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

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

Abstract

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 

Notes

Acknowledgments

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.).

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© 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

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