Analysis of Mitochondrial Membrane Fusion GTPase OPA1 Expressed by the Silkworm Expression System

  • Tadato BanEmail author
  • Naotada Ishihara
Part of the Methods in Molecular Biology book series (MIMB, volume 2159)


Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid–silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.

Key words

Mitochondria Membrane fusion GTPase protein Optic atrophy 1(OPA1) In vitro reconstitution Proteoliposome Fluorescence resonance energy transfer- (FRET-) based lipid mixing assay Baculovirus expression system Silkworm 



We thank Dr. T. Oka (Rikkyo University) and Dr. K. Maenaka (Hokkaido University) for advice on the silkworm expression system and Dr. J. Mima (Osaka University) for advice on the preparation of assay with proteoliposomes. This work is supported by JSPS KAKENHI grant number 18K06096, MEXT-Supported Program for the Strategic Research Foundation at Private Universities, the Takeda Science Foundation (T.B.), the Naito Foundation (T.B.) and the Ichiro Kanehara Foundation (T.B.).


  1. 1.
    Ishihara N, Otera H, Oka T, Mihara K (2013) Regulation and physiologic functions of GTPases in mitochondrial fusion and fission in mammals. Antioxid Redox Signal 19:389–399CrossRefGoogle Scholar
  2. 2.
    Labbe K, Murley A, Nunnari J (2014) Determinants and functions of mitochondrial behavior. Annu Rev Cell Dev Biol 30:357–391CrossRefGoogle Scholar
  3. 3.
    Mishra P, Chan DC (2016) Metabolic regulation of mitochondrial dynamics. J Cell Biol 212:379–387CrossRefGoogle Scholar
  4. 4.
    McNew JA, Sondermann H, Lee T, Stern M, Brandizzi F (2013) GTP-dependent membrane fusion. Annu Rev Cell Dev Biol 29:529–550CrossRefGoogle Scholar
  5. 5.
    MacVicar T, Langer T (2016) OPA1 processing in cell death and disease—the long and short of it. J Cell Sci 129:2297–2306CrossRefGoogle Scholar
  6. 6.
    Ishihara N, Fujita Y, Oka T, Mihara K (2006) Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J 25:2966–2977CrossRefGoogle Scholar
  7. 7.
    Tondera D, Grandemange S, Jourdain A, Karbowski M, Mattenberger Y, Herzig S, Da Cruz S, Clerc P, Raschke I, Merkwirth C, Ehses S, Krause F, Chan DC, Alexander C, Bauer C, Youle R, Langer T, Martinou JC (2009) SLP-2 is required for stress-induced mitochondrial hyperfusion. EMBO J 28:1589–1600CrossRefGoogle Scholar
  8. 8.
    Ban T, Ishihara T, Kohno H, Saita S, Ichimura A, Maenaka K, Oka T, Mihara K, Ishihara N (2017) Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat Cell Biol 19:856–863CrossRefGoogle Scholar
  9. 9.
    Song Z, Chen H, Fiket M, Alexander C, Chan DC (2007) OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. J Cell Biol 178:749–755CrossRefGoogle Scholar
  10. 10.
    Anand R, Wai T, Baker MJ, Kladt N, Schauss AC, Rugarli E, Langer T (2014) The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission. J Cell Biol 204:919–929CrossRefGoogle Scholar
  11. 11.
    Ban T, Heymann JA, Song Z, Hinshaw JE, Chan DC (2010) OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum Mol Genet 19:2113–2122CrossRefGoogle Scholar
  12. 12.
    Ban T, Kohno H, Ishihara T, Ishihara N (2018) Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion. Biochim Biophys Acta 1859:951–957CrossRefGoogle Scholar
  13. 13.
    Wickner W, Schekman R (2008) Membrane fusion. Nat Struct Mol Biol 15:658–664CrossRefGoogle Scholar
  14. 14.
    Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Sollner TH, Rothman JE (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772CrossRefGoogle Scholar
  15. 15.
    Orso G, Pendin D, Liu S, Tosetto J, Moss TJ, Faust JE, Micaroni M, Egorova A, Martinuzzi A, McNew JA, Daga A (2009) Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature 460:978–983CrossRefGoogle Scholar
  16. 16.
    Kato T, Kajikawa M, Maenaka K, Park EY (2010) Silkworm expression system as a platform technology in life science. Appl Microbiol Biotechnol 85:459–470CrossRefGoogle Scholar
  17. 17.
    Kajikawa M, Sasaki-Tabata K, Fukuhara H, Horiuchi M, Okabe Y, Maenaka K (2012) Silkworm baculovirus expression system for molecular medicine. J Biotechnol Biomaterial S9:005CrossRefGoogle Scholar
  18. 18.
    Scott BL, Van Komen JS, Liu S, Weber T, Melia TJ, McNew JA (2003) Liposome fusion assay to monitor intracellular membrane fusion machines. Methods Enzymol 372:274–300CrossRefGoogle Scholar
  19. 19.
    Rigaud JL, Levy D (2003) Reconstitution of membrane proteins into liposomes. Methods Enzymol 372:65–86CrossRefGoogle Scholar
  20. 20.
    Mima J, Wickner W (2009) Complex lipid requirements for SNARE- and SNARE chaperone-dependent membrane fusion. J Biol Chem 284:27114–27222CrossRefGoogle Scholar
  21. 21.
    Ardail D, Privat JP, Egret-Charlier M, Levrat C, Lerme F, Louisot P (1990) Mitochondrial contact sites. Lipid composition and dynamics. J Biol Chem 265:18797–18802PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Protein Biochemistry, Institute of Life ScienceKurume UniversityFukuokaJapan
  2. 2.Department of Biological Science, Graduate School of ScienceOsaka UniversityOsakaJapan

Personalised recommendations