Topology control by a conserved cysteine pair in the OMM-protein CCDC127 enables MICOS interaction
Mitochondrial disulfide relay substrates beyond the canonical substrates remain incompletely defined. Revisiting the human MIA40 interactome with enhanced depth, we identified CCDC127 as a previously unrecognized substrate candidate. CCDC127 contains a single transmembrane segment and a conserved C-terminal helical bundle domain (CHB). Comprehensive proteomic and biochemical analyses revealed that, contrary to earlier reports, CCDC127 adopts an Nout-Cin topology in the outer mitochondrial membrane (OMM) with its CHB residing in the intermembrane space (IMS). CCDC127 undergoes oxidation by the disulfide relay, forming a long-range intramolecular disulfide bond between C174 and C219. Loss of these cysteines disrupts correct OMM insertion, inverts transmembrane topology and triggers proteasome-dependent degradation, establishing the disulfide as a key determinant of CCDC127 maturation. Interactome analyses identified MICOS components--particularly the MIC60/MIC19 module--as major partner proteins required for the stability of large oligomeric CCDC127 complexes. CCDC127 deficiency impaired cellular proliferation, influenced phospholipid levels, and caused grossly altered cristae morphology. Together, CCDC127 emerges as a MICOS-associated OMM protein essential for mitochondrial membrane organization and lipid homeostasis.