Two conserved cysteine triads in human Ero1alpha cooperate for efficient disulfide bond formation in the endoplasmic reticulum.

Human Ero1alpha is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation. To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations. Our results reveal that two essential cysteine triads (Cys85-Cys94-Cys99 and Cys391-Cys394-Cys397) cooperate in electron transfer, with Cys94 likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys394, Cys397, and to a lesser extent Cys99). Replacing the first cysteine in either triad (Cys85 or Cys391) generates mutants with weaker activity. In addition, mutating either Cys85 or Cys391, but not Cys397, reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys85 and Cys391, are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.