Fig. 1.

Truncation of the conserved negatively charged C-terminal region of human MIA40 decreases its cellular levels. a Conservation of the C-terminal domain of human MIA40. Human MIA40 consists of three parts, an N-terminal domain important for interaction with AIFM1 and MIA40 import, a core domain important for interaction with its substrates and their oxidation, and a negatively charged C-terminal region (indicated in light blue) with unknown function. The negative charges in the C-terminal region are clustered into three clusters (indicated in dark blue), and corresponding truncation mutants were employed for further studies. The logo plot was derived from alignments of MIA40 from 86 species. b Localization of HA-tagged MIA40 variants. HEK293 cells stably expressing the indicated MIA40-HA variants were incubated for 24 h with 1 μg ml⁻¹ doxycycline to induce expression. Cells were fixed, permeabilized, and stained using a primary antibody against the HA epitope (HA, green) and Mitotracker (red). Nuclei were stained with DAPI (blue). Cells were analyzed by fluorescence microscopy. All MIA40 variants localize to mitochondria. Scale bar, 15 μm. N = 11–15 cells, 2 biological replicates. c Complementation of MIA40 CRISPR-Cas9 clone with truncated MIA40 variants and test of MIA40 substrate levels. Cell lines were stably transfected with inducible plasmids harboring the indicated variants of MIA40 or with the empty vector (Mock). Expression of MIA40 variants was induced for 7 days with 30 μg ml⁻¹ cumate in glucose-containing medium. Cells were lysed, and the levels of the indicated proteins were analyzed by SDS-PAGE and immunoblotting (gray background: MIA40 substrates). Expression of MIA40^WT and all truncation variants, but not MIA40^C53S, complemented the loss of endogenous MIA40. Quantification using Image Lab. Data from 2 to 3 experiments were combined and standard deviations are presented if n > 2. d Complementation of MIA40 CRISPR-Cas9 clone with truncated MIA40 variants and test of proliferation. Cell lines were stably transfected with inducible plasmids harboring the indicated variants of MIA40 or with the empty vector (Mock). Expression of MIA40 variants was induced with 30 μg ml⁻¹ cumate in galactose-containing medium. Cells were analyzed by PrestoBlue cell viability reagent at the indicated times. Fold viability is presented as the viability data of induced cells divided by the data of non-induced cells of the same cell line. MIA40^C53S cannot rescue the growth of cells depleted of MIA40, while MIA40^WT and MIA40^Δ108 can. Data from 6 experiments were combined and standard deviations are presented. e Steady-state levels of MIA40 variants. HEK293 cells stably and inducibly expressing different MIA40 variants were lysed 24 h after induction of MIA40 expression (1 μg ml⁻¹ doxycycline, DOX), and then analyzed by immunoblotting. Truncated variants of MIA40 are present at strongly decreased levels compared to MIA40^WT(black arrowhead, endogenous MIA40, blue arrowhead, MIA40^Δ108). Quantification using Image Lab. Data from 3 experiments were combined and standard deviations are presented. f–h Steady-state levels of MIA40 variants. As e, except that different C-terminal extensions were fused to MIA40^Δ108 (f, g) and MIA40^Δ131 (f, h) and expressed stably, inducibly in HEK293 cells (induction with 1 μg ml⁻¹ doxycycline, DOX for 24 h). Either the missing endogenous amino acid residues were added back (light blue bar), or we exchanged the negative amino acid residues in these add-back parts with positive (red bar, pos.) or neutral (gray bar, neu.) amino acid residues. Alternatively, we only added the most C-terminal amino acid residues of wild type MIA40, “GSS” (black bar). Only add-back of the endogenous parts containing the negative charges restored MIA40 levels. Quantification using Image Lab. Data from 2 experiments were combined. Black arrowhead, endogenous MIA40; red arrowhead, signal of MIA40^Δ131 fused to the C-terminal region with negatively charged residues mutated to positively charged residues