Glutathione is a ubiquitous molecule found in all parts of the cell where it fulfils a range of functions from detoxification to protection from oxidative damage. It provides the main redox buffer for cells and as such has been implicated in the formation of native disulphide bonds. However, the discovery of the enzyme Ero1 has called into question the exact role of glutathione in this process. In this review, we discuss the arguments for and against a role for glutathione in facilitating disulphide-bond formation and consider its role in protecting the cell from endoplasmic-reticulum-generated oxidative stress.

At least 17 members of the protein disulphide isomerase (PDI) family of oxidoreductases are present in the endoplasmic reticulum (ER) of mammalian cells. They are thought to catalyse disulphide formation to aid folding or to regulate protein function; however, little is known about their individual functions. Here, we show that some proteins that enter the ER are clients for single oxidoreductases, whereas others are clients for several PDI-like enzymes. We previously identified potential substrates for ERp57, and here identify substrates for ERp18 and ERp46. In addition, we analysed the specificity of substrates towards PDI, ERp72, ERp57, ERp46, ERp18 and P5. Strikingly, ERp18 shows specificity towards a component of the complement cascade, pentraxin-related protein PTX3, whereas ERp46 has specificity towards peroxiredoxin-4, a thioredoxin peroxidase. By contrast, most PDI family members react with Ero1. Moreover, P5 forms a non-covalent complex with immunoglobulin heavy chain binding protein (BiP) and shows specificity towards BiP client proteins. These findings highlight cooperation between BiP and P5, and demonstrate that individual PDI family members recognise specific substrate proteins. Supplementary material available online at

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.

The discovery that the flavoprotein oxidase, Erv2p, provides oxidizing potential for disulfide bond formation in yeast, has led to investigations into the roles of the mammalian homologues of this protein. Mammalian homologues of Erv2p include QSOX (sulfhydryl oxidases) from human lung fibroblasts, guinea-pig endometrial cells and rat seminal vesicles. In the present study we show that, when expressed in mammalian cells, the longer version of human QSOX1 protein (hQSOX1a) is a transmembrane protein localized primarily to the Golgi apparatus. We also present the first evidence showing that hQSOX1a can act in vivo as an oxidase. Overexpression of hQSOX1a suppresses the lethality of a complete deletion of ERO1 (endoplasmic reticulum oxidase 1) in yeast and restores disulfide bond formation, as assayed by the folding of the secretory protein carboxypeptidase Y.