Structural evolution of the MTCH family of mitochondrial insertases

Taylor A. Stevens; Zhilin Luo; Camryn Lee; Masami Hazu; Erini G. Galatis; Alison J. Inglis; Alina Guna; Rebecca M. Voorhees

doi:10.64898/2026.02.17.705849

QED claims analysis

Summary

The MTCH family of mitochondrial outer membrane insertases evolved from solute carrier 25 transporters through loss of a transmembrane helix that created a lipid-accessible hydrophilic groove, with insertase activity naturally attenuated by conserved hydrophobic residues whose positioning regulates function, representing one of three convergent evolutionary solutions for outer membrane protein insertion across eukaryotic kingdoms.

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Main Claim 1 –

MTCH2 Defines Holozoan Outer-Membrane Insertase Family Diverged from SLC25

Supporting evidence

Distant MTCH Homologs Rescue Insertase Function in Human Cells
Purified MTCH Insertase Activity Reconstitutes Outer-Membrane Protein Insertion In Vitro
Outer-Membrane SLC25A46 Lacks Insertase Activity, Unlike MTCH

Main Claim 2 –

Transmembrane Helix Loss Created MTCH Hydrophilic Groove Stabilized by C-Terminus

Supporting evidence

MTCH2 Has Five Transmembrane Helices with Defined Orientation
MTCH2 C-Terminal Domain Is Required for Protein Stability
MTCH2 Is Cytoplasm-Open Without Canonical Transporter Salt Bridges

Main Claim 3 –

Groove-Lining Hydrophobic Residues Attenuate MTCH2 Insertase Activity

Supporting evidence

Alanine Scans Show Many Groove Mutations Hyperactivate MTCH2
MTCH Insertase Attenuation Mechanism Is Conserved Across Species
Class I Mutations Hyperactivate MTCH2 by Widening Groove Geometry
Class II Mutations Reposition Inhibitory Hydrophobics via Conformational Change
Hyperactive MTCH2 Mutants Increase Intrinsic Insertion in Proteoliposomes
Known MTCH2 Partners Do Not Explain Insertase Activity Attenuation
ARMC1 Overexpression Partially Inhibits MTCH2 via C-Terminal Domain

Main Claim 4 –

Outer-Membrane Insertases Evolved Independently, Converging on Hydrophilic Grooves

Supporting evidence

Unrelated Insertases Share Five-TM Hydrophilic-Groove Architecture
Plant and Protist Insertases Functionally Replace Human MTCH2

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.

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