Fig. 4.

FAM136A cysteines determine its stability.

A. Scheme of FAM136A cysteine variants. For detailed analysis of cysteines in FAM136A, we generated FAM136A variants: one lacking all cysteines (9CA), one lacking the cysteines 35, 39, 53, 57 (CA-1), and another one lacking the cysteines 82, 86, 110, 114 (CA-2).

B. Immunofluorescence analysis to localize FAM136A cysteine variants. HEK293 cells stably and inducibly expressing the indicated FAM136A-HA variants were fixated, permeabilized, and stained using a primary antibody against the HA epitope (HA) and mitotracker. Cells were analysed by fluorescence microscopy. FAM136A-HA WT localizes to mitochondria, while the cysteines variants do not, indicating that cysteines are critical for FAM136A localization. Bar corresponds to 10 μm

C. In organello import assay with FAM136A cysteine variant to test for its dependence on its cysteines. Experiment was performed as described in Fig. 3A. To test for the dependence of FAM136A on its cysteines, a wild-type and a cysteine-to-alanine mutant of FAM136A (9CA, all cysteines mutated) were compared. Import of the cysteine variant was strongly impaired (one-sample t-test). N = 3 replicates

D. Protein levels in HEK293 cell lines expressing different FAM136A variants. Lysates from different cells were analysed by reducing SDS-PAGE and immunoblotting. Signals were quantified using ImageLab, and the amount of protein was plotted. All FAM136A variants lacking cysteines were present at very low levels (one-way ANOVA with post hoc Tukey HSD test). N = 3 replicates.

E. FAM136A contains a patch of hydrophobic amino acids around cysteine 35 that might serve as MISS/ITS facilitating MIA40 interaction.

F. In organello import assay with FAM136A MISS/ITS variant. Experiment was performed as described in Fig. 3A. To test for the dependence of FAM136A on its MISS/ITS, a wild-type (WT), a M28A/L31A/M32A and a M28S/L31S/M32S mutant of FAM136A were compared. Import of the MISS/ITS variant was impaired (one-sample t-test). N = 3 replicates.