Inactivation of mammalian Ero 1α is catalysed by specific protein disulfide isomerases

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as protein disulfide isomerase and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide. Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactive the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen, nor hydrogen peroxide was able to oxidise Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family such as PDI or ERp46 were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory disulfides in Ero1α and that the PDI substrate binding domain is crucial to allow electron transfer between the two enzymes. These results demonstrate a simple feedback mechanism of regulation of mammalian Ero1α involving its primary substrate