Mitochondrial fusion defects caused by CMT2A disease-associated variants of Mfn2

The peripheral neuropathy, Charcot Marie Tooth Syndrome Type 2A (CMT2A) is caused by amino acid substitutions in a component of the mitochondrial outer membrane fusion machine, mitofusin 2 (Mfn2). Mfn2 is predicted to exist in two conformational states, extended and closed; this would require that hinge regions mediate changes in the relative position of the two helical bundle domains (HB1 and HB2). Several disease-associated substitutions are located near the hinge that connects HB1 and HB2. We characterized these Mfn2 variants to assess the role of this domain in mitofusin-mediated fusion. While a fusion defect in cells was not evident, our cell-free mitochondrial fusion assay revealed a fusion deficit, which was compensated for by the addition of a cytosolic fraction. Consistent with this, all four variants had decreased nucleotide-dependent assembly. Given the partial loss of function associated with single substitution, we assessed fusion activity of variants with two substitutions in the hinge region. A variant with substitutions in both HB1 and HB2 was more severely impacted, with less fusion activity than a variant with two substitutions in HB1. These data support a model where conformational changes in Mfn2 require contribution from both helical bundle domains to support nucleotide-dependent assembly.