Mfn2 requires Hinge 1 integrity for efficient nucleotide-dependent assembly and membrane fusion

Abstract

Mitofusins are members of the dynamin-related protein family, large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains including two extended helical bundles that are connected by a flexible linker. The role of this linker (Hinge 1) in mitofusin-mediated membrane fusion is not well understood. We have characterized four variants with amino acid substitutions within this region of Mfn2. While a defect was not apparent in cells, a fusion deficiency was observed in vitro, and was rescued by the addition of cytosolic fraction. All four variants had decreased nucleotide-dependent assembly, which was improved by the addition of Bax. Assembly of mitofusins across two membranes was unaffected as formation of the trans complex was similar to wild type for all variants. We further demonstrate that variants with substitutions in both helical bundles are more severely impaired than any single mutant, suggesting that both helical bundles contribute to this function. Our data are consistent with a model where this region contributes to conformational changes that are important for assembly.