Two conserved cysteine triads in human Ero1α cooperate for efficient disulfide bond formation in the endoplasmic reticulum

Human Ero1α 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 Cys ⁹⁹). Replacing the first cysteine in either triad (Cys⁸⁵ or Cys³⁹¹) generates mutants with weaker activity. In addition, mutating either Cys⁸⁵ or Cys³⁹¹, but not Cys ³⁹⁷, 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 Ero1α function, possibly stabilizing a platform for efficient PDI oxidation.