Cyclophilin D is a new non-canonical substrate of the mitochondrial intermembrane space assembly pathway.

Mitochondrial protein import is essential for organelle function and cellular homeostasis. While Cyclophilin D (CypD) is a well-characterized regulator of the mitochondrial permeability transition pore (MPTP) and resides in the matrix, the mechanisms underlying its import remain poorly defined. In this study, we identify CypD as a novel non-canonical substrate of the mitochondrial intermembrane space assembly (MIA) pathway mediated by the oxidoreductase Mia40. Structural analysis revealed conserved cysteine pairs in CypD that are compatible with disulfide bond formation. Using in vitro pull-down assays, we demonstrate a redox-sensitive interaction between CypD and Mia40, which was further confirmed by co-immunoprecipitation and proximity ligation assays. Expression of CypD cysteine mutants in cells revealed that residues Cys82 and Cys203 are critical for Mia40-dependent interaction and protein stability. Notably, expression of the Cys203Ala mutant significantly reduced cell viability, suggesting a key functional role for this residue. Functional experiments showed that depletion of Mia40 leads to a significant reduction in mitochondrial CypD levels, a result that was confirmed in a series of leukemia cell lines with variable Mia40 expression. Our results shed light on a previously unrecognized import mechanism for CypD and expand the known substrate repertoire of Mia40, demonstrating that the MIA pathway also contributes to the import of mitochondrial matrix proteins. This work highlights the functional versatility of the MIA pathway beyond the intermembrane space and reveals an additional regulatory level in mitochondrial proteostasis with implications for cell death signaling and mitochondrial pathophysiology.