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Accessory subunits are integral for assembly and function of human mitochondrial complex I

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes1. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP2. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson’s disease and ageing3,4,5. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear1,6. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

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Figure 1: Analysis of complex I assembly in knockout cell lines.
Figure 2: Metabolic and proteomic analysis of representative complex I accessory subunit-knockout lines.
Figure 3: Subunit stability correlates with structural modules.
Figure 4: Analysis of complex I assembly factors including DMAC1 and ATP5SL.

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Gene Expression Omnibus

Data deposits

Data are available via ProteomeXchange under accession PXD004666, and the NCBI Gene Expression Omnibus (GEO) under accession GSE84913.

References

  1. Sazanov, L. A. A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat. Rev. Mol. Cell Biol. 16, 375–388 (2015)

    Article  CAS  PubMed  Google Scholar 

  2. Lapuente-Brun, E. et al. Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Science 340, 1567–1570 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Vafai, S. B. & Mootha, V. K. Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374–383 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Morais, V. A. et al. PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 344, 203–207 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Miwa, S. et al. Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice. Nat. Commun. 5, 3837 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Hirst, J. Mitochondrial complex I. Annu. Rev. Biochem. 82, 551–575 (2013)

    Article  CAS  PubMed  Google Scholar 

  7. Vinothkumar, K. R., Zhu, J. & Hirst, J. Architecture of mammalian respiratory complex I. Nature 515, 80–84 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zickermann, V. et al. Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I. Science 347, 44–49 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Zhu, J., Vinothkumar, K. R. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Baradaran, R., Berrisford, J. M., Minhas, G. S. & Sazanov, L. A. Crystal structure of the entire respiratory complex I. Nature 494, 443–448 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lazarou, M., McKenzie, M., Ohtake, A., Thorburn, D. R. & Ryan, M. T. Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol. Cell. Biol. 27, 4228–4237 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Runswick, M. J., Fearnley, I. M., Skehel, J. M. & Walker, J. E. Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria. FEBS Lett. 286, 121–124 (1991)

    Article  CAS  PubMed  Google Scholar 

  14. Rodenburg, R. J. Mitochondrial complex I-linked disease. Biochim. Biophys. Acta 1857, 938–945 (2016)

    Article  CAS  PubMed  Google Scholar 

  15. Stroud, D. A. et al. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2. Hum. Mol. Genet. 24, 5404–5415 (2015)

    Article  CAS  PubMed  Google Scholar 

  16. Quirós, P. M., Langer, T. & López-Otín, C. New roles for mitochondrial proteases in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16, 345–359 (2015)

    Article  PubMed  CAS  Google Scholar 

  17. Andrews, B., Carroll, J., Ding, S., Fearnley, I. M. & Walker, J. E. Assembly factors for the membrane arm of human complex I. Proc. Natl Acad. Sci. USA 110, 18934–18939 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sánchez-Caballero, L., Guerrero-Castillo, S. & Nijtmans, L. Unraveling the complexity of mitochondrial complex I assembly: A dynamic process. Biochim. Biophys. Acta 1857, 980–990 (2016)

    Article  PubMed  CAS  Google Scholar 

  19. Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2015)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Stroud, D. A., Formosa, L. E., Wijeyeratne, X. W., Nguyen, T. N. & Ryan, M. T. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I. J. Biol. Chem. 288, 1685–1690 (2013)

    Article  CAS  PubMed  Google Scholar 

  21. Formosa, L. E. et al. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I. Hum. Mol. Genet. 24, 2952–2965 (2015)

    Article  CAS  PubMed  Google Scholar 

  22. Mimaki, M., Wang, X., McKenzie, M., Thorburn, D. R. & Ryan, M. T. Understanding mitochondrial complex I assembly in health and disease. Biochim. Biophys. Acta 1817, 851–862 (2012)

    Article  CAS  PubMed  Google Scholar 

  23. DuBridge, R. B. et al. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 7, 379–387 (1987)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Heide, H. et al. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex. Cell Metab. 16, 538–549 (2012)

    Article  CAS  PubMed  Google Scholar 

  25. Vogel, R. O. et al. Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits. J. Biol. Chem. 282, 7582–7590 (2007)

    Article  CAS  PubMed  Google Scholar 

  26. Huttlin, E. L. et al. The BioPlex Network: a systematic exploration of the human interactome. Cell 162, 425–440 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Reljic´, B. & Stroud, D. A. Screening strategies for TALEN-mediated gene disruption. Methods Mol. Biol. 1419, 231–252 (2016)

    Article  PubMed  Google Scholar 

  29. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protocols 8, 2281–2308 (2013)

    Article  CAS  PubMed  Google Scholar 

  30. Sander, J. D. et al. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 38, W462–8 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–7 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Morgenstern, J. P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Acín-Pérez, R., Fernández-Silva, P., Peleato, M. L., Pérez-Martos, A. & Enriquez, J. A. Respiratory active mitochondrial supercomplexes. Mol. Cell 32, 529–539 (2008)

    Article  PubMed  CAS  Google Scholar 

  34. McKenzie, M., Lazarou, M., Thorburn, D. R. & Ryan, M. T. Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis. Anal. Biochem. 364, 128–137 (2007)

    Article  CAS  PubMed  Google Scholar 

  35. Wittig, I., Braun, H. P. & Schägger, H. Blue native PAGE. Nat. Protocols 1, 418–428 (2006)

    Article  CAS  PubMed  Google Scholar 

  36. Schägger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379 (1987)

    Article  PubMed  Google Scholar 

  37. Ryan, M. T., Voos, W. & Pfanner, N. Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215 (2001)

    Article  CAS  PubMed  Google Scholar 

  38. Dunning, C. J. et al. Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease. EMBO J. 26, 3227–3237 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Jänicke, A., Vancuylenberg, J., Boag, P. R., Traven, A. & Beilharz, T. H. ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18, 1289–1295 (2012)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Harrison, P. F. et al. PAT-seq: a method to study the integration of 3′-UTR dynamics with gene expression in the eukaryotic transcriptome. RNA 21, 1502–1510 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Frazier, A. E. & Thorburn, D. R. Biochemical analyses of the electron transport chain complexes by spectrophotometry. Methods Mol. Biol. 837, 49–62 (2012)

    Article  CAS  PubMed  Google Scholar 

  42. Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014)

    Article  CAS  PubMed  Google Scholar 

  43. Johnston, A. J. et al. Insertion and assembly of human Tom7 into the preprotein translocase complex of the outer mitochondrial membrane. J. Biol. Chem. 277, 42197–42204 (2002)

    Article  CAS  PubMed  Google Scholar 

  44. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008)

    Article  CAS  PubMed  Google Scholar 

  45. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  46. Gagnon-Bartsch, J. A. & Speed, T. P. Using control genes to correct for unwanted variation in microarray data. Biostatistics 13, 539–552 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  47. Leek, J. T. & Storey, J. D. Capturing heterogeneity in gene expression studies by surrogate variable analysis. PLoS Genet. 3, 1724–1735 (2007)

    Article  CAS  PubMed  Google Scholar 

  48. Münch, C. & Harper, J. W. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534, 710–713 (2016)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  49. Wrobel, L. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  50. Hubner, N. C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001)

    Article  ADS  CAS  MATH  PubMed  PubMed Central  Google Scholar 

  52. Merico, D., Isserlin, R., Stueker, O., Emili, A. & Bader, G. D. Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One 5, e13984 (2010)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  53. Stroud, D. A. et al. Composition and topology of the endoplasmic reticulum-mitochondria encounter structure. J. Mol. Biol. 413, 743–750 (2011)

    Article  CAS  PubMed  Google Scholar 

  54. Gebert, N. et al. Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane. Mol. Cell 44, 811–818 (2011)

    Article  CAS  PubMed  Google Scholar 

  55. Richter, V. et al. Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission. J. Cell Biol. 204, 477–486 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ostergaard, E. et al. Respiratory chain complex I deficiency due to NDUFA12 mutations as a new cause of Leigh syndrome. J. Med. Genet. 48, 737–740 (2011)

    Article  CAS  PubMed  Google Scholar 

  58. Assouline, Z. et al. A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome. Biochim. Biophys. Acta 1822, 1062–1069 (2012)

    Article  CAS  PubMed  Google Scholar 

  59. Haack, T. B. et al. Mutation screening of 75 candidate genes in 152 complex I deficiency cases identifies pathogenic variants in 16 genes including NDUFB9. J. Med. Genet. 49, 83–89 (2012)

    Article  CAS  PubMed  Google Scholar 

  60. Kirby, D. M. et al. NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J. Clin. Invest. 114, 837–845 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. van den Bosch, B. J. et al. Defective NDUFA9 as a novel cause of neonatally fatal complex I disease. J. Med. Genet. 49, 10–15 (2012)

    Article  CAS  PubMed  Google Scholar 

  62. Hoefs, S. J. et al. NDUFA10 mutations cause complex I deficiency in a patient with Leigh disease. Eur. J. Hum. Genet. 19, 270–274 (2011)

    Article  PubMed  Google Scholar 

  63. Angebault, C. et al. Mutation in NDUFA13/GRIM19 leads to early onset hypotonia, dyskinesia and sensorial deficiencies, and mitochondrial complex I instability. Hum. Mol. Genet. 24, 3948–3955 (2015)

    Article  CAS  PubMed  Google Scholar 

  64. Haack, T. B. et al. Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing. J. Med. Genet. 49, 277–283 (2012)

    Article  CAS  PubMed  Google Scholar 

  65. Shehata, B. M. et al. Exome sequencing of patients with histiocytoid cardiomyopathy reveals a de novo NDUFB11 mutation that plays a role in the pathogenesis of histiocytoid cardiomyopathy. Am. J. Med. Genet. A. 167A, 2114–2121 (2015)

    Article  PubMed  CAS  Google Scholar 

  66. Fernandez-Moreira, D. et al. X-linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathy. Ann. Neurol. 61, 73–83 (2007)

    Article  CAS  PubMed  Google Scholar 

  67. Peralta, S. et al. Partial complex I deficiency due to the CNS conditional ablation of Ndufa5 results in a mild chronic encephalopathy but no increase in oxidative damage. Hum. Mol. Genet. 23, 1399–1412 (2014)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Curtis, P. Faou, M. Lazarou, B. Porebski, L. Twigg, R. Schittenhelm (Monash Biomedical Proteomics Platform), A. Barugahare and P. Harrison (Monash Bioinformatics Platform), Monash Micro Imaging and the Micromon NGS Facility for assistance. We acknowledge funding from NHMRC Project Grants (1068056, 1107094) and fellowships (1070916 to D.A.S., 541920 to A.E.F., 1022896 to D.R.T.), the Australian Mitochondrial Disease Foundation and the Victorian Government’s Operational Infrastructure Support Program.

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Authors and Affiliations

Authors

Contributions

D.A.S. and M.T.R. conceived the project and wrote the manuscript; D.A.S., D.R.T. and M.T.R. designed the experiments; D.A.S., E.E.S., L.E.F., B.R., M.G.D., L.D.O. and M.T.R. generated and analysed knockout lines; D.A.S. performed proteomic experiments; A.E.F. and T.S. performed enzymology; T.H.B. undertook transcript analysis; A.S. developed normalization algorithms.

Corresponding authors

Correspondence to David A. Stroud or Michael T. Ryan.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks J. Hirst, B. Lightowlers and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Assembly analysis of the complex I/III/IV supercomplex in knockout cell lines.

Mitochondria were solubilized in digitonin and complexes separated by BN–PAGE followed by immunoblotting using the indicated antibodies. An antibody against complex V (CV) subunit ATP5A was used as loading control. #, subcomplexes; *, non-specific. SC, supercomplex.

Extended Data Figure 2 Steady-state levels of respiratory chain complexes I–IV and supercomplex forms in the 28 complex I accessory subunit knockout lines generated in this study.

NDUFA9KO has been analysed previously20, whereas the NDUFAB1KO is described in Extended Data Fig. 3. Mitochondria were solubilized in Triton X-100 (TX100) or digitonin (DIG) and analysed by BN–PAGE and immunoblotting with antibodies against NDUFA9 (complex I), SDHA (complex II), UQCRC1 (complex III) and COX4 (complex IV). In Triton X-100 samples, some complex III–IV supercomplex is retained. #, secondary clone later identified be an incomplete knockout.

Extended Data Figure 3 Generation and analysis of NDUFAB1-knockout cell lines.

a, Scheme detailing knockout strategy of genomic NDUFAB1 using doxycycline (DOX)-inducible expression of CRISPR/Cas9-resistant NDUFAB1 (NDUFAB1*Flag) or yACP1Flag. b, NDUFAB1 knockouts complemented with NDUFAB1Flag (NDUFAB1*-2) cells were cultured in media lacking DOX for the indicated times. Isolated mitochondria were analysed by BN–PAGE (Triton X-100) or SDS–PAGE and immunoblotting with the indicated antibodies. c, Brightfield images of cells grown ± DOX, or +DOX in glucose or galactose cell culture medium. Scale bars, 25 μm. Representative results from 4 independent experiments. d, SILAC-labelled mitochondria from DOX-treated HEK293T or NDUFAB1Flag (NDUFAB1*-2) cells were solubilized in Triton X-100 and incubated with anti-Flag affinity gel. Elutions were mixed and analysed by liquid chromatography–mass spectrometry (LC–MS). Proteins enriched with NDUFAB1 include complex I subunits and LYRM proteins. P values are from an unpaired single-sided t-test. n = 3 biological replicates; light grey dots, not significant (P >0.05). e, Mitochondria isolated from NDUFAB1 knockouts complemented with yACP1Flag or NDUFAB1Flag were solubilized in Triton X-100 and analysed by BN–PAGE and immunoblotting with the indicated antibodies.

Extended Data Figure 4 Analysis of N-module accessory subunits.

a, Mitochondria were isolated from cell lines, solubilized in Triton X-100 and analysed by BN–PAGE and immunoblotting for N-module subunit NDUFV1 or non N-module subunit NDUFA9. ‡, complex lacking N-module; N*, subcomplex containing N-module. SDHA was used as a loading control. b, Mitochondria were solubilized in digitonin and analysed by BN–PAGE and immunoblotting for NDUFAF2. †, NDUFAF2 associated complex I. c, [35S]methionine-labelled proteins were imported into the indicated mitochondria, solubilized in digitonin and analysed by BN–PAGE and autoradiography. 10% of the input lysate was analysed by SDS–PAGE and autoradiography. CISC, complex I supercomplex; *, non-specific band. d, Mitochondria isolated from NDUFV3-knockout cells complemented with NDUFV3Flag were solubilized in Triton X-100 or digitonin and complexes bound to anti-Flag affinity gel. Eluted proteins were analysed by LC–MS. P values are from an unpaired single-sided t-test. n = 3 biological replicates; light grey dots, not significant (P > 0.05).

Extended Data Figure 5 Proteomic analysis of knockout cell lines.

a, Relative levels of proteins in representative accessory subunit knockout cell lines, clustered according to Euclidean distance. Column order is as in Fig. 2b. The inset shows complex I subunit-specific clusters. b, Volcano plot depicting proteins regulated in representative accessory subunit knockout cell lines containing respiration defects (NDUFA2, NDUFA8, NDUFS5, NDUFC1, NDUFB10, NDUFB11 and NDUFB7 knockouts). Proteins found to be regulated in a cell line with a severe complex IV defect15 are shaded light blue (down) and green (up), suggesting their response is due to general defects in respiration. Inset, volcano plot depicting the relative level of proteins in a complex IV knockout cell line. P values are from an unpaired t-test; n = 8 independent means comprised each of 3 biological replicates (main panel), n = 3 (inset) biological replicates; light grey dots, not significant (P > 0.05, <1.5-fold change). Data are reproduced in Supplementary Table 6. c, Proteins affected >2-fold in levels in respiration-deficient subunit knockout cell lines. Colour key according to b. Bold, proteins listed in MitoCarta2.0. d, Proteins associated with GO terms and groups outlined in Fig. 2d.

Extended Data Figure 6 Mapping of complex I subunit levels onto the structure.

a, Subunit levels in complex I accessory subunit knockout lines were mapped to homologous subunits in the bovine single-particle electron cryo-microscopy structure of complex I (ref. 9) as in Fig. 3b. Both sides of complex I are shown. Median ratio data used in the preparation of this figure can be found in Supplementary Table 7. b, Opposite side view of Fig. 3c. n.d., dark grey shading on the structures, subunits not quantified. Subunits not clustered to modules removed for clarity.

Extended Data Figure 7 mRNA expression levels in selected accessory subunit knockout lines.

Transcripts were measured for nuclear-encoded complex I subunit genes along with control genes from complex II (SDHA), complex III (UQCRC1, UQCRFS1), complex IV (COX4L1, NDUFA4), complex V (ATP5B, ATP5H) and mt-ribosome (MRPS2, MRPL46) in knockout lines (performed in duplicate).

Extended Data Figure 8 Analysis of assembly factor knockout lines.

a, Mitochondrial proteins from the indicated cell lines were separated by SDS–PAGE and subjected to western blot analysis. b, Volcano plots showing fold changes versus P values for the mitochondrial proteins in assembly factor knockout cell lines. P values are from an unpaired t-test; n = 3 biological replicates; coloured dots are according to the key at bottom right. n.s., not significant (P > 0.05). c, Subunit levels mapped to homologous subunits in the bovine single-particle electron cryo-microscopy structure as in Fig. 3b. n.d., dark grey shading on the structures, subunits not quantified. Both sides of complex I are shown.

Extended Data Figure 9 Characterization of DMAC1 and ATP5SL.

a, ATP5SL-knockout mitochondria were solubilized in Triton X-100 or digitonin and analysed by BN–PAGE and immunoblotting with the indicated antibodies. b, As in a using DMAC1-knockout mitochondria. c, Volcano plots showing fold changes versus P values for the mitochondrial proteins in ATP5SL and DMAC1 knockout cell lines. P values are from an unpaired t-test; n = 3 biological replicates; coloured dots represent complex I subunits depicted in the key; n.s., P > 0.05. d, Subunit levels mapped to homologous subunits in the bovine single-particle electron cryo-microscopy structure as for Fig. 3b. n.d., dark grey shading on the structures, not quantified. Both sides of complex I are shown. e, Mitochondria isolated from DMAC1 cells complemented with DMAC1Flag were resuspended in isotonic buffer, hypoosmotic swelling buffer, or Triton X-100 followed by proteinase K (PK) incubation where indicated. Alternately, mitochondria were treated with 100 mM Na2CO3 and membrane-integral (pellet) and soluble or peripherally attached (supernatant, SN) proteins were separated by ultracentrifugation. Samples were analysed by SDS–PAGE and immunoblotting for TOMM20 (outer mitochondrial membrane protein); MIC10 (integral inner membrane protein exposed to intermembrane space); NDUFAF1 (matrix, soluble); and NDUFS2 (matrix, peripheral). f, DMAC1-knockout cells complemented with DMAC1Flag were analysed by immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm. Representative result from 3 independent experiments. g, Cells were pulsed with [35S]methionine for 1 h and chased for the indicated times. Isolated mitochondria were solubilized in Triton X-100 and analysed by 2D-PAGE and autoradiography. , 600 kDa complex; #, subcomplex containing ND1 and ND2.

Extended Data Table 1 Pathogenic mutations in complex I accessory subunit genes in patients with mitochondrial disease

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, uncropped scans with size marker indications and Supplementary Table 1, detailed information on targeting strategies and resulting indels detected in knockout cell lines generated in this study. (PDF 28333 kb)

Supplementary Data

This file contains Supplementary Tables 2-12, representing the proteomic data generated in this study and a list of primer sequences used for mRNA expression level analysis. (XLSX 18192 kb)

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Stroud, D., Surgenor, E., Formosa, L. et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature 538, 123–126 (2016). https://doi.org/10.1038/nature19754

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