FIG. 7.
Tim9p and Tim10p, but not Tim12p and Tom40p, can be cross-linked to an AAC translocation intermediate in strain Δ70k. (A) Radiolabeled AAC was synthesized in vitro and imported into wild-type (WT) or Δ70k mitochondria. A fraction of the import reaction mixture was removed as an untreated control (−BMH), and the remainder was subjected to cross-linking with 0.5 mM BMH for 5 min on ice. After quenching, an aliquot was removed for direct analysis (+BMH). The remainder was denatured with SDS, and equal aliquots were incubated with protein A-Sepharose beads containing immobilized rabbit IgGs monospecific for Tim9p (α9), Tim12p (α12), and Tom40p (α40). After centrifugation, bound proteins were eluted with SDS-containing sample buffer and analyzed by SDS-PAGE and fluorography. The arrow marked “AAC” denotes the position of monomeric AAC. S, standard (as defined in the Fig. 6 legend). (B) As in panel A, except that antibodies against Tim10p were used for immunoprecipitation. (C) As in panel A, with isolated mitochondria from strain Tim9S67C, which expresses Tim9S67Cp, Tim8p, Tim10p, and Tim13p (Table 1), and wild-type mitochondria. Antibodies against Tim9p were used for immunoprecipitation (IP). (D) As in panel B, except that the cross-linker MBS (0.1 mM) was used.