The endoplasmic reticulum (ER) plays a major role in regulating synthesis, folding, and orderly transport of proteins. It is also essentially involved in various cellular signaling processes, primarily by its function as a dynamic Ca(2+) store. Compared to the cytosol, oxidizing conditions are found in the ER that allow oxidation of cysteine residues in nascent polypeptide chains to form intramolecular disulfide bonds. However, compounds and enzymes such as PDI that catalyze disulfide bonds become reduced and have to be reoxidized for further catalytic cycles. A number of enzymes, among them products of the ERO1 gene, appear to provide oxidizing equivalents, and oxygen appears to be the final oxidant in aerobic living organisms. Thus, protein oxidation in the ER is connected with generation of reactive oxygen species (ROS). Changes in the redox state and the presence of ROS also affect the Ca(2+) homeostasis by modulating the functionality of ER-based channels and buffering chaperones. In addition, a close relationship exists between oxidative stress and ER stress, which both may activate signaling events leading to a rebalance of folding capacity and folding demand or to cell death. Thus, redox homeostasis appears to be a prerequisite for proper functioning of the ER.

Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core.

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b domain and show the existence of an intradomain linker between the b and a domains. We have expressed the recombinant b domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in fulllength PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.Native disulfide bond formation in the endoplasmic reticulum is a complex process that is rate-limiting in the biogenesis of many outer membrane and secreted proteins. Native disulfide bond formation can occur via multiple parallel pathways, and there is evidence that a large number of different gene families and redox carriers may play a role in the supply of redox equivalents for protein disulfide bond formation. What is clear is that the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is latestage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure. These steps are thought to be catalyzed only by proteins belonging to the protein disulfide isomerase (PDI) family. PDI 1 was the first catalyst of protein folding identified over 40 years ago (1), but despite probably being the most widely studied protein folding catalyst, significant details of the mechanisms of action of this critical enzyme are still unclear. In all eukaryotes, there exists a species-dependent PDI family of enzymes; for example, in humans (2), ERp72, ERp57, P5, PDIp, PDIr, ERp44 (3), ERp28/29 (4), ERdj5 (5), and ERp18 (6) have been reported to date. Functional characterization and differentiation between these family members is far from complete. PDI is a multifunctional, multidomain enzyme. The domain structure of PDI has been determined by theoretical (7) and experimental (8 -11) procedures and comprises two catalytic domains, a and a, separated by two homologous non-catalytic domains, b and b, plus a C-terminal region designated as c. In addition, it has bee...

Protein disulfide isomerases (PDIs) catalyse the formation of native disulfide bonds in protein folding pathways. The key steps involve disulfide formation and isomerization in compact folding intermediates. The high-resolution structures of the a and b domains of PDI are now known, and the overall domain architecture of PDI and its homologues can be inferred. The isolated a and a′ domains of PDI are good catalysts of simple thiol-disulfide interchange reactions but require additional domains to be effective as catalysts of the rate-limiting disulfide isomerizations in protein folding pathways. The b′ domain of PDI has a specific binding site for peptides and its binding properties differ in specificity between members of the PDI family. A model of PDI function can be deduced in which the domains function synergically: the b′ domain binds unstructured regions of polypeptide, while the a and a′ domains catalyse the chemical isomerization steps.

The tripeptide glutathione is the most abundant thiol/ disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo. The endoplasmic reticulum (ER)1 lumen of uni-and multicellular eukaryotes is the compartment in which nascent secretory proteins fold to attain their native structure. The luminal environment favors the maturation of secretory proteins by providing chaperones; for example, immunoglobulin heavy chain-binding protein, folding catalysts such as protein disulfide isomerase (PDI), and an oxidizing environment suitable for native disulfide bond formation. There are numerous studies on different aspects of the molecular chaperones and disulfide isomerases (for examples, see Ref. 1-10), but there have been only limited studies directly concerning the redox environment of the ER lumen. This is because of the difficulties of developing suitable methods for characterizing organelle microenvironments. One landmark study (11) using a probe thiol peptide confirmed that glutathione is the principal redox buffer in the ER lumen and reported that the ratio of reduced (GSH) to oxidized (GSSG) glutathione in the secretory pathway is between 1:1 and 3:1. This is considerably more oxidizing than the cytosolic ratio of 30:1-100:1 (11) and similar to the optimum for in vitro folding of disulfide bond-containing proteins; in most cases refolding in vitro is carried out with [GSH] at ϳ2 mM (range, 1-5 mM) and [GSSG] at ϳ0.5 mM (range 0.1-2 mM) after the initial optimization of oxidative refolding of lysozyme (12).The redox environment of the ER lumen is of great importance for the production of secretory proteins, and a role for glutathione in maintaining appropriate redox conditions in this compartment has been conjectured for many years (13-14). There is indirect evidence for the involvement of glutathione in protein disulfide bond formation in vivo (15), but some basic questions still require an answer. First, how are net oxidizing equivalents delivered to the ER lumen to maintain the observed oxidizing environment? The study of Hwang et al. (1...