Structural evolution of the MTCH family of mitochondrial insertases

Abstract 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. Teaser Structure of human MTCH2 reveals conserved features necessary to maintain mitochondrial proteome integrity across eukaryotes. Competing Interest Statement R.M.V. is a consultant and equity holder of Gate Bioscience Footnotes ↵‡ Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, CT, USA ↵§ Amgen, Thousand Oaks, CA, USA Additional data shown in Fig. 3, Fig. 4, Fig. S1, Fig. S7, Fig. S10, Fig. S15, Fig. S20, Fig. S24, and Table S6; Introduction, results, and discussion sections revised; Methods section moved from supplement to main text.