Knock-in reconstitution studies reveal an unexpected role of Cys65 in regulating APE1/Ref-1 subcellular trafficking and function.

APE1 protects cells from oxidative stress via BER pathway and as a redox transcriptional co-activator. It is required for tumor progression/metastasis and its upregulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect between these activities, we performed reconstitution experiments by using wild type and various APE1 mutants. Our results newly suggest that the redox function is responsible for cell proliferation through the involvement of Cys(65) in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with E3330, a specific inhibitor of APE1 redox function through increased Cys(65) oxidation, confirm that Cys(65) controls APE1 subcellular trafficking and provide the basis for a new role of this residue.