Tim50 mutants with mutations in the coiled-coil region in the IMS. (A) Amino acid sequences of the IMS domains of wild-type control (WT) and mutant Tim50 (top) and coiled-coil regions in Tim50 (bottom) predicted as in Fig. 1 A. Mutation points are shown in black. (B) Serial dilutions of wild-type and tim50 mutant cells were plated on SCD (−Trp) and YPGlycerol and grown at the indicated temperature for 2 and 3 d, respectively. (C) Mitochondria were isolated from wild-type and tim50 mutant cells grown at 23°C in lactate medium. Indicated amounts of mitochondrial proteins were analyzed for indicated proteins by SDS-PAGE followed by immunoblotting. (D) ΔΨ of wild-type and tim50-279,282,286 mitochondria measured as in Fig. 1 D. (E) Mitochondria were isolated from wild-type and tim50 mutant cells after cultivation in lactate medium at 23°C. Radiolabeled precursor proteins were incubated with those mitochondria at 25°C for indicated times, and import reactions were analyzed as in Fig. 2. p, precursor form; i, processing-intermediate form; m, mature form.