Plasmids and cell lines
For plasmids and cell lines used in this study, see Additional file 5: Tables S1-S3. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) complete containing 4.5 g l−1 glucose, 10% fetal bovine serum (FCS), and 500 μg ml−1 penicillin/streptomycin at 37 °C under 5% CO2. For the generation of stable, doxycycline (DOX)-inducible cell lines, the HEK293 cell line-based Flp-In™ T-REx™-293 cell line was used with the Flp-In™ T-REx™ system (Invitrogen). Positive clones were selected with DMEM complete containing 100 μg ml−1 hygromycin and 10 μg ml−1 blasticidin. The expression of stable cell lines was induced for at least 16 h with 1 mg ml−1 doxycycline. For the stable complementation of the HEK293T MIA40 CRISPR cell line #2 [15], the different MIA40 constructs were inserted into the cumate-inducible PB-CuO-MCS-IRES-GFP-EF1-CymR-Puro vector (System Biosciences). Positive clones were selected with DMEM complete containing 2 μg ml−1 puromycin and expression of the cell line was induced with 30 mg ml−1 cumate for 5–7 days.
Western blot analysis and antibodies
For analyzing steady-state protein levels, cells were washed once with ice-cold PBS, harvested in Laemmli buffer (2% SDS, 60 mM Tris-HCl pH 6.8, 10% glycerol, 0.0025% bromophenol blue) containing 50 mM dithiothreitol (DTT), and lysed by boiling for 5 min at 96 °C and sonification (60% amplitude, 5 cycles). Protein samples were analyzed by SDS-PAGE and immunoblotting. The addition of 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowed for visualization of migrated proteins and as a loading control. For antibodies used in this study, see Additional file 5: Table S1.
Cell treatments with MG132/emetine
To inhibit the proteasome, cells were treated with MG132 (Sigma-Aldrich) or with DMSO as control. For western blot analysis, cells were treated for 16 h with DMEM complete containing 1 μM MG132 and doxycyclin. In pulse or pulse-chase experiments, cells were treated with 5 μM MG132, which was added to all media except for the chase medium. For the inhibition of the cytosolic ribosome, cells were treated for 0, 2, 4, or 8 h with 100 μg ml−1 emetine (Sigma-Aldrich).
In vitro protein expression and purification
Experiments were performed as described in [36]. For recombinant protein expression, MIA40 variants were cloned into the pGEX-6p-1 (GE Healthcare) expression vector containing N-terminal cleavable GST tag. The proteins were heterologously expressed in E. coli Rosetta2™ (DE3) cells and lysed by French pressure in lysis buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.2 mM PMSF). After clearing centrifugation, GST-fused MIA40 proteins were purified using GSTrap 4B columns (GE Healthcare). The GST-tag was cleaved off using previously immobilized recombinant PreScission protease. Further purification and removal of the GST was achieved via size exclusion chromatography using a Superdex 16/600 75 pg High Load column (GE Healthcare). The proteins were stored in 20 mM TRIS pH 7.2 and 100 mM NaCl at 4 °C.
Circular dichroism (CD) spectrometry
Prior to the record of the CD spectra, purified MIA40 variants were dialyzed to 100 mM KPi pH 7.2 buffer. For estimation of the change in the extent of secondary structure upon unfolding, a series of 13 spectra (each an accumulation of 5 individual spectra with a data pitch of 1 nm) was recorded in a range of temperature between 40 and 100 °C at 5 °C increments.
The ellipticity θd was normalized to the protein concentration (c), the number of residues (nr), the cuvette diameter (l), and the molecular weight (M).
$$ {\theta}_{mr}={\theta}_d\cdot \frac{M}{c\cdot l\cdot {n}_r} $$
The resulting molar ellipticity θmr was plotted over the wavelength λ. For temperature stability determination, spectra were recorded between 40 and 95 °C. For TM determination, spectra at 206 and 225 nm were assessed.
In vitro direct redox state assay
To analyze the direct redox state, recombinantly expressed MIA40 proteins were reduced with 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 15 min at either 4 or 96 °C. Water instead of TCEP was added to the steady-state and unmodified samples. Free thiols were modified with the maleimide compound mmPEG24 at a final concentration of 2 mM for 1 h at RT. The unmodified controls were incubated with water. Samples were analyzed by SDS-PAGE and Coomassie staining.
In vitro oxidation kinetic with purified proteins
This assay was performed as described in [15]. The radiolabeled MIA40 substrates human COX19 and NDUFA8 were synthesized in vitro using the TNT Quick Coupled Transcription/Translation System (Promega) under normoxic conditions. 1 mM DTT was added during the synthesis to keep the proteins in a reduced state. Redox state was verified by alkylation assays. The oxidation reaction was started by adding a dilution of 1:40 of the radioactive lysate to 5 μM recombinant MIA40 protein diluted in reaction buffer (20 mM TRIS pH 7.2, 200 mM NaCl, 1 mM EDTA). After indicated times, the reaction was stopped by adding 10% trichloroacetic acid (TCA). After TCA precipitation, the pellets were resolved in Laemmli buffer containing 5 mM mmPEG24 and incubated for 1 h at RT. Samples were analyzed by SDS-PAGE and autoradiography.
In organello import assay
For the isolation of crude mitochondria, HEK293 cells were washed in PBS and resuspended in homogenization buffer (220 mM mannitol, 70 mM sucrose, 5 mM HEPES/KOH pH 7.4, 1 mM EGTA) prior to homogenization with a rotating Teflon potter (Potter S, Braun). The homogenate was cleared of debris and nuclei by centrifugation at 600g for 5 min at 4 °C. The supernatant was centrifuged at 8000g for 10 min at 4 °C to obtain mitochondrial fractions. Mitochondria were washed in homogenization buffer and 100 μg was taken per import reaction. Radiolabeled precursor proteins were synthesized for 1 h at 30 °C using the SP6 promoter TNT Quick Coupled Transcription/Translation System (Promega) containing 20 μCi [35S]-methionine. Import assays were performed as described previously [31]. Briefly, protein import was initiated by incubating precursor protein with crude mitochondria at 30 °C in the presence or absence of CCCP (1 mM). Import was stopped after 10, 30, or 60 min by placing mitochondria on ice. All samples were treated with proteinase K (20 μg ml−1) for 20 min to degrade non-imported precursor protein. Mitochondria were then washed in homogenization buffer containing PMSF (1 mM) and resuspended in Laemmli buffer for analysis by SDS-PAGE and autoradiography.
Immunoprecipitation
For native immunoprecipitation, cells were once washed and then incubated for 10 min at 4 °C in ice-cold PBS containing 20 mM NEM (N-Ethylmaleimide). After sedimentation (5 min, 500×g, 4 °C), cells were resuspended in 3 ml native lysis buffer (100 mM NaPi pH 8.1, 1% Triton X-100) per 10 cm plate and incubated for 1 h on ice for efficient native lysis. From here on, the immunoprecipitation was performed the same for the following experiments: native immunoprecipitation, pulse assay, and oxidative protein folding assay and organelle fractionation assay. Cell debris was sedimented (1 h, 20,000×g, 4 °C) and the supernatant was subjected to immunoprecipitation with equilibrated monoclonal anti-HA agarose conjugate beads (HA beads; Sigma-Aldrich) at 4 °C overnight under gentle shaking. The samples were washed five times in wash buffer A containing Triton X-100 (100 mM NaPi pH 8.1, 1% Triton X-100, 250 mM NaCl) and once in wash buffer B without Triton X-100 (100 mM NaPi pH 8.1, 250 mM NaCl). Immunoprecipitated proteins were eluated from dried HA beads by adding Laemmli buffer and subsequent boiling for 7 min at 96 °C. Samples were analyzed by SDS-PAGE or Tris-Tricine-PAGE.
In cellulo pulse assay
Cells were starved in cysteine- and methionine-free medium (Sigma-Aldrich) for 15 min at 37 °C. Newly synthesized proteins were pulse-labeled for 5, 10, 20, and 40 min at 37 °C in cysteine- and methionine-free medium containing 100 μCi ml−1 EasyTag EXPRESS 35S Protein Labeling Mix (PerkinElmer). To inhibit the proteasome, the starvating and pulse medium was supplemented with 5 μM MG132 (Sigma-Aldrich) or with DMSO as control. Pulse labeling was stopped by removing the medium and adding ice-cold PBS. After sedimentation (5 min, 500×g, 4 °C), the cells were resuspended in 250 μl denaturing lysis buffer A (30 mM Tris pH 8.1, 100 mM NaCl, 5 mM EDTA, 2% SDS) and incubated for 15 min at 96 °C. 750 µl denaturing lysis buffer B (30 mM Tris pH 8.1, 100 mM NaCl, 5 mM EDTA, 2.5% Triton X-100) was added and the samples were incubated for 1 h on ice. From here on, the immunoprecipitation was performed as described above. Samples were analyzed by SDS-PAGE and autoradiography.
In cellulo oxidation kinetics assay
The assay to follow the oxidative protein folding of proteins in HEK293 cells was performed as described in [14]. Cells were starved in cysteine- and methionine-free medium (Sigma-Aldrich) for 15 min at 37 °C. Newly synthesized proteins were pulse-labeled for 5 min at 37 °C in cysteine- and methionine-free medium containing 200 μCi ml−1 EasyTag EXPRESS 35S Protein Labeling Mix (PerkinElmer). To inhibit the proteasome, the starvating and pulse medium was supplemented with 5 μM MG132 (Sigma-Aldrich) or with DMSO as control. Pulse labeling was stopped by removing the medium and adding chase medium containing 20 mM methionine. The chase was performed for 0, 10, 20, 40, and 90 min at 37 °C and stopped by adding ice-cold 8% trichloroacetic acid (TCA). After TCA precipitation, the samples were resolved in modification buffer (0.2 M Tris pH 7.5, 6 M urea, 10 mM EDTA, 2% SDS) and the proteins were modified with a final concentration of 15 mM mmPEG12 for 1 h at RT. Reduced control samples were treated with 2 mM Tris (2-carboxyethyl)phosphine (TCEP) for 5 min at 96 °C previous to modification. Oxidized control samples were resolved in modification buffer without mmPEG12. After modification, 250 μl denaturing lysis buffer A was added and the samples were incubated or 15 min at 96 °C. Afterward, 750 μl denaturing lysis buffer B was added and the samples were incubated for 1 h on ice. From here on, the immunoprecipitation was performed as described above. Samples were analyzed by Tris-Tricine-PAGE and autoradiography.
In cellulo translocation assay
To follow the posttranslational import of newly synthesized proteins into mitochondria of HEK293 cells, this assay was performed as the oxidative protein folding assay with the following modifications and as described previously [14]. After the case, cells were fractionated for 30 min at 4 °C in ice-cold fractionation buffer (20 mM 4-(2-hydroxyethyl)-1-pipera- zineethanesulfonic acid [HEPES] pH 7.4, 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl, 1 mM DTT, 0.003% digitonin). By centrifugation at 10,000×g, 4 °C, the samples were fractionated into a cytosolic and mitochondrial fraction. The latter was digested for 20 min at 4 °C with ice-cold trypsin buffer (20 mM HEPES, pH 7.4, 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl, 1 mM DTT, 25 μg ml−1 trypsin). TCA with a final concentration of 8% was added to all samples. After TCA precipitation and immunoprecipitation, the samples were analyzed by SDS-PAGE and autoradiography.
In cellulo inverse redox state assay
The inverse redox state assay was performed as described previously [37]. In short, to block reduced thiols, cells were once washed and then incubated for 10 min at 4 °C in ice-cold PBS containing 15 mM NEM (N-Ethylmaleimide). Oxidized controls were pretreated for 10 min at 37 °C in warm PBS containing 10 mM diamide prior to the NEM blockage reaction. Unmodified, min. and max. controls were washed and incubated for 10 min at 4 °C in ice-cold PBS without NEM. Thiol-exchange reactions were stopped by the addition of 8% ice-cold TCA. After TCA precipitation, the samples were resolved in Laemmli buffer containing 5 mM TCEP by sonification and afterward incubated for 15 min at 45 °C. After TCEP reduction, the samples were modified with mmPEG24 (15 μM final concentration) for 1 h at room temperature. Min. shift samples were modified with NEM instead. For unmodified samples, the same amount of water was added. Samples were analyzed by Tris-Tricine-PAGE and immunoblotting.
Immunofluorescence
Immunofluorescence experiments were performed as described in [36]. In short, HEK293 Flp-In T-REx cells expressing HA-tagged MIA40 variants were cultured on poly-L-lysine-coated coverslips. Cells were stained with Mitotracker red (Thermo Fisher) for 1 h. After cell fixation with 4% paraformaldehyde for 15 min, cells were permeabilized with blocking buffer (10 mM HEPES pH 7.4, 3% BSA, 0.3% Triton X-100) for 1 h. Cells were washed and incubated with primary (anti-HA, 3F10, Roche) and secondary antibodies (anti-rat, AlexaFluor 488) for 16 h at 4 °C and 1 h at RT, respectively. After washing in PBS, nuclei were stained with 1 μg ml−1 DAPI for 15 min. Coverslips were mounted onto microscope slides using mounting medium (30% glycerol, 12% polyvinyl alcohol, 60 mM TRIS, 2.5% 1,4-diazabicyclo-2,2,2-octan) and dried overnight in the darkness. Cells and pictures were analyzed by confocal fluorescence microscopy (Leica Microsystems TCS SP8; Inverse, DMi 8 CS; PL Apo 63x/1.40 Oil CS2, LAS X) and Fiji, respectively [38].
Digitonin fractionation
For analyzing the subcellular localization, doxycyclin-induced (16 h) cells were trypsinized and washed in 200 μl ice-cold fractionation buffer (20 mM HEPES pH 7.4, 250 mM Sucrose, 50 mM KCl, 2.5 mM MgCl, 1 mM DTT). The cells were then resuspended in 800 μl fractionation buffer containing either 0, 0.003, 0.005, 0.01, 0.03, 0.05, 0.1, or 0.3% digitonin (PanReac AppliChem). 25 U Benzonase® Nuclease (Sigma-Aldrich) was added for DNA degradation. The samples were incubated for 30 min on ice while being inverted every 5 min. The supernatant and the pellet fraction was then separated by centrifugation at 9000g for 10 min at 4 °C. Proteins of the supernatant were precipitated in ice-cold TCA. The pellet was resuspended in 800 μl fractionation buffer containing 25 μg ml−1 trypsin and the samples were incubated for 30 min on ice while being inverted every 5 min. The proteins of the pellet fraction were then precipitated in ice-cold TCA. After TCA precipitation, the samples were resolved in modification buffer (0.2 M Tris pH 7.5, 6 M urea, 10 mM EDTA, 2% SDS). The samples were sonicated until the pellets were entirely dissolved, supplemented with Laemmli buffer for analysis by SDS-PAGE and immunoblotting.
Viability assay
For analyzing the cell viability, cells were adapted and cultured in galactose-containing medium (DMEM supplemented with 4.5 g l−1 galactose, 2 mM L-glutamine, 1 mM sodium pyruvate, 1× nonessential amino acids, 50 mg l−1 uridine, 10% FCS and 500 μg ml−1 penicillin/streptomycin) at 37 °C under 5% CO2. Twenty-four hours after the cell seeding (day 0), the viability of the cells was determined by treating the cells for 1 h at 37 °C with the PrestoBlue™ Cell Viability Reagent (Invitrogen by Thermo Fisher). The fluorescence was measured at excitation and emission wavelengths of 560 and 590 nm, respectively. After the measurement, the cells were cultured in galactose-containing medium containing 30 mg ml−1 cumate. Control cells were treated without cumate. After 1, 3, 5, and 7 days the viability of the cells was measured again. Fluorescence values of cells treated with cumate were divided by fluorescence values of cells treated without cumate.
Conservation analysis and sequence logo
The primary sequence alignment of the MIA40 C-terminus (isoform 1, CHCHD4.1) was performed by using the plastp protein-protein BLAST algorithm (NCBI) and full-length protein sequences provided by NCBI. The alignment and sequence logo were generated by Jalview [39] with the help of the algorithm of ClustalOWS [40]. Sequences from 86 species were aligned for Fig. 1a. The conservation is depicted as a sequence logo where the relative size of the respective letters indicates the frequency in the sequences. The percentage of negatively charged amino acids was calculated and the presence of patches of at least four negatively charged aa in a row was analyzed for the C-termini. Protein sequences of 36 MIA40 substrates were analyzed manually for the occurrence of negatively charged amino acids.
Three-dimensional structure and secondary structure prediction
The three-dimensional NMR structure of the core of MIA40 [2K3J] [21] was illustrated by using the protein data bank RCSB PDB. The secondary structure of the N- and C-terminus of MIA40 was predicted with JPred4 [41]. Arrows represent beta-sheets and boxes stand for alpha-helices.
Quantification and statistical analysis
Immunoblot signals and intensities of autoradiograms were quantified using Image Lab Software (Bio-Rad Laboratories GmbH) and ImageQuant TL (GE Healthcare), respectively. Error bars in figures represent standard deviation and the n-number of experiments is noted in each figure legend.