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

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.