A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outer-membrane tethering and fusion

J Biol Chem. 2019 May 17;294(20):8001-8014. doi: 10.1074/jbc.RA118.006347. Epub 2019 Apr 1.

Abstract

Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. In Mfn1 and Mfn2 paralogs, a conserved phenylalanine (Phe-202 (Mfn1) and Phe-223 (Mfn2)) located in the GTPase domain on a conserved β strand is part of an aromatic network in the core of this domain. To gain insight into the poorly understood mechanism of Mfn-mediated membrane fusion, here we characterize a Mitofusin mutant variant etiologically linked to Charcot-Marie-Tooth syndrome. From analysis of mitochondrial structure in cells and mitochondrial fusion in vitro, we found that conversion of Phe-202 to leucine in either Mfn1 or Mfn2 diminishes the fusion activity of heterotypic complexes with both Mfn1 and Mfn2 and abolishes fusion activity of homotypic complexes. Using coimmunoprecipitation and native gel analysis, we further dissect the steps of mitochondrial fusion and demonstrate that the mutant variant has normal tethering activity but impaired higher-order nucleotide-dependent assembly. The defective coupling of tethering to membrane fusion observed here suggests that nucleotide-dependent self-assembly of Mitofusin is required after tethering to promote membrane fusion.

Keywords: Charcot-Marie-Tooth disease (CMT); GTPase; dynamin related protein (DRP); fusion protein; membrane fusion; mitochondria; mitochondrial network; organelle; structure-function; tethering.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism*
  • Membrane Fusion*
  • Mice
  • Mitochondria / genetics
  • Mitochondria / metabolism*
  • Mitochondrial Dynamics*
  • Mitochondrial Membranes / metabolism*
  • Protein Domains

Substances

  • GTP Phosphohydrolases
  • Mfn1 protein, mouse
  • Mfn2 protein, mouse

Associated data

  • PDB/5GOE