Purification of Functional F-ATP Synthase from Blue Native PAGE

  • Chiara Galber
  • Giulia Valente
  • Sophia von Stockum
  • Valentina GiorgioEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)


In the presence of Ca2+, F-ATP synthase preparations eluted from Blue Native gels generate electrophysiological currents that are typical of an inner mitochondrial membrane mega-channel, the permeability transition pore. Here we describe an experimental protocol for purification of F-ATP synthase that allows to maintain the enzyme assembly and activity that are essential for catalysis and channel formation.

Key words

Blue Native PAGE F-ATP synthase Lipid bilayer Gel purification Permeability transition 



We would like to thank Paolo Bernardi (University of Padova), Giovanna Lippe (University of Udine), and Ildikò Szabò (University of Padova) for their helpful suggestions. This work was supported by AIRC grant MFAG 2017 20316 to V.G.


  1. 1.
    Walker JE (2013) The ATP synthase: the understood, the uncertain and the unknown. Biochem Soc Trans 41:1–16CrossRefGoogle Scholar
  2. 2.
    Rees DM, Leslie AGW, Walker JE (2009) The structure of the membrane extrinsic region of bovine ATP synthase. Proc Natl Acad Sci U S A 106:21597–21601CrossRefGoogle Scholar
  3. 3.
    Zhou A, Rohou A, Schep DG, Bason JV, Montgomery MG, Walker JE, Grigorieffniko N, Rubinstein JL (2015) Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. elife 4:1–15Google Scholar
  4. 4.
    Hiroyuki N, Ryohei Y, Yoshida Masasuke KK (1997) Noji 1997 pdf. Direct observation of the rotation of F1-ATPase. Nature 386:299–302CrossRefGoogle Scholar
  5. 5.
    Senior AE (2007) ATP synthase: motoring to the finish line. Cell 130:220–221CrossRefGoogle Scholar
  6. 6.
    Kühlbrandt W, Davies KM (2016) Rotary ATPases: a new twist to an ancient machine. Trends Biochem Sci 41:106–116CrossRefGoogle Scholar
  7. 7.
    Strauss M, Hofhaus G, Schröder RR, Kühlbrandt W (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 27:1154–1160CrossRefGoogle Scholar
  8. 8.
    Davies KM, Strauss M, Daum B, Kief JH, Osiewacz HD, Rycovska A, Zickermann V, Kuhlbrandt W (2011) Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc Natl Acad Sci 108:14121–14126CrossRefGoogle Scholar
  9. 9.
    Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabó I, Lippe G, Bernardi P (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci U S A 110:5887–5892CrossRefGoogle Scholar
  10. 10.
    Carraro M, Giorgio V, Sileikyte J, Sartori G, Forte M, Lippe G, Zoratti M, Szabò I, Bernardi P (2014) Channel formation by yeast F-ATP synthase and the role of dimerization in the mitochondrial permeability transition. J Biol Chem 289:15980–15985CrossRefGoogle Scholar
  11. 11.
    Von Stockum S, Giorgio V, Trevisan E, Lippe G, Glick GD, Forte MA, Da-Rè C, Checchetto V, Mazzotta G, Costa R, Szabò I, Bernardi P (2015) F-ATPase of drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca 2+-induced Ca2+ release. J Biol Chem 290:4537–4544CrossRefGoogle Scholar
  12. 12.
    Bernardi P, Rasola A, Forte M, Lippe G (2015) The mitochondrial permeability transition pore: channel formation by F-ATP synthase, integration in signal transduction, and role in pathophysiology. Physiol Rev 95:1111–1155CrossRefGoogle Scholar
  13. 13.
    Giorgio V, Guo L, Bassot C, Petronilli V, Bernardi P (2018) Calcium and regulation of the mitochondrial permeability transition. Cell Calcium 70:56–63CrossRefGoogle Scholar
  14. 14.
    Giorgio V, Burchell V, Schiavone M, Bassot C, Minervini G, Petronilli V, Argenton F, Forte M, Tosatto S, Lippe G, Bernardi P (2017) Ca 2+ binding to F-ATP synthase β subunit triggers the mitochondrial permeability transition. EMBO Rep 18:1065–1076CrossRefGoogle Scholar
  15. 15.
    Wittig I, Schägger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5:4338–4346CrossRefGoogle Scholar
  16. 16.
    Zerbetto E, Vergani LD-SF (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064CrossRefGoogle Scholar
  17. 17.
    Wittig I, Carrozzo R, Santorelli FM, Schägger H (2006) Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation. Biochim Biophys Acta Bioenerg 1757:1066–1072CrossRefGoogle Scholar
  18. 18.
    Meyer B, Wittig I, Trifilieff E, Karas M, Schägger H (2007) Identification of two proteins associated with mammalian ATP synthase. Mol Cell Proteomics 6:1690–1699CrossRefGoogle Scholar
  19. 19.
    Giorgio V, Bisetto E, Franca R, Harris DA, Passamonti S, Lippe G (2010) The ectopic FOF1 ATP synthase of rat liver is modulated in acute cholestasis by the inhibitor protein IF1. J Bioenerg Biomembr 42:117–123CrossRefGoogle Scholar
  20. 20.
    He J, Ford HC, Carroll J, Douglas C, Gonzales E, Ding S, Fearnley IM, Walker JE (2018) Assembly of the membrane domain of ATP synthase in human mitochondria. Proc Natl Acad Sci 115:2988–2993CrossRefGoogle Scholar
  21. 21.
    Reisinger V, Eichacker LA (2008) Solubilization of membrane protein complexes for blue native PAGE. J Proteome 71:277–283CrossRefGoogle Scholar
  22. 22.
    Ko YH, Delannoy M, Hullihen J, Chiu W, Pedersen PL (2003) Mitochondrial ATP synthasome: Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP. J Biol Chem 278:12305–12309CrossRefGoogle Scholar
  23. 23.
    Rehling P, Model K, Brandner K, Kovermann P, Sickmann A, Meyer HE, Wagner R, Truscott KN (2003) Mitochondrial inner membrane by a twin-pore translocase. Science 229:1747–1751CrossRefGoogle Scholar
  24. 24.
    Schägger H, Cramer WA, Vonjagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217:220–230CrossRefGoogle Scholar
  25. 25.
    Bisetto E, Di Pancrazio F, Simula MP, Mavelli ILG (2007) Mammalian ATPsynthase monomer versus dimer profiled by blue native PAGE and activity stain. Electrophoresis 28:3178–3185CrossRefGoogle Scholar
  26. 26.
    Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287–295CrossRefGoogle Scholar
  27. 27.
    Yamada A, Yamamoto T, Yoshimura Y, Gouda S, Kawashima S, Yamazaki N, Yamashita K, Kataoka M, Nagata T, Terada H, Pfeiffer DR, Shinohara Y (2009) Ca2+−induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions. Biochim Biophys Acta Bioenerg 1787:1486–1491CrossRefGoogle Scholar
  28. 28.
    Nicolli A, Basso E, Petronilli V, Wenger RM, Bernardi P (1996) Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, a cyclosporin A-sensitive channel. JBC 271:2185–2196CrossRefGoogle Scholar
  29. 29.
    Chatzianastasiou A, Bibli S-I, Andreadou I, Efentakis P, Kaludercic N, Wood ME, Whiteman M, Di Lisa F, Daiber A, Manolopoulos VG, Szabó C, Papapetropoulos George Livanos AP (2016) Cardioprotection by H 2 S donors: nitric oxide-dependent and -independent mechanisms. J Pharmacol Exp Ther 358:431–440CrossRefGoogle Scholar
  30. 30.
    Giorgio V, Bisetto E, Soriano ME, Dabbeni-Sala F, Basso E, Petronilli V, Forte MA, Bernardi P, Lippe G (2009) Cyclophilin D modulates mitochondrial F0F1-ATP synthase by interacting with the lateral stalk of the complex. J Biol Chem 284:33982–33988CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chiara Galber
    • 1
  • Giulia Valente
    • 1
  • Sophia von Stockum
    • 2
    • 3
  • Valentina Giorgio
    • 1
    Email author
  1. 1.Neuroscience Institute and Department of Biomedical SciencesCNR and University of PaduaPaduaItaly
  2. 2.Department of BiologyUniversity of PaduaPaduaItaly
  3. 3.Fondazione Ospedale San CamilloIRCCSVeneziaItaly

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