incubation for 3 h at 4°C, the beads were pelleted and proteins in the pellets were subjected to SDS-PAGE, blotting and immunodecoration with antibodies against AAC. The amount of added AAC was taken as 100%.