No 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.

Mitochondrial structure can be maintained at steady state or modified in response to changes in cellular physiology. This is achieved by the coordinated regulation of dynamic properties including mitochondrial fusion, division, and transport. Disease states, including neurodegeneration, are associated with defects in these processes. In vertebrates, two mitofusin paralogues, Mfn1 and Mfn2, are required for efficient mitochondrial fusion. The mitofusins share a high degree of homology and have very similar domain architecture, including an amino terminal GTPase domain and two extended helical bundles that are connected by flexible regions. Mfn1 and Mfn2 are nonredundant and are both required for mitochondrial outer membrane fusion. However, the molecular features that make these proteins functionally distinct are poorly defined. By engineering chimeric proteins composed of Mfn1 and Mfn2, we discovered a region that contributes to isoform-specific function (mitofusin isoform-specific region [MISR]). MISR confers unique fusion activity and mitofusin-specific nucleotide-dependent assembly properties. We propose that MISR functions in higher-order oligomerization either directly, as an interaction interface, or indirectly through conformational changes.

Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A–associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.

Mitochondrial structure can be maintained at steady state or modified in response to changes in cellular physiology. This is achieved by the coordinated regulation of dynamic properties including mitochondrial fusion, division and transport. Disease states, including neurodegeneration, are associated with defects in these processes. In vertebrates, two Mitofusin paralogs, Mfn1 and Mfn2, are required for efficient mitochondrial fusion. The Mitofusins share a high degree of homology and have very similar domain architecture, including an amino terminal GTPase domain and two extended helical bundles that are connected by flexible regions. Mfn1 and Mfn2 are non-redundant and are both required for mitochondrial outer membrane fusion.However, the molecular features that make these proteins functionally distinct are poorly defined. By engineering chimeric proteins composed of Mfn1 and Mfn2, we discovered a region that contributes to isoform-specific function (Mitofusin Isoform Specific Region -MISR). MISR confers unique fusion activity and Mitofusin specific nucleotide-dependent assembly properties. We propose that MISR functions in higher order oligomerization either directly, as an interaction interface, or indirectly through conformational changes.