Structural evolution of the MTCH family of mitochondrial insertases

We demonstrate that MTCH2 is the defining member of a large family of mitochondrial outer membrane (OM) insertases. MTCH insertases are conserved across holozoa and have diverged from the solute carrier 25 transporters. The cryoelectron microscopy structure of the 33 kDa human MTCH2 revealed that evolution of its insertase activity required loss of a transmembrane helix, which created a lipid-accessible hydrophilic groove stabilized by its unique, structured C-terminus. Mutational analyses showed that MTCH insertase activity is attenuated, while experimental structures and reconstitution of hyperactive mutants demonstrated that the hydrophobicity, charge, and size of the residues that line its groove regulated MTCH function. Leveraging the MTCH2 structure, we identified the plant OM insertase, and proposed a universal mechanism for OM insertion across all kingdoms of life. TeaserStructure of human MTCH2 reveals conserved features necessary to maintain mitochondrial proteome integrity across eukaryotes.