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