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

Methods Mol Biol. 2020;2159:115-127. doi: 10.1007/978-1-0716-0676-6_9.

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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bombyx / genetics
  • Fluorescence Resonance Energy Transfer
  • GTP Phosphohydrolases / chemistry*
  • GTP Phosphohydrolases / genetics*
  • GTP Phosphohydrolases / isolation & purification
  • Gene Expression*
  • Humans
  • Membrane Fusion*
  • Mitochondrial Membranes / chemistry*
  • Mitochondrial Membranes / metabolism
  • Proteolipids
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification

Substances

  • Proteolipids
  • Recombinant Proteins
  • proteoliposomes
  • GTP Phosphohydrolases
  • OPA1 protein, human