Extended Data Fig. 7. Yeast expressing Tim17^D17A_D76A and analysis of Tim17^D17A_D76A mitochondria.

a, Graphical depiction of the approach to generate Tim17 mutants by placing chromosomal TIM17 under the control of the galactose promotor (pGAL-TIM17) in a TOM22 deletion background. This strain was transformed with single copy pFL(TRP) plasmids, encoding WT TOM22 and the desired TIM17 alleles. After elimination of the pYEP(URA)-TOM22 cover plasmid by 5-Fluoroorotic acid (5-FOA) treatment, the pFL plasmid is maintained, as the yeast cells require the TOM22 gene for viability. pGal, galactose inducible promoter; TRP, tryptophan; URA, uracil; Mut., tim17 mutant. b, Analysis of Tim17^WT protein depletion by shifting the pGAL-TIM17^WT strain from galactose to fermentable glycerol medium for 21 h. Protein levels in yeast whole cell extracts (WCE) were analysed by SDS-PAGE and immunodecoration. c, Growth analysis of tom22Δ, pGal-TIM17^WT + pFL39-TOM22 yeast strains expressing Tim17^WT (blue), Tim17^D17A_D76A (red) or no Tim17/‘empty’ (yellow) in glycerol-containing media at 25 °C. Arrow indicates the time before the growth of the strain expressing Tim17^D17A_D76A is affected compared to the WT strain. d, Protein amounts of WT* and Tim17^D17A_D76A mitochondria isolated from yeast cells shifted to non-fermentable media (YPG) at 23 °C, analysed by SDS-PAGE and immunodecoration against the indicated antibodies. WT*, WT strain with Tim17 protein levels comparable to Tim17^D17A_D76A mutant e, Protein complexes of isolated Tim17 WT and Tim17^D17A_D76A mutant yeast mitochondria were analysed by BN-PAGE and immunodecoration. III₂IV, III₂IV₂, respiratory chain supercomplexes. f, TIM23 complex isolation from digitonin-solubilised Tim17^WT + Tim21^ProtA or Tim17^D17A_D76A + Tim21^ProtA mitochondria. Bound protein complexes were analysed by SDS-PAGE and immunodecoration with the indicated antibodies. ProtA, Protein A; Load, 1%; Eluate 100%. g, Mitochondrial membrane potential of WT and Tim17^D17A_D76A mitochondria isolated from yeast cells grown in non-fermentable glycerol medium. Assessment of the membrane potential of isolated mitochondria as described in Extended Data Fig. 2d. h, Import of radiolabelled metabolite carrier ADP/ATP carrier (Aac2) into isolated WT and Tim17^D17A_D76A mitochondria followed by BN-PAGE and autoradiography. AAC₍₂₎, assembled ADP/ATP carrier_(oligomer). i, Import of radiolabelled pSu9-DHFR (top) and F₁β (Atp2, bottom) into WT and Tim17^D17A_D76A mitochondria isolated as described in Extended Data Fig. 2f.

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