A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1 alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1 alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry-between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1 alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1 alpha-Cys131Ala or the isoform Ero1 beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.