Tim23 mutants with mutations in the coiled-coil region in the IMS. (A) Amino acid sequences of the IMS domains of Tim23 mutants in tim23 mutant strains (top) and coiled-coil regions in Tim23 (bottom) predicted by COILS (http://www.ch.embnet.org/software/COILS_form.html). Mutation points are shown in black. (B) Serial dilutions of tim23 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 control (WT) and tim23 mutant cells grown at 30°C. Indicated amounts of mitochondrial proteins were analyzed for indicated proteins by SDS-PAGE followed by immunoblotting. (D) ΔΨ of wild-type control (WT) and tim23 mitochondria measured by DiSC3(5) (Sims et al., 1974). mito., mitochondria; val., valinomycin.
