Mutations in the FAD binding domain cause stress-induced misoxidation of the endoplasmic reticulum oxidoreductase Ero1b

Disulfide bond catalysis is an essential component of protein biogenesis in the secretory pathway, from yeast through to man. In the endoplasmic reticulum (ER), protein-disulfide isomerase (PDI) catalyzes the oxidation and isomerization of disulfide bonds and is re-oxidized by an endoplasmic reticulum oxidoreductase (ERO). The elucidation of ERO function was greatly aided by the genetic analysis of two ero mutants, whose impairment results from point mutations in the FAD binding domain of the Ero protein. The ero1-1 and ero1-2 yeast strains have conditional and dithiothreitol-sensitive phenotypes, but the effects of the mutations on the behavior of Ero proteins has not been reported. Here, we show that these Gly to Ser and His to Tyr mutations do not prevent the dimerization of Ero1 or the non-covalent interaction of Ero1 with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1 heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1 susceptible to misoxidation and aggregation, particularly during a temperature or redox stress. We conclude that the Ero FAD binding domain is critical for conformational stability, allowing Ero proteins to withstand stress conditions that cause client proteins to misfold

Expression, interactions and dynamics of the oxidoreductase Ero1Lbeta

Protein oxidation in the Endoplasmic Reticulum (ER) is catalysed by Endoplasmic reticulum oxidoreductases (EROs) that donate disulfide bonds to (and accept electrons from) Protein Disulfide Isomerase (PDI). Eros are essential for viability and protein secretion in yeast. Objective: we have studied the mammalian Ero1p homolog, Ero1beta to in order to understand the expression, interaction partners and ultimately function of EROs in multicellular organisms. Methods: We have used immunohistochemistry to examine the localization of Ero1beta and other ER chaperones in mouse and human tissues. Western blotting, pulse chase analysis and co-immunoprecipitation was used in combination with non-reducing SDS-PAGE to determine the oxidation status, half life and disulfide-dependent complex formation of Ero1beta and its partners. Conclusion: We find that Ero1beta is highly expressed in certain secretory tissues such as stomach and pancreas. Ero1beta is not expressed in most cell lines in the absence of an unfolded protein response. Like Ero1alpha, Ero1beta interacts with PDI and can also form disulfide dependent dimers that depend on the CXXCXXC active site. These dimers may have a role in regulating disulfide bond formation

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Erolb

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1 and Ero1. Ero1 is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1 can form homodimers and mixed heterodimers with Ero1, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1 protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1 occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1 expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1α, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1α in cells arises from the formation of a disulphide bond—identified by mass spectrometry—between the active-site Cys94 (connected to Cys99 in the active enzyme) and Cys131. Competition between substrate thiols and Cys131 creates a feedback loop where activation of Ero1α is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1α-Cys131Ala or the isoform Ero1β, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis