Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors

  1. Sven Dennerlein
  2. Sabine Poerschke
  3. Silke Oeljeklaus
  4. Cong Wang
  5. Ricarda Richter-Dennerlein
  6. Johannes Sattmann
  7. Diana Bauermeister
  8. Elisa Hanitsch
  9. Stefan Stoldt
  10. Thomas Langer
  11. Stefan Jakobs
  12. Bettina Warscheid
  13. Peter Rehling  Is a corresponding author
  1. Department of Cellular Biochemistry, University Medical Center Göttingen, Germany
  2. Biochemistry and Functional Proteomics, Institute of Biology II, University of Freiburg, Germany
  3. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Germany
  4. Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany
  5. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Germany
  6. Department of Neurology, University Medical Center Göttingen, Germany
  7. Department of Mitochondrial Proteostasis, Max Planck Institute for Biology of Ageing, Germany
  8. Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Translational Neuroinflammation and Automated Microscopy, Germany
  9. Max Planck Institute for Biophysical Chemistry, Germany
6 figures, 1 table and 3 additional files

Figures

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%).

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).

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).

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%).

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 %).

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).

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.

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).

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HEK293-Flp-InTM T-RexTM (HEK293T) Cell LineThermo Fisher ScientificRRID:CVCL_U421
Cell line (H. sapiens)HEK293-Flp-InTM T-RexTM (HEK293T)-TMEM223−/−This paperN/ACell line generated as described in Materials and methods
Transfected construct (H. sapiens)pX330-TMEM223 gRNAThis paperN/ACloning described in Materials and methods
Transfected construct (H. sapiens)pEGFPN1CloneTechN/A
Transfected construct (H. sapiens)pCDNA5-mL62-FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-mL62-FLAG
Transfected construct (H. sapiens)pCDNA5-MITRAC7-FLAGDennerlein et al., 2015 (Cell Rep.)N/AConstruct generated by mutagenesis of pCDNA5-MITRAC7-FLAG
Transfected construct (H. sapiens)pCDNA5-C12ORF62-FLAGRichter-Dennerlein et al., 2016 (Cell)N/AConstruct generated by mutagenesis of pCDNA5-C12ORF62-FLAG
Transfected construct (H. sapiens)pCDNA5-MITRAC12- FLAGAich et al., 2018 (eLife)N/AConstruct generated by mutagenesis of pCDNA5-MITRAC12-FLAG
Transfected construct (H. sapiens)pCDNA5-C12ORF73- FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-C12ORF73-FLAG
Transfected construct (H. sapiens)pCDNA5-SMIM4-FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-SMIM4-FLAG
Transfected construct (H. sapiens)pCDNA5-UQCC1-FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-UQCC1-FLAG
Transfected construct (H. sapiens)pCDNA5-UQCC2-FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-UQCC2-FLAG
Transfected construct (H. sapiens)pCDNA5-UQCC3-FLAGThis paperN/AConstruct generated by mutagenesis of pCDNA5-UQCC3-FLAG
AntibodyTMEM223rabbit polyclonalSelf madePRAB4850(1:500)
AntibodySMIM4rabbit polyclonalSelf madePRAB5494(1:500)
AntibodymS40 (MRPS18B) rabbit polyclonalProteinTechRRID:AB_2146368(1:1000)
AntibodyTMEM177rabbit polyclonalSelf madePRAB4988(1:1000)
AntibodyTIM44rabbit polyclonalSelf madePRAB5142(1:4000)
AntibodyCOX6A rabbit polyclonalSelf madePRAB3282(1:1000)
AntibodySLIRP rabbit polyclonalSelf madePRAB3813(1:500)
AntibodyCYTB rabbit polyclonalSelf madePRAB5151(1:1000)
AntibodyC12ORF73rabbit polyclonalSelf madePRAB5105(1:500)
AntibodyPHB1rabbit polyclonalProteinTechRRID:AB_2164476(1:1000)
AntibodyuL3m rabbit polyclonalProteinTechRRID:AB_10639509(1:1000)
AntibodyuS14m rabbit polyclonalProteinTechRRID:AB_2878240(1:2000)
AntibodybS16m rabbit polyclonalProteinTechRRID:AB_2180166(1:5000)
AntibodyYME1L rabbit polyclonalSelf madePRAB5113(1:500)
AntibodySLP2rabbit polyclonalProteinTechRRID:AB_2286822(1:1000)
AntibodyAFG3L2rabbit polyclonalSelf madePRAB5149(1:500)
AntibodyMITRAC12rabbit polyclonalSelf madePRAB3761(1:1000)
AntibodyC12ORF62rabbit polyclonalSelf madePRAB4844(1:500)
AntibodyMITRAC7rabbit polyclonalSelf madePRAB4843(1:500)
AntibodyCOX1rabbit polyclonalSelf madePRAB2035(1:2000)
AntibodyCOX4-1rabbit polyclonalSelf madePRAB1522(1:2000)
AntibodyuL23m rabbit polyclonalSelf madePRAB1716(1:500)
AntibodyuL1m rabbit polyclonalSelf madePRAB4969(1:500)
AntibodyTOM70rabbit polyclonalSelf madePRAB3280(1:1000)
AntibodyTACO1rabbit polyclonalSelf madePRAB3627(1:500)
AntibodyMITRAC15rabbit polyclonalSelf madePRAB4814(1:500)
AntibodyFLAG rabbit polyclonalSigma-AldrichRRID:AB_259529(1:2000)
AntibodyTIM21rabbit polyclonalSelf madePRAB3674(1:2000)
AntibodySDHAMouse monoclonalSelf madePRAB4978(1:2000)
AntibodyRieske rabbit polyclonalSelf madePRAB1512(1:2000)
AntibodyATP5B rabbit polyclonalSelf madePRAB4826(1:10000)
AntibodyTIM44rabbit polyclonalProteinTechRRID:AB_2204679(1:2500)
AntibodyNDUFB8rabbit polyclonalSelf madePRAB3764(1:500)
AntibodyNDUFA9rabbit polyclonalSelf madePRAB1524(1:500)
AntibodyTIM23rabbit polyclonalSelf madePRAB1527(1:2000)
AntibodySCO2rabbit polyclonalSelf madePRAB4982(1:500)
AntibodyFAM36A rabbit polyclonalSelf madePRAB4490(1:500)
AntibodySURF1rabbit polyclonalSelf madePRAB1528(1:1000)
Recombinant DNA reagentQuikChange Site-Directed Mutagenesis KitAgilent210515
Recombinant DNA reagentKOD Hot Start DNA PolymeraseMerck71086-3
Recombinant DNA reagentFirst Strand cDNA Synthesis KitThermo Fisher ScientificK1612
Commercial assay or kitHuman Complex IV Activity KitAbcamab109910
Chemical compound, drugGeneJuiceMerck70967-3
Chemical compound, drugAnti-FLAG M2 Affinity GelSigma-AldrichA2220
Chemical compound, drugTRIzolThermo Fisher Scientific15596026
Chemical Compound, drugProtein-A SepharoseTM CL-4BGE Healthcare17-0963-03
Chemical Compound, drug[35 S]methionineHartmann AnalyticSCM-01
Chemical compound, drugEmetine dihydrochloride hydrateSigma-Aldrich219282
Software, algorithmImageQuantTL 7.0 softwareGE HealthcareRRID:SCR_014246
Software, algorithmImageJ 1.47vNIHRRID:SCR_003070
Software, algorithmGeneiousBiomatters LtdRRID:SCR_010519
Software, algorithmPrism5GraphPad SoftwareRRID:SCR_015807

Additional files

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
Supplementary file 2

Mass spectrometric analyses of mL62 and SMIM4 containing complexes.

https://cdn.elifesciences.org/articles/68213/elife-68213-supp2-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/68213/elife-68213-transrepform1-v1.pdf

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  1. Sven Dennerlein
  2. Sabine Poerschke
  3. Silke Oeljeklaus
  4. Cong Wang
  5. Ricarda Richter-Dennerlein
  6. Johannes Sattmann
  7. Diana Bauermeister
  8. Elisa Hanitsch
  9. Stefan Stoldt
  10. Thomas Langer
  11. Stefan Jakobs
  12. Bettina Warscheid
  13. Peter Rehling
(2021)
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