The A88R/E, S140R, and L148H BTHS mutants are recognized as defective by the i-AAA protease quickly after import. (A and B) In vitro import of ³⁵S-radiolabeled wt Taz1 or the BTHS mutant Taz1 precursor into wt (A) or Δyme1 (B) mitochondria. The nonimported precursor was removed with trypsin, mitochondria were reisolated, and the OM was ruptured by osmotic shock in the absence or presence of proteinase K (Prot. K). C, control (2.5% of precursor proteins + 100 µg mitochondria). The samples were resolved by SDS-PAGE, the bottom half of the gel was analyzed by phosphoimaging, and the top half was immunoblotted for markers of the OM (Tom70p), membranes facing the IMS (Dld1p), and the matrix (α-ketoglutarate dehydrogenase [KDH]). The percentage of each precursor imported at each time point was determined after phosphoimaging. Significant differences were determined by one-way ANOVA with Holm–Sidak pairwise comparisons and are indicated. Relative molecular masses are shown on the left. (C) Comparison of import into wt versus Δyme1 mitochondria. Significant differences between the percentage of each precursor imported into wt versus Δyme1 mitochondria were determined at each time point by Student t tests, with significant differences indicated. The four BTHS mutants being characterized in the present study are shown in red. Values are means ± SEM (n = 5–7 for import into wt mitochondria, and n = 4 for import into Δyme1 mitochondria).