Letter | Published:

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

Nature volume 538, pages 123126 (06 October 2016) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

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. 1.

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

  2. 2.

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

  3. 3.

    & Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374–383 (2012)

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , & Architecture of mammalian respiratory complex I. Nature 515, 80–84 (2014)

  8. 8.

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

  9. 9.

    , & Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016)

  10. 10.

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

  11. 11.

    , , & Crystal structure of the entire respiratory complex I. Nature 494, 443–448 (2013)

  12. 12.

    , , , & Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol. Cell. Biol. 27, 4228–4237 (2007)

  13. 13.

    , , & Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria. FEBS Lett. 286, 121–124 (1991)

  14. 14.

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

  15. 15.

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

  16. 16.

    , & New roles for mitochondrial proteases in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16, 345–359 (2015)

  17. 17.

    , , , & Assembly factors for the membrane arm of human complex I. Proc. Natl Acad. Sci. USA 110, 18934–18939 (2013)

  18. 18.

    , & Unraveling the complexity of mitochondrial complex I assembly: A dynamic process. Biochim. Biophys. Acta 1857, 980–990 (2016)

  19. 19.

    , & MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2015)

  20. 20.

    , , , & 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)

  21. 21.

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

  22. 22.

    , , , & Understanding mitochondrial complex I assembly in health and disease. Biochim. Biophys. Acta 1817, 851–862 (2012)

  23. 23.

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

  24. 24.

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

  25. 25.

    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)

  26. 26.

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

  27. 27.

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

  28. 28.

    & Screening strategies for TALEN-mediated gene disruption. Methods Mol. Biol. 1419, 231–252 (2016)

  29. 29.

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

  30. 30.

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

  31. 31.

    , , , & CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–7 (2014)

  32. 32.

    & 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)

  33. 33.

    , , , & Respiratory active mitochondrial supercomplexes. Mol. Cell 32, 529–539 (2008)

  34. 34.

    , , & Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis. Anal. Biochem. 364, 128–137 (2007)

  35. 35.

    , & Blue native PAGE. Nat. Protocols 1, 418–428 (2006)

  36. 36.

    & 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)

  37. 37.

    , & Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215 (2001)

  38. 38.

    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)

  39. 39.

    , , , & ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18, 1289–1295 (2012)

  40. 40.

    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)

  41. 41.

    & Biochemical analyses of the electron transport chain complexes by spectrophotometry. Methods Mol. Biol. 837, 49–62 (2012)

  42. 42.

    , , , & Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014)

  43. 43.

    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)

  44. 44.

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

  45. 45.

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

  46. 46.

    & Using control genes to correct for unwanted variation in microarray data. Biostatistics 13, 539–552 (2012)

  47. 47.

    & Capturing heterogeneity in gene expression studies by surrogate variable analysis. PLoS Genet. 3, 1724–1735 (2007)

  48. 48.

    & Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534, 710–713 (2016)

  49. 49.

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

  50. 50.

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

  51. 51.

    , & Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001)

  52. 52.

    , , , & Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One 5, e13984 (2010)

  53. 53.

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

  54. 54.

    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)

  55. 55.

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

  56. 56.

    , & NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

  57. 57.

    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)

  58. 58.

    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)

  59. 59.

    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)

  60. 60.

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

  61. 61.

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

  62. 62.

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

  63. 63.

    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)

  64. 64.

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

  65. 65.

    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)

  66. 66.

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

  67. 67.

    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)

Download references

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.

Author information

Author notes

    • Boris Reljic

    Present address: Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia.

Affiliations

  1. Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, 3800, Melbourne, Australia

    • David A. Stroud
    • , Elliot E. Surgenor
    • , Luke E. Formosa
    • , Marris G. Dibley
    • , Laura D. Osellame
    • , Traude H. Beilharz
    •  & Michael T. Ryan
  2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University 3086, Melbourne, Australia

    • Luke E. Formosa
    •  & Boris Reljic
  3. Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne 3052, Australia

    • Ann E. Frazier
    • , Tegan Stait
    •  & David R. Thorburn
  4. Department of Pediatrics, University of Melbourne, Melbourne 3052, Australia

    • Ann E. Frazier
    •  & David R. Thorburn
  5. Victorian Clinical Genetics Services, Royal Children’s Hospital 3052, Melbourne, Australia

    • David R. Thorburn
  6. Department of Mathematics and Statistics, La Trobe University 3086, Melbourne Australia

    • Agus Salim

Authors

  1. Search for David A. Stroud in:

  2. Search for Elliot E. Surgenor in:

  3. Search for Luke E. Formosa in:

  4. Search for Boris Reljic in:

  5. Search for Ann E. Frazier in:

  6. Search for Marris G. Dibley in:

  7. Search for Laura D. Osellame in:

  8. Search for Tegan Stait in:

  9. Search for Traude H. Beilharz in:

  10. Search for David R. Thorburn in:

  11. Search for Agus Salim in:

  12. Search for Michael T. Ryan in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

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

Supplementary information

PDF files

  1. 1.

    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.

Excel files

  1. 1.

    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.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature19754

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing