Extended Data Fig. 6: Mutational analysis on the cavities of Tim17 and Tim23.

a, As in Fig. 2f, but testing mutations on aromatic residues lining the Tim17 cavity (Phe65, Phe72, and Trp68) of Tim17. b and c, Expression levels of WT and indicated mutants were measured by immunoblotting. All Tim17 variants were expressed under the endogenous promoter from a CEN/ARS plasmid. 3-phosphoglycerate kinase (Pgk1) was used as a loading control. d, As in Fig. 2f, but the experiments were performed with a 2 μ plasmid expressing indicated mutants of HA-tagged Tim17 under a cyanamide-inducible DDI2 promoter. Cells were spotted on synthetic complete without leucine (SC[–Leu]) plates supplemented with varying concentrations of cyanamide. The plates contain doxycycline (Dox), where indicated. e–g, As in b and c, but measuring expression levels of Tim17 under the DDI2 promoter in the presence of varying concentrations of cyanamide. As a control, expression of WT Tim17 under the endogenous promoter (endo) from a CEN/ARS plasmid was included. h, Co-immunoprecipitation of the essential subunits of the TIM23 complex with Tim17 mutants. Mitochondria expressing indicated Tim17 variants (WT, D17N/E126Q [mut1] and D76N/E126Q [mut2]) were solubilized with digitonin, and Tim17 was pulled down with anti-Spot-tag nanobody beads. The samples were analyzed by immunoblotting with indicated antibodies. i, WT mitochondria (50 μg protein) were incubated with 1 μg of purified Cyb2Δ-DHFR-His in a 100-μL reaction volume. Where indicated, ATP and NADH (2 mM each) and/or methotrexate (MTX; 2 μM) were included. After 20-min incubation at 25 °C, mitochondria were washed and resuspended in a hypo-osmotic buffer solution. After splitting the reactions, mitoplasts were treated with proteinase K (+PK) where indicated. The samples were quenched with phenylmethylsulfonyl fluoride and analyzed by SDS-PAGE and immunoblotting (IB) with anti-His-tag antibody. p, precursor form of Cyb2Δ-DHFR-His; m, mature (presequence-cleaved) form of Cyb2Δ-DHFR-His. j, Additional controls including a reaction in the presence of 2 μM valinomycin (−Ψ_m). Note that indicated amounts of Cyb2Δ-DHFR-His were added to 100-uL reactions and that all reactions contained MTX. k, As in Fig. 2h, but import reactions were subjected to blue native-PAGE (BN-PAGE). l, As in Fig. 2c, but view into the Tim23 cavity. The view is similar to the right panels of Fig. 2a,b. m, As in Fig. 2f, but testing mutations of acidic, polar, and aromatic amino acids lining the Tim23 cavity. Data in a–j and m are representative of three independent experiments. Data in k is representative of two independent experiments.