Identification of a new functional domain in the Mitofusin proteins
Sloat S.R., Engelhart E.A., Hoppins S.C.
University of Washington, Department of Biochemistry, Seattle, WA, USA
Mitochondrial membrane fusion is mechanistically unique and poorly understood. In vertebrates, mitochondrial outer membrane fusion requires Mitofusin1 and Mitofusin2 (Mfn1 and Mfn2), which function in place of a single outer membrane fusion protein in most other organisms. Despite their substantial similarity and shared role in mitochondrial fusion, Mfn1 and Mfn2 are functionally distinct. In fact, mitochondrial fusion is more efficient with Mfn1 and Mfn2 in a heterotypic complex, compared to either homotypic complex. Surprisingly, we have found that mutations in Mfn2 predicted to be catalytic dead are functional in a heterotypic complex with Mfn1, but not a homotypic complex. The same mutations in Mfn1 are non-functional in both heterotypic and homotypic complexes, indicating that only Mfn2 can function without enzymatic activity. To identify domains in Mfn1 and Mfn2 that are contributing to this important mechanistic distinction, we generated chimeric Mitofusin proteins and tested their function. We identified a small domain adjacent to the amino terminal GTPase domain, which we call GAD (GTPase adjacent domain), that contributes to Mitofusin-specific behavior. When we replace Mfn2-GAD with Mfn1-GAD, the chimeric protein (Mfn2-1GAD) rescues mitochondrial morphology in Mfn2 null cells. Interestingly, in contrast to Mfn2, rescue by Mfn2-1GAD is dependent on the catalytic activity of the GTPase domain. These results indicate that the GAD domain is altering the behavior of the Mfn2 GTPase domain to a more Mfn1-like state, where catalytic dead mutations are non-functional. Together, our data indicate that we have identified an important domain in the middle region of the Mitofusin proteins that confers Mitofusin-specific activity.