Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin. Role of protein-disulphide isomerase

Native disulphide-bonded prolactin (band III) was distinguished from reduced prolactin (band II) and intermediate unstable disulphide-linked conformations by: (a) faster mobility of the former in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and (b) high-pressure liquid chromatography analyses of tryptic-digested peptides derived from prolactin in various conformations during its refolding pathway from reduced, unfolded to native conformation. The electrophoretic separation has been used to examine the state of disulphide bonding in newly synthesised prolactin translated from bovine pituitary mRNA in a rabbit reticulocyte translation system supplemented with nuclease-treated dog pancreatic microsomal membranes. The formation of correct disulphide pairing in prolactin (band III), synthesised in the in vitro translation system in the presence of pancreatic microsomes, required the presence of a thiol oxidant such as oxidised glutathione during the translation. The action of thiol oxidants on the in vitro biosynthesised and microsomally processed prolactin were both dose-dependent and catalytic; non-thiol oxidants such as NAD+ and NADP+ were ineffective. Examination of the time course of addition of oxidised glutathione to translating lysates showed that efficient and correct disulphide pairing in newly biosynthesised prolactin occurred when the oxidant was present co-translationally, but much lower yields of correctly disulphide-bonded prolactin were obtained when the oxidant was added after translation and processing were complete. The presence of protein-disulphide isomerase in dog pancreatic microsomes, employed in the in vitro translation system to process preprolactin, was demonstrated by (a) two-dimensional polyacrylamide gel electrophoresis of the membrane proteins, and (b) enzymic activity to accelerate reactivation of scrambled ribonuclease. Protein-disulphide isomerase activity was latent in intact microsomal vesicles, full activity being expressed upon sonication. A procedure has been devised to prepare pancreatic microsomal vesicles depleted of protein-disulphide isomerase which are active in processing and segregating in vitro biosynthesised prolactin. These membranes in the presence of low concentrations of oxidised glutathione are less active but in the presence of saturating levels of oxidised glutathione are fully competent in forming correct disulphide bridges in newly synthesised prolactin.     

The latency of rat liver microsomal protein disulphide-isomerase.

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.     

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 min) pulse, increased in concentration to peak at 60 min, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (t 1/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.     

Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides

Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.     

Characterization of the ribosomal binding site in rat liver rough microsomes: Ribophorins I and II,two integral membrane proteins related to ribosome binding

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: (i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; (ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detegent Kyro EOB; (iii) in intact rough microsomes ribophorins can be crosslinked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and “rough-inverted” vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents sugest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.     

Soluble constituents of the ER lumen are required for GPI anchoring of a model protein.

Transfer of a glycosylphosphatidylinositol (GPI) anchor to proteins carrying a C-terminal GPI-directing signal sequence occurs after protein translocation across the endoplasmic reticulum (ER). We describe the translocation and GPI modification of a model protein, preprominiPLAP, in ER microsomes depleted of lumenal content by high pH washing. In untreated microsomes preprominiPLAP was processed to prominiPLAP and GPI-anchored miniPLAP. Both products were fully translocated, since they resisted proteinase K treatment of the microsomes, and both behaved as membrane proteins by the carbonate extraction criterion. Microsomes depleted of lumenal content were able to translocate and process preprominiPLAP to give protease-protected prominiPLAP, but were unable to convert prominiPLAP to miniPLAP. Loss of GPI anchoring capacity occurred with a wash of pH > 9.5. If the alkaline wash was performed after formation of prominiPLAP conversion to miniPLAP was relatively unimpaired. The results indicate that constituents of the ER lumen, possibly chaperones interacting with the proprotein and/or the GPI anchor precursor, are required in the initial steps of GPI anchoring.     

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

ON THE SPATIAL ARRANGEMENT OF PROTEINS IN MICROSOMAL MEMBRANES FROM RAT LIVER

Rat liver rough microsomes were labeled enzymatically with ¹²⁵I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [³H]-NaBH₄ after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.     

Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila

Legionella pneumophila is an intracellular pathogen that replicates in a unique vacuole that avoids endocytic maturation. Previous studies have shown host vesicles attached to the L. pneumophila-containing vacuole (LCV) minutes after uptake. Here we examine the origin and content of these vesicles by electron microscopy (EM). Our data demonstrate that the attached vesicles are derived from endoplasmic reticulum (ER) based the presence of the resident ER proteins glucose-6-phosphatase, protein disulphide isomerase (PDI) and proteins having the ER-retention signal lysine-aspartic acid-glutamic acid-leucine (KDEL). After tethering occurred, ER markers inside of attached vesicles were delivered into the lumen of the LCV, indicating ER fusion. Treatment of cells with brefeldin A did not interfere with the attachment of ER vesicles with the LCV, suggesting that tethering of these vesicles does not require activities mediated by ADP-ribosylation factor (ARF). ER vesicles were not tethered to the LCV in cells producing the Sar1H79G protein, indicating that vesicles produced by the Sar1/CopII system are necessary for vesicle attachment. From these data we conclude that formation of the organelle that supports L. pneumophila replication is a two-stage process that involves remodelling of the LCV by early secretory vesicles produced by the Sar1/CopII system, followed by attachment and fusion of ER.     

Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum

Disulphide formation in the endoplasmic reticulum (ER) is catalysed by members of the protein disulphide isomerase (PDI) family. These enzymes can be oxidized by the flavoprotein ER oxidoreductin 1 (Ero1), which couples disulphide formation with reduction of oxygen to form hydrogen peroxide (H(2)O(2)). The H(2)O(2) produced can be metabolized by ER-localized peroxiredoxin IV (PrxIV). Continuous catalytic activity of PrxIV depends on reduction of a disulphide within the active site to form a free thiol, which can then react with H(2)O(2). Here, we demonstrate that several members of the PDI family are able to directly reduce this PrxIV disulphide and in the process become oxidized. Furthermore, we show that altering cellular expression of these proteins within the ER influences the efficiency with which PrxIV can be recycled. The oxidation of PDI family members by PrxIV is a highly efficient process and demonstrates how oxidation by H(2)O(2) can be coupled to disulphide formation. Oxidation of PDI by PrxIV may therefore increase efficiency of disulphide formation by Ero1 and also allows disulphide formation via alternative sources of H(2)O(2).     

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.     

Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes

Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.     

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY : I. Permeability Changes Induced by Low Detergent Concentrations

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.     

Disulphide production by Ero1���CPDI relay is rapid and effectively regulated

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady‐state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1‐deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed‐disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI‐family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1‐ and glutathione disulphide‐mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.     

ERp19 and ERp46, new members of the thioredoxin family of endoplasmic reticulum proteins

Using a proteomic analysis of the luminal environment of the endoplasmic reticulum (ER), we have identified 141 proteins, of which 6 were previously unknown. Two newly discovered ER luminal proteins, designated ERp19 and ERp46, are related to protein disulphide isomerase. Western and Northern blot analyses revealed that both ERp19 and ERp46 and their respective mRNAs are highly expressed in the liver as compared with other tissues. Both proteins were enriched in purified liver ER vesicles and were localized specifically to the ER in McA-RH7777 hepatocytes. Functional analysis with yeast complementation studies showed that ERp46 but not ERp19 can substitute for protein disulphide isomerase function in vivo.     

Crystal structures of human Ero1�� reveal the mechanisms of regulated and targeted oxidation of PDI

In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine‐tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b′‐domain through its substrate‐binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.     

Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.     

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.