Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria

  • Luke E. Formosa
  • Annette Hofer
  • Christin Tischner
  • Tina Wenz
  • Michael T. RyanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)


In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Key words

Mitochondria Mitochondrial DNA (mtDNA) Oxidative phosphorylation (OXPHOS) Respiratory chain Complex assembly Pulse labeling Pulse-chase labeling Blue-native PAGE (BN-PAGE) 


  1. 1.
    Hoogenraad NJ, Ward LA, Ryan MT (2002) Import and assembly of proteins into mitochondria of mammalian cells. Biochim Biophys Acta 1592:97–105CrossRefPubMedGoogle Scholar
  2. 2.
    Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465CrossRefPubMedGoogle Scholar
  3. 3.
    Grollman AP (1966) Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics. Proc Natl Acad Sci U S A 56:1867–1874CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Schneider-Poetsch T, Ju J, Eyler DE, Dang Y, Bhat S, Merrick WC, Green R, Shen B, Liu JO (2010) Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6:209–217CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gay DA, Sisodia SS, Cleveland DW (1989) Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation. Proc Natl Acad Sci U S A 86:5763–5767CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kardalinou E, Zhelev N, Hazzalin CA, Mahadevan LC (1994) Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction. Mol Cell Biol 14:1066–1074CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hazzalin CA, Le Panse R, Cano E, Mahadevan LC (1998) Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction. Mol Cell Biol 18:1844–1854CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Tsuchida T, Kato T, Yamada A, Kawamoto K (2002) Cycloheximide induces apoptosis of astrocytes. Pathol Int 52:181185CrossRefGoogle Scholar
  9. 9.
    Ledda-Columbano GM, Faa PCG, Manenti G, Columbano A (1992) Rapid induction of apoptosis in rat liver by cycloheximide. Am J Pathol 140:545–549PubMedPubMedCentralGoogle Scholar
  10. 10.
    Sugiana C, Pagliarini D, McKenzie M, Kirby D, Salemi R, Abu-Amero K, Dahl H-HM, Hutchison W, Vascotto K, Smith S, Newbold R, Christodoulou J, Calvo S, Mootha V, Ryan M, Thorburn D (2008) Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease. Am J Human Genet 83:468–546CrossRefGoogle Scholar
  11. 11.
    Dunning CJ, McKenzie M, Sugiana C, Lazarou M, Silke J, Connelly A, Fletcher JM, Kirby DM, Thorburn DR, Ryan MT (2007) Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease. EMBO J 26:3227–3237CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    McKenzie M, Tucker EJ, Compton AG, Lazarou M, George C, Thorburn DR, Ryan MT (2011) Mutations in the gene encoding C8orf38 block complex i assembly by inhibiting production of the mitochondria-encoded subunit ND1. J Mol Biol 41:413–426CrossRefGoogle Scholar
  13. 13.
    Stroud D, Formosa L, Wijeyeratne X, Nguyen T, Ryan M (2013) 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 288:1685–1690CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Bai Y, Attardi G (1998) The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme. EMBO J 17:4848–4858CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AM, Elsas LJ, Nikoskelainen EK (1988) Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242:1427–1430CrossRefPubMedGoogle Scholar
  16. 16.
    McKenzie M, Lazarou M, Thorburn DR, Ryan MT (2007) Analysis of mitochondrial subunit assembly into respiratory chain complexes using blue native polyacrylamide gel electrophoresis. Anal Biochem 364:128–137CrossRefPubMedGoogle Scholar
  17. 17.
    Chen JJ, Jones ME (1976) The cellular location of dihydroorotate dehydrogenase: relation to de novo biosynthesis of pyrimidines. Arch Biochem Biophys 176:82–90CrossRefPubMedGoogle Scholar
  18. 18.
    King MP, Attardi G (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246:500–503CrossRefPubMedGoogle Scholar
  19. 19.
    Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  20. 20.
    Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231CrossRefPubMedGoogle Scholar
  21. 21.
    Wittig I, Braun H-P, Schägger H (2006) Blue native PAGE. Nat Protoc 1:418–428CrossRefPubMedGoogle Scholar
  22. 22.
    Chomyn A (1996) In vivo labeling and analysis of human mitochondrial translation products. Method Enzymol 264:197–211CrossRefGoogle Scholar
  23. 23.
    Clarke S (1976) A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase. J Biol Chem 251:950–961PubMedGoogle Scholar
  24. 24.
    Fernández-Silva P, Acín-Pérez R, Fernández-Vizarra E, Pérez-Martos A, Enriquez JA (2007) In vivo and in organello analyses of mitochondrial translation. Method Cell Biol 80:571–588Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Luke E. Formosa
    • 1
    • 3
  • Annette Hofer
    • 2
  • Christin Tischner
    • 2
  • Tina Wenz
    • 2
  • Michael T. Ryan
    • 3
    Email author
  1. 1.Department of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceLa Trobe UniversityMelbourneAustralia
  2. 2.Institute for Genetics and Cluster of Excellence: Cellular Stress Responses in Aging-Associated Diseases (CECAD)University of CologneCologneGermany
  3. 3.Department of Biochemistry and Molecular BiologyMonash UniversityMelbourneAustralia

Personalised recommendations