Sequences flanking the transmembrane segments facilitate mitochondrial localization and membrane fusion by mitofusin

Significance Mitochondria constantly connect through membrane fusion. The merging of the outer mitochondrial membrane requires mitofusin (MFN) proteins. MFN is a membrane-anchored GTPase, but whether it is sufficient to achieve fusion, and if so how, is largely unknown. We have taken advantage of a similar GTPase named atlastin (ATL), which mediates fusion of the endoplasmic reticulum (ER), as its mechanism is better understood. Domain swapping experiments show that MFN is capable of fusing membranes, even on the ER. The C-terminal tail of MFN contains an amphipathic helix that promotes fusion. MFN is properly inserted into the mitochondrial membrane with the help of the helix and neighboring hydrophobic residues. These findings provide insight into how mitochondria fuse.
Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.