Two conserved cysteine triads in human Ero1α 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 (Cys(85)-Cys(94)-Cys(99) and Cys(391)-Cys(394)-Cys(397)) cooperate in electron transfer, with Cys(94) likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys(394), Cys(397), and to a lesser extent Cys(99)). Replacing the first cysteine in either triad (Cys(85) or Cys(391)) generates mutants with weaker activity. In addition, mutating either Cys(85) or Cys(391), but not Cys(397), reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys(85) and Cys(391), are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.