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. 2025 Oct 8;87:103884. doi: 10.1016/j.redox.2025.103884

Fig. 3.

Fig. 3

IMS import of FAM136A depends on the mitochondrial disulphide relay

A-C.In organello import assays with FAM136A. In vitro translated radioactive FAM136A was incubated with mitochondria isolated either from MIA40 knockdown (KD, B) or AIFM1 knockout (KO, C) HEK293 cells. Non-imported proteins were removed by treatment with Proteinase K. An import reaction was performed on mitochondria treated with CCCP to dissipate the mitochondrial membrane potential (–ΔΨ). Imported proteins were analysed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL, and the amount of imported protein was plotted. FAM136A import is strongly dependent on both MIA40 and AIFM1 (one-sample t-test). In addition, it relies on the mitochondrial membrane potential for import. Import of COX19 served as positive control. N = 3 replicates.

D,E. Levels of endogenous FAM136A in cells lacking MIA40 (MIA40 KD) (D) or AIFM1 (AIFM1 KO) (E). Protein levels in HEK293 cell lines depleted of MIA40 (D) or AIFM1 (E) or overexpressing the respective protein were compared to protein levels in wild‐type HEK293 cells. Lysates were analysed by reducing SDS‐PAGE and immunoblotting. Signals were quantified using ImageLab, and the amount of protein was plotted. TCE staining served as loading control. FAM136A levels were decreased in cells lacking MIA40 or AIFM1 significantly (one-way ANOVA with post hoc Tukey HSD test). Black and white circles represent the indicated treatment and not‐treated samples. N = 5 (D) or 3 (E) replicates

F. Steady-state levels of FAM136A in HEK293 cells stably expressing different variants of MIA40. Expression of MIA40 variants lacking either both cysteines of the redox active CPC motif (SPS) or only C53 (SPC) or with a mutation in the chaperone site of MIA40 (F68E) was induced for 5 days in glucose-containing medium. Cells were lysed, and protein levels were analysed by SDS-PAGE and immunoblot against the indicated proteins. Overexpression of MIA40 variants decreases FAM136A steady state levels. (one-way ANOVA with post hoc Tukey HSD test). N = 2.