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 2μ 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/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 (−Ψm). 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. 2 a and 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.