Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation–enhancing α-mannosidase–like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.

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Significance: Disulfide bond formation is an essential reaction involved in the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells utilize various disulfide oxidoreductases and redox-active cofactors to accelerate this oxidative reaction, and higher eukaryotes have diversified and refined these disulfide-introducing cascades over the course of evolution. Recent Advances: In the past decade, atomic resolution structures have been solved for an increasing number of disulfide oxidoreductases, thereby revealing the structural and mechanistic basis of cellular disulfide bond formation systems. Critical Issues: In this review, we focus on the evolution, structure, and regulatory mechanisms of endoplasmic reticulum oxidoreductin 1 (Ero1) family enzymes, the primary disulfide bond-generating catalysts in the endoplasmic reticulum (ER). Detailed comparison of Ero1 with other oxidoreductases, such as Prx4, QSOX, Erv1/2, and disulfide bond protein B (DsbB), provides important insight into how this ER-resident flavoenzyme acts in a regulated and specific manner to maintain redox and protein homeostasis in eukaryotic cells. Future Directions: Currently, it is presumed that multiple pathways in addition to that mediated by Ero1 cooperate to achieve oxidative folding of many secretory and membrane proteins in mammalian cells. The important open question is how each oxidative pathway works distinctly or redundantly in response to various cellular conditions. Antioxid. Redox Signal. 16, 790–799.

Oxidative protein folding in the endoplasmic reticulum is supported by efficient electron relays driven by enzymatic reactions centering on the ERO1-protein-disulfide isomerase (PDI) pathway. A controlled in vitro oxygen consumption assay was carried out to analyze the ERO1-PDI reaction. The results showed the pH-dependent oxidation of PDI by ERO1α. Among several possible disulfide bonds regulating ERO1α activity, Cys(94)-Cys(131) and Cys(99)-Cys(104) disulfide bonds are dominant regulators by excluding the involvement of the Cys(85)-Cys(391) disulfide in the regulation. The fine-tuned species specificity of the ERO1-PDI pathway was demonstrated by functional in vitro complementation assays using yeast and mammalian oxidoreductases. Finally, the results provide experimental evidence for the intramolecular electron transfer from the a domain to the a' domain within PDI during its oxidation by ERO1α.