Aims: Ero1 flavoproteins catalyze oxidative folding in the endoplasmic reticulum (ER), consuming oxygen and generating hydrogen peroxide (H 2 O 2 ). The ER-localized glutathione peroxidase 7 (GPx7) shows protein disulfide isomerase (PDI)-dependent peroxidase activity in vitro. Our work aims at identifying the physiological role of GPx7 in the Ero1a/PDI oxidative folding pathway and at dissecting the reaction mechanisms of GPx7. Results: Our data show that GPx7 can utilize Ero1a-produced H 2 O 2 to accelerate oxidative folding of substrates both in vitro and in vivo. H 2 O 2 oxidizes Cys57 of GPx7 to sulfenic acid, which can be resolved by Cys86 to form an intramolecular disulfide bond. Both the disulfide form and sulfenic acid form of GPx7 can oxidize PDI for catalyzing oxidative folding. GPx7 prefers to interact with the a domain of PDI, and intramolecular cooperation between the two redox-active sites of PDI increases the activity of the Ero1a/GPx7/PDI triad. Innovation: Our in vitro and in vivo evidence provides mechanistic insights into how cells consume potentially harmful H 2 O 2 while optimizing oxidative protein folding via the Ero1a/GPx7/PDI triad. Cys57 can promote PDI oxidation in two ways, and Cys86 emerges as a novel noncanonical resolving cysteine. Conclusion: GPx7 promotes oxidative protein folding, directly utilizing Ero1a-generated H 2 O 2 in the early secretory compartment. Thus, the Ero1a/ GPx7/PDI triad generates two disulfide bonds and two H 2 O molecules at the expense of a single O 2 molecule. Antioxid. Redox Signal. 20, 545-556.

ERp44 mediates thiol-dependent retention in the early secretory pathway, forming mixed disulphides with substrate proteins through its conserved CRFS motif. Here, we present its crystal structure at a resolution of 2.6 Å . Three thioredoxin domains-a, b and b 0 -are arranged in a clover-like structure. A flexible carboxy-terminal tail turns back to the b 0 and a domains, shielding a hydrophobic pocket in domain b 0 and a hydrophobic patch around the CRFS motif in domain a. Mutational and functional studies indicate that the C-terminal tail gates the CRFS area and the adjacent hydrophobic pocket, dynamically regulating protein quality control.

ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.