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. 2023 Aug 1;621(7979):627–634. doi: 10.1038/s41586-023-06477-8

Fig. 4. Tim17-dependent presequence translocation is initiated at the negative charged patch.

Fig. 4

a, Growth analysis of yeast strains expressing wild-type or tim17 variants with the indicated double or triple negative charge mutation on non-fermentable glycerol (YPG) medium at 19 °C. b, Mitochondrial protein complexes were analysed by BN–PAGE as described in Fig. 1f. WT*, mitochondria isolated from tom22Δ, pGal-TIM17WT + pFL39-TOM22 yeast cells with levels of Tim17 comparable with Tim17D17A_D76A mutant mitochondria. c, Protein import into wild-type and Tim17D17A_D76A mitochondria was analysed as described for Fig. 3c.d, Radiolabelled b2(167)Δ-DHFR was imported and accumulated in import sites of isolated mitochondria in the presence of MTX as indicated. Samples were analysed by BN–PAGE and autoradiography. TOM–TIM, TOM–TIM23 preprotein stabilized supercomplex. e, Accumulation of radiolabelled b2(220)-DHFR in TOM–TIM23 import sites of isolated mitochondria as in Fig. 3c, followed by treatment with proteinase (Prot.) K.