Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors
- Department of Cellular Biochemistry, University Medical Center Göttingen, Germany
- Biochemistry and Functional Proteomics, Institute of Biology II, University of Freiburg, Germany
- Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Germany
- Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany
- Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Germany
- Department of Neurology, University Medical Center Göttingen, Germany
- Department of Mitochondrial Proteostasis, Max Planck Institute for Biology of Ageing, Germany
- Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Translational Neuroinflammation and Automated Microscopy, Germany
- Max Planck Institute for Biophysical Chemistry, Germany
Figures
Figure 1
TMEM223 and SMIM4 interact with the mitochondrial ribosome.
(A) Mitochondria isolated from cells expressing mL62FLAG were subjected to co-immunoprecipitation. Natively isolated complexes were separated by sucrose density gradient ultracentrifugation. Fractions (1–15) were analyzed by western blotting, using indicated antibodies against components of the 39S mtLSU (mL62, uL1m, uL3m, and uL23m) and 28S mtSSU (uS14m and bS16m) subunits. (B) Mitochondria were isolated from wild-type (WT) and mL62FLAG-expressing cells, cultured in SILAC-medium, and subjected to co-immunoisolation. Eluates were analyzed by quantitative mass spectrometry (LC-MS/MS) (n=4). Ribosomal proteins of the mtLSU and mtSSU are indicated in red and blue, respectively. Dashed lines indicate a p-value of 0.05 and mean mL62FLAG/WT ratios. (C) Scheme of proteins identified in (B). (D) Complexes containing mL62FLAG were purified as in (A) and (B) and analyzed by western blotting (Total, 0.75%; Eluate, 100%).
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Figure 1—source data 1
Data of Figure 1A and D.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig1-data1-v1.zip
Figure 2 with 1 supplement
TMEM223 is a mitochondrial membrane protein.
(A) Membrane topology of TMEM223. The predicted transmembrane spans (TM1 and TM2) with corresponding amino acids (aa) are indicated. IMS: intermembrane space; IMM: inner mitochondrial membrane. (B) and (C) Submitochondrial localization of TMEM223. Wild-type (WT) mitochondria were treated with Proteinase K (PK) under iso-osmotic (Mito), hyper-osmotic conditions (swelling, MP), or solubilized with Triton X-100 (TX-100) (B). The unspecific band is marked with an asterisk. Mitochondrial proteins were extracted in sodium carbonate containing buffer at different pH (total, T; pellet, P; soluble fraction, S) (C). (D) Protein steady-state levels in TMEM223−/− cells. Mitochondrial lysates from WT and TMEM223−/− cells were analyzed by western blotting using indicated antibodies and protein amounts were quantified using ImageQuant software (mean ± SEM, n=3). (E) Isolated mitochondria from WT and TMEM223−/− cells were solubilized in DDM-containing buffer, separated on 2.5–10% (Complex I) or 4–13% (Complexes II–V) BN-PAGE and analyzed by western blotting. OXPHOS complexes were detected with indicated antibodies and amounts quantified using ImageQuant software(mean ± SEM, n=3).
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Figure 2—source data 1
Data of Figure 2B–E.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig2-data1-v1.zip
Figure 2—figure supplement 1
Loss of TMEM223 increases the level cytochrome c reductase.
(A) BN-PAGE loading control corresponding to Figure 2E. Isolated mitochondria were subjected to BN-PAGE analyses (Figure 2E) or to SDS-PAGE and western blotting to confirm equal loading of the BN-PAGE. (B) Isolated mitochondria from TMEM223−/− and control cells were solubilized using digitonin buffer, separated on 2.5–10% (complexes I, III and IV) or 4–13% (complex II) BN-PAGE, analyzed by western blotting, and complexes were detected with indicated antibodies (mean ± SEM, n=3). A fraction of the isolated mitochondria was subjected to SDS-PAGE and western blotting analysis as a loading control (right panel). (C) Steady-state protein analysis of selected cytochrome c reductase related proteins in TMEM223−/− cells. Mitochondria were isolated, subjected to western blotting, and probed with indicated antibodies (left panel). The steady-state amount of the indicated proteins was quantified using ImageQuant software (mean ± SEM, n=3). (D) Stability of cytochrome c reductase related proteins. Control cells and TMEM223−/− cells were treated with thiamphenicol (final 50 µg/ml) for 48 hr and cell lysates subjected to western blotting. Signals were quantified with ImageQuant software and graphed (mean ± SEM, n=3).
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Figure 2—figure supplement 1—source data 1
Data of Figure 2—figure supplement 1A, B, C.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig2-figsupp1-data1-v1.zip
Figure 3
TMEM223 is involved in cytochrome c oxidase biogenesis.
(A) Cytochrome c oxidase activity was measured in cellular extracts from wild-type (WT) and TMEM223−/− photometrically (mean ± SEM, n=4). (B, C) Mitochondrial protein synthesis in TMEM223−/− (B) or siRNA mediated TMEM223 depleted (C) cells. Cells were grown in the presence of [35S]methionine for 1 hr to monitor synthesis of mitochondrial-encoded proteins. Cell lysates were subjected to SDS-PAGE and analyzed by digital autoradiography and western blotting (lower panel in (B)). Newly synthesized mitochondrial proteins were quantified, using the ImageQuant software, calculated as percentage of WT and internally standardized to ATP6 (mean ± SEM; n=3). (D–G) TMEM223 interacts with early cytochrome c oxidase assembly factors. Mitochondria isolated from WT, C12ORF62FLAG (D), MITRAC12FLAG (E), CMC1 (F), or MITRAC7FLAG (G) cells were subjected to co-immunoisolation and samples analyzed by western blotting (Total, 0.75%; Eluate, 100%).
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Figure 3—source data 1
Data of Figure 3B, D, E, F, G.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig3-data1-v1.zip
Figure 4
SMIM4 is a mitochondrial protein, interacting with cytochrome c reductase and mitochondrial quality control proteins.
(A) Schematic presentation of SMIM4 membrane topology. The predicted transmembrane domain (AA 20–41) is indicated. IMS: intermembrane space; IMM, inner mitochondrial membrane. (B) Immunofluorescence microscopy of HEK293T cells expressing SMIM4FLAG. Cells were induced with doxycycline for 24 hr (+Dox). SMIM4FLAG was labeled using a FLAG-specific antiserum. As a mitochondrial marker, a TOM22 specific antiserum was used. SMIM4FLAG and TOM22 were imaged with STED microscopy, PicoGreen by confocal microscopy. DNA was labeled via Quant-iT PicoGreen dsDNA reagent. Scale bars: 10 µm (overview), 500 nm (magnification). (C) Submitochondrial localization of SMIM4FLAG. Mitochondria isolated from SMIM4FLAG-expressing cells were treated with proteinase K (PK) either under iso-osmotic (Mito), hyperosmotic (swelling, mitoplasts [MP]) conditions. Unspecific band is marked with an asterisk. (D) SMIM4 is an integral membrane protein. Mitochondrial proteins were extracted using sodium carbonate (at indicated pH), or Triton X-100 (TX-100). Samples (total, T; pellet, P; soluble fraction, S) were analyzed by western blotting using antibodies. (E) Mitochondrial extracts from wild-type (WT) and SMIM4FLAG-expressing cells cultured in SILAC medium were subjected to native-immunoprecipitation, and analyzed by quantitative mass spectrometry (LC-MS/MS) (n=4). Cytochrome c reductase assembly factors and components of the mitochondrial quality control system are indicated in black. The dashed horizontal line indicates a p-value of 0.05, the solid vertical line a mean SMIM4FLAG/WT ratio of 10. (F) Samples obtained by co-immunoprecipitation of WT or SMIM4FLAG-containing mitochondrial lysates were analyzed by western blotting (Total, 1.5%; Eluate, 100 %).
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Figure 4—source data 1
Data of Figure 4C, D, F.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig4-data1-v1.zip
Figure 5 with 1 supplement
Loss of SMIM4 or C12ORF73 affects cytochrome c reductase biogenesis.
(A) FLAG-immunoisolation of C12ORF73FLAG. Samples were analyzed by SDS-PAGE and western blotting (Total, 1.5%; Eluate, 100 %). (B) Western blot analyses of SMIM4 or C12ORF73 depleted cells. HEK293T cells were treated with indicated siRNAs and cultured either in glucose- or galactose-containing media for 72 hr. Cell extracts were subjected to SDS-PAGE separation and western blotting. (C) Loss of SMIM4 or C12ORF73 affects cell growth. HEK293T cells were transfected with siRNAs as in Figure 6B. Cells were cultured either in glucose- or galactose-containing media for 72 hr; cell counts are presented as percentage relative to non-targeting siRNA-treated cells (siNT; indicated as dashed line) (mean ± SEM, n=3). (D, E) BN-PAGE analyses of mitochondrial protein complexes upon SMIM4 (B) or C12ORF73 ablation (C). Mitochondria were solubilized with DDM (N-Dodecyl-beta-Maltoside) and subjected to BN-PAGE followed by western blot analyses. OXPHOS complex levels were quantified using the ImageQuant software and graphed (mean ± SEM, n=3).
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Figure 5—source data 1
Data of Figure 5A,B, D, E.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig5-data1-v1.zip
Figure 5—figure supplement 1
C12ORF73 is an inner mitochondrial protein in human.
(A) STED super-resolution light microcopy images of TOM22 and C12ORF73FLAG in HEK293T cells. TOM22 and C12ORF73FLAG were labeled by specific antisera. Detection via secondary antibodies custom labeled with the dyes ALEXA Fluor594 or Abberior STAR RED, respectively. Scale bars: 10 µm (overview), 500 nm (magnification). (B, C) Submitochondrial localization of C12ORF73. (B) Sodium carbonate buffers with indicated pH were used to extract mitochondrial membrane proteins (total, T; pellet, P; supernatant fraction, S). (C) Different amounts of Proteinase K (PK) were added under iso-osmotic (Mito), and hyper-osmotic (MP) conditions or during Triton X-100 (TX-100) treatment. (D, E) Immunoisolation of UQCRQFLAG or RIESKE. Purified mitochondria were solubilized in digitonin-containing buffer and subjected to immunoisolation with FLAG- (D) or anti-RIESKE antibodies, respectively (E). Eluted proteins were analyzed by western blotting using indicated antibodies (Total, 0.5%; Eluate, 100%). (F, G) BN-PAGE loading controls corresponding to Figure 5D and E. Isolated mitochondria of the BN-PAGE experiment were subjected to SDS-PAGE and western blotting.
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Figure 5—figure supplement 1—source data 1
Data of Figure 5—figure supplement 1B–D.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Data of Figure 5—figure supplement 1E–G.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig5-figsupp1-data2-v1.zip
Figure 6
SMIM4 and C12ORF73 promote cytochrome c reductase assembly.
(A) SMIM4 and C12ORF73 co-migrate with cytochrome c reductase assembly intermediates. Wild-type mitochondrial lysates were subjected to BN-PAGE, followed by second dimension SDS-PAGE and western blotting. (B) C12ORF73FLAG isolates cytochrome c reductase assembly intermediates. Mitochondria isolated from C12ORF73FLAG-expressing cells were solubilized and subjected to co-immunoisolation. Natively eluted complexes were separated by BN-PAGE and subjected to second dimension SDS-PAGE followed by western blot analyses using indicated antibodies. (C) Immunoprecipitations of UQCC1FLAG, UQCC2FLAG, and UQCC3FLAG. Eluates were analyzed by SDS-PAGE followed by western blotting with indicated antibodies. (D) Mitochondrial translation products were labeled with [35S]methionine for 1 hr prior to co-immunoprecipitation using anti-SMIM4, -C12ORF73, or control antibodies (αIgGcon). Eluates were separated by SDS-PAGE followed by western blotting, and analyzed by digital autoradiography (upper panel) and immunodetection (lower panel) (Total, 2%; Eluate, 100%). (E) Mitochondria isolated from control or C12ORF73-depleted cells were lysed in digitonin-containing buffer and complexes separated by BN-PAGE followed by second dimension SDS-PAGE. Cytochrome c reductase sub-assembly complexes were monitored with indicated antibodies (red arrow mark cytochrome c reductase subcomplexes in C12ORF73-deficient samples).
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Figure 6—source data 1
Data of Figure 6A-E.
- https://cdn.elifesciences.org/articles/68213/elife-68213-fig6-data1-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293-Flp-InTM T-RexTM (HEK293T) Cell Line | Thermo Fisher Scientific | RRID:CVCL_U421 | |
| Cell line (H. sapiens) | HEK293-Flp-InTM T-RexTM (HEK293T)-TMEM223−/− | This paper | N/A | Cell line generated as described in Materials and methods |
| Transfected construct (H. sapiens) | pX330-TMEM223 gRNA | This paper | N/A | Cloning described in Materials and methods |
| Transfected construct (H. sapiens) | pEGFPN1 | CloneTech | N/A | |
| Transfected construct (H. sapiens) | pCDNA5-mL62-FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-mL62-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-MITRAC7-FLAG | Dennerlein et al., 2015 (Cell Rep.) | N/A | Construct generated by mutagenesis of pCDNA5-MITRAC7-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-C12ORF62-FLAG | Richter-Dennerlein et al., 2016 (Cell) | N/A | Construct generated by mutagenesis of pCDNA5-C12ORF62-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-MITRAC12- FLAG | Aich et al., 2018 (eLife) | N/A | Construct generated by mutagenesis of pCDNA5-MITRAC12-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-C12ORF73- FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-C12ORF73-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-SMIM4-FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-SMIM4-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-UQCC1-FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-UQCC1-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-UQCC2-FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-UQCC2-FLAG |
| Transfected construct (H. sapiens) | pCDNA5-UQCC3-FLAG | This paper | N/A | Construct generated by mutagenesis of pCDNA5-UQCC3-FLAG |
| Antibody | TMEM223rabbit polyclonal | Self made | PRAB4850 | (1:500) |
| Antibody | SMIM4rabbit polyclonal | Self made | PRAB5494 | (1:500) |
| Antibody | mS40 (MRPS18B) rabbit polyclonal | ProteinTech | RRID:AB_2146368 | (1:1000) |
| Antibody | TMEM177rabbit polyclonal | Self made | PRAB4988 | (1:1000) |
| Antibody | TIM44rabbit polyclonal | Self made | PRAB5142 | (1:4000) |
| Antibody | COX6A rabbit polyclonal | Self made | PRAB3282 | (1:1000) |
| Antibody | SLIRP rabbit polyclonal | Self made | PRAB3813 | (1:500) |
| Antibody | CYTB rabbit polyclonal | Self made | PRAB5151 | (1:1000) |
| Antibody | C12ORF73rabbit polyclonal | Self made | PRAB5105 | (1:500) |
| Antibody | PHB1rabbit polyclonal | ProteinTech | RRID:AB_2164476 | (1:1000) |
| Antibody | uL3m rabbit polyclonal | ProteinTech | RRID:AB_10639509 | (1:1000) |
| Antibody | uS14m rabbit polyclonal | ProteinTech | RRID:AB_2878240 | (1:2000) |
| Antibody | bS16m rabbit polyclonal | ProteinTech | RRID:AB_2180166 | (1:5000) |
| Antibody | YME1L rabbit polyclonal | Self made | PRAB5113 | (1:500) |
| Antibody | SLP2rabbit polyclonal | ProteinTech | RRID:AB_2286822 | (1:1000) |
| Antibody | AFG3L2rabbit polyclonal | Self made | PRAB5149 | (1:500) |
| Antibody | MITRAC12rabbit polyclonal | Self made | PRAB3761 | (1:1000) |
| Antibody | C12ORF62rabbit polyclonal | Self made | PRAB4844 | (1:500) |
| Antibody | MITRAC7rabbit polyclonal | Self made | PRAB4843 | (1:500) |
| Antibody | COX1rabbit polyclonal | Self made | PRAB2035 | (1:2000) |
| Antibody | COX4-1rabbit polyclonal | Self made | PRAB1522 | (1:2000) |
| Antibody | uL23m rabbit polyclonal | Self made | PRAB1716 | (1:500) |
| Antibody | uL1m rabbit polyclonal | Self made | PRAB4969 | (1:500) |
| Antibody | TOM70rabbit polyclonal | Self made | PRAB3280 | (1:1000) |
| Antibody | TACO1rabbit polyclonal | Self made | PRAB3627 | (1:500) |
| Antibody | MITRAC15rabbit polyclonal | Self made | PRAB4814 | (1:500) |
| Antibody | FLAG rabbit polyclonal | Sigma-Aldrich | RRID:AB_259529 | (1:2000) |
| Antibody | TIM21rabbit polyclonal | Self made | PRAB3674 | (1:2000) |
| Antibody | SDHAMouse monoclonal | Self made | PRAB4978 | (1:2000) |
| Antibody | Rieske rabbit polyclonal | Self made | PRAB1512 | (1:2000) |
| Antibody | ATP5B rabbit polyclonal | Self made | PRAB4826 | (1:10000) |
| Antibody | TIM44rabbit polyclonal | ProteinTech | RRID:AB_2204679 | (1:2500) |
| Antibody | NDUFB8rabbit polyclonal | Self made | PRAB3764 | (1:500) |
| Antibody | NDUFA9rabbit polyclonal | Self made | PRAB1524 | (1:500) |
| Antibody | TIM23rabbit polyclonal | Self made | PRAB1527 | (1:2000) |
| Antibody | SCO2rabbit polyclonal | Self made | PRAB4982 | (1:500) |
| Antibody | FAM36A rabbit polyclonal | Self made | PRAB4490 | (1:500) |
| Antibody | SURF1rabbit polyclonal | Self made | PRAB1528 | (1:1000) |
| Recombinant DNA reagent | QuikChange Site-Directed Mutagenesis Kit | Agilent | 210515 | |
| Recombinant DNA reagent | KOD Hot Start DNA Polymerase | Merck | 71086-3 | |
| Recombinant DNA reagent | First Strand cDNA Synthesis Kit | Thermo Fisher Scientific | K1612 | |
| Commercial assay or kit | Human Complex IV Activity Kit | Abcam | ab109910 | |
| Chemical compound, drug | GeneJuice | Merck | 70967-3 | |
| Chemical compound, drug | Anti-FLAG M2 Affinity Gel | Sigma-Aldrich | A2220 | |
| Chemical compound, drug | TRIzol | Thermo Fisher Scientific | 15596026 | |
| Chemical Compound, drug | Protein-A SepharoseTM CL-4B | GE Healthcare | 17-0963-03 | |
| Chemical Compound, drug | [35 S]methionine | Hartmann Analytic | SCM-01 | |
| Chemical compound, drug | Emetine dihydrochloride hydrate | Sigma-Aldrich | 219282 | |
| Software, algorithm | ImageQuantTL 7.0 software | GE Healthcare | RRID:SCR_014246 | |
| Software, algorithm | ImageJ 1.47v | NIH | RRID:SCR_003070 | |
| Software, algorithm | Geneious | Biomatters Ltd | RRID:SCR_010519 | |
| Software, algorithm | Prism5 | GraphPad Software | RRID:SCR_015807 |
Additional files
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Supplementary file 1
Mass spectrometric analyses of mL62 and SMIM4 containing complexes.
Isolated complexes of mL62FLAG (Supplementary file 1) or from SMIM4FLAG (Supplementary file 2) were subjected to quantitative mass spectrometric analyses (compare Figures 1B and 4E). MS raw data were analyzed with MaxQuant/Andromeda (see Materials and methods section). Analyzed datasets are presented as Excel files.
- https://cdn.elifesciences.org/articles/68213/elife-68213-supp1-v1.xlsx
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Supplementary file 2
Mass spectrometric analyses of mL62 and SMIM4 containing complexes.
- https://cdn.elifesciences.org/articles/68213/elife-68213-supp2-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68213/elife-68213-transrepform1-v1.pdf
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Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors
eLife 10:e68213.
https://doi.org/10.7554/eLife.68213
