Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax

Abstract

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 dynamics in health and disease

“…By elevating fission (DRP1 overexpression) in R364W mutant flies, mitochondrial morphology, distribution and locomotor defects were reversed. Similarly, mutations proximal to the hinge (1) region between HB1/2 domains were fusion-competent [100]. Surprisingly, defects were noted when fusion competency assay was conducted on isolated mitochondria with mutations within the hinge 1 region.…”

Mitochondrial Surveillance by Cdc48/p97: MAD vs. Membrane Fusion

“…Interestingly, these regions map to mutations associated with CMT2A [204], suggesting pathogenic relevance. Their importance as a driving force for membrane merging could be confirmed with subsequent functional analysis, allowing further dissection of the fusion mechanism [75,76,194,195,[205][206][207][208][209][210][211][212]. Consistent with the self-assembly properties of DRPs, mitofusins can be found in multiple oligomerization states [184,207] (Figure 4C).…”

“…Upon GTP hydrolysis, several conformational changes occur, which are critical to complete fusion. Bending at the hinge 1 constricts mitofusins, likely assisted by cytosolic factors [212]. This constriction is facilitated by dynamic changes at a trilateral salt bridge, present at hinge 2a [75,195,198].…”

“…Interestingly, these regions map to mutations associated with CMT2A [ 204 ], suggesting pathogenic relevance. Their importance as a driving force for membrane merging could be confirmed with subsequent functional analysis, allowing further dissection of the fusion mechanism [ 75 , 76 , 194 , 195 , 205 , 206 , 207 , 208 , 209 , 210 , 211 , 212 ].…”

“…Several possible assembly models have been proposed [ 207 , 208 , 209 ], likely involving interaction via the G–G interface and intermolecular hydrophobic interactions between the HR2 domains [ 195 , 217 , 218 ]. Although the exact triggers facilitating trans oligomerization are unknown, studies in mammals suggest that cytosolic signaling regulates Mfn2 oligomerization, e.g., via the oxidative state and the apoptosis regulator BAX [ 197 , 212 , 219 , 220 ]. The C-terminal HR domain was also suggested to assist trans oligomerization [ 217 , 218 , 221 ], a model under debate.…”