Glutathione Is Required to Regulate the Formation of Native Disulfide Bonds within Proteins Entering the Secretory Pathway
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m406912200
ISSN1083-351X
AutoresSeema Chakravarthi, Neil J. Bulleid,
Tópico(s)Hemoglobin structure and function
ResumoThe formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum. The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum. The endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; DTT, dithiothreitol; BSO, buthionine sulfoximine; CHO, chinese hamster ovary; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; tPA, tissue type plasminogen; PDI, protein-disulfide isomerase; AMS, acetamidomaleimidylstilbene-disulfonic acid. provides an environment that allows the oxidative folding and post-translational modification of proteins entering the secretory pathway. The compartmentalization of the ER away from the cytosol allows the correct redox conditions to be established (1Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1608) Google Scholar), which in turn enables a distinct set of folding catalysts to facilitate the formation of native disulfide bonds. A growing family of ER oxidoreductases is thought to be responsible for catalyzing the formation, isomerization, and reduction of disulfide bonds (2Sevier C.S. Kaiser C.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 836-847Crossref PubMed Scopus (586) Google Scholar). These oxidoreductases contain active sites homologous to the active site found in the cytosolic reductase thioredoxin, characterized by a pair of cysteine residues (CXXC) that shuttle between the disulfide and dithiol form (3Ferrari D.M. Soling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). The reactions that these enzymes catalyze require the individual active sites to be maintained in either the oxidized disulfide form, for disulfide bond formation, or the reduced dithiol form, for isomerization or reduction of disulfide bonds (4Freedman R.B. Curr. Opin. Struct. Biol. 1995; 5: 85-91Crossref PubMed Scopus (85) Google Scholar). How the active sites are maintained in either their reduced or oxidized state and how the ER maintains an environment conducive to disulfide bond formation, isomerization, and reduction has been the subject of intense speculation over the past 40 years (5Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 6Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Recently, components of the oxidative pathway have been identified, however, we know very little about the reductive pathway even though it is clear that disulfide bond isomerization and reduction are essential for cell viability (7Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). It is now firmly established that the oxidoreductase PDI catalyzes the formation of disulfide bonds within the eukaryotic ER (8Holst B. Tachibana C. Winther J.R. J. Cell Biol. 1997; 138: 1229-1238Crossref PubMed Scopus (71) Google Scholar, 9Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). During disulfide bond formation an intrachain disulfide bond between the cysteine residues within the active site is able to accept two electrons from the polypeptide chain substrate resulting in the reduction of the PDI active site. Ero1p has been shown to be responsible for the oxidation of PDI in yeast, a defect in Ero1p leads to a lack of disulfide bond formation demonstrating the crucial role played by Ero1p in the oxidative folding pathway (5Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 6Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). There is growing evidence to suggest that Ero1p is an FAD-dependent oxidase that is able to pass electrons from PDI to the ultimate electron acceptor oxygen (10Tu B.P. Ho-Schleyer S.C. Travers K.J. Weissman J.S. Science. 2000; 290: 1571-1574Crossref PubMed Scopus (355) Google Scholar). However, Ero1p can also catalyze the oxidation of PDI under anaerobic conditions suggesting the possibility that alternative electron acceptors could substitute for oxygen under these conditions (11Sevier C.S. Cuozzo J.W. Vala A. Aslund F. Kaiser C.A. Nat. Cell Biol. 2001; 3: 874-882Crossref PubMed Scopus (155) Google Scholar). Although a clear mechanism exists to oxidize PDI, in mammalian cells most of the ER oxidoreductases, including PDI appear to be in a predominantly reduced form at steady state (12Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (216) Google Scholar). This would suggest that a pathway exists to maintain these proteins in a reduced state within the cell. That PDI is predominantly reduced in mammalian cells contrasts with the situation in yeast where PDI is clearly predominantly oxidized (9Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). The yeast ER maybe more oxidizing than the mammalian ER explaining this discrepancy; however, despite the differences it is clear that PDI in mammalian cells can be oxidized by Ero1 and catalyzes disulfide bond formation (12Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (216) Google Scholar). At least one other ER oxidoreductases, ERp57, does not appear to be a substrate for Ero1 as judged by a lack of oxidation in cells overexpressing Ero1 (12Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (216) Google Scholar). ERp57 has also been shown to act as a reductase, at least in vitro, to allow the breaking of non-native disulfide bonds within MHC-class I heavy chain (13Antoniou A.N. Ford S. Alphey M. Osborne A. Elliott T. Powis S.J. EMBO J. 2002; 21: 2655-2663Crossref PubMed Scopus (91) Google Scholar). Hence there is a requirement for both an oxidative pathway for disulfide bond formation and a reductive pathway to allow reduction and isomerization of non-native disulfides and for these pathways to co-exist in the same intracellular compartment. The mechanism for maintaining ER oxidoreductases in a reduced state could involve a protein-mediated process such as exists in the Escherichia coli periplasm where DsbD maintains the main enzyme catalyzing disulfide isomerization DsbC in a reduced state (14Stewart E.J. Katzen F. Beckwith J. EMBO J. 1999; 18: 5963-5971Crossref PubMed Scopus (127) Google Scholar). Alternatively a glutathione buffer may be involved, eliminating the requirement for a separate protein-mediated pathway for their reduction. Maintaining a pool of GSH within the ER could be brought about by continuous transport from the cytosol where glutathione reductase maintains a high concentration of GSH. Indeed some evidence exists to suggest the presence of a transport system that allows the selective passage of GSH rather than GSSG (15Banhegyi G. Lusini L. Puskas F. Rossi R. Fulceri R. Braun L. Mile V. di Simplicio P. Mandl J. Benedetti A. J. Biol. Chem. 1999; 274: 12213-12216Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Also, elimination of glutathione from yeast cells by removal of the enzyme involved in the first step of synthesis, γglutamylcysteine synthetase, did not prevent disulfide bond formation but did render the cells more prone to hyperoxidation, suggesting a direct or indirect role for glutathione in maintaining a redox balance in the yeast ER (16Cuozzo J.W. Kaiser C.A. Nat. Cell Biol. 1999; 1: 130-135Crossref PubMed Scopus (256) Google Scholar). Hence the possibility remains that reduction of ER oxidoreductases can be bought about either directly by glutathione or by a separate enzyme catalyzed pathway. To address this point we have chosen to study the folding and disulfide bond formation of human tissue type plasminogen activator (tPA). This protein contains 17 disulfide bonds and is secreted from recombinant cell lines as a mixture of two glycoforms that differ in their extent of core N-linked glycosylation. We have previously shown that conditions preventing disulfide bond formation, such as addition of the reducing agent DTT to culture medium of living cells, lead to complete glycosylation of a sequon that would otherwise undergo variable glycosylation in untreated cells (17Allen S. Naim H.Y. Bulleid N.J. J. Biol. Chem. 1995; 270: 4797-4804Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The extent of glycosylation of tPA is therefore intimately linked to the rate of protein folding and disulfide bond formation. The close temporal relationship between glycosylation and folding of tPA allows us to evaluate the affect of altering the redox conditions on both the rate of disulfide bond formation and also the rate of protein folding. Here we present evidence establishing that a rate-limiting step in the oxidative pathway and protein folding is the oxidation of PDI. We also show that a high concentration of intracellular glutathione is required to ensure the formation of native disulfide bonds. Cell Lines and Treatment—tPA-expressing CHO cells (ATCC CRL-9606) were cultured in HAM F-12 medium supplemented with 2 mml-glutamine and 10% fetal bovine serum (tissue culture media and supplements obtained from Invitrogen) plus appropriate antibiotics at 37 °C. In the buthionine sulfoximine (BSO, Sigma) experiments, 0.5 mm BSO was added to the media 6 h after seeding. Cells were cultured in the presence of BSO for 16 h. During the cell labeling experiments with cells cultured in the presence of BSO, 0.5 mm BSO was added to the starve, pulse, and chase media. Generation of Stable Transfected Cell Lines—pcDNA3.1-ERO1-Lαmyc vector, a kind gift from Prof. Roberto Sitia (San Raffaele University, Milano, Italy), was linearized with SmaI and transfected into tPA-expressing CHO cells using FuGENE-6 transfection reagent (Roche Diagnostics). Briefly, 2 μg of plasmid DNA was mixed together with various amounts of FuGENE-6, incubated at room temperature for 20 min, and added in a dropwise manner to 6 × 104 CHO cells maintained in 6-well dishes. 48 h after transfection, cells from each well were seeded into 150-mm dishes in the selection medium, which consisted of growth medium containing 0.4 mg/ml G418 (Sigma). The cells were grown in the selection medium for 2-3 weeks, and the neomycin-resistant clones were selected. The stably transfected cells were maintained in the selection growth medium and overexpression of Ero1-Lαmyc was examined by Western blot analysis. Immunofluorescence—Cells were grown on coverslips to 50-70% confluency, washed twice in PBS, and fixed in methanol for 10 min at -20 °C followed by a brief rehydration with PBS. Cells were stained with primary antibodies, mouse anti-c-Myc hybridoma supernatant, clone 9E10 (Sigma) and rabbit anti-calnexin polyclonal antibody (Stressgen Bioreagents), diluted in PBS, for 20 min. After three 5-min washes in PBS, cells were incubated with the secondary antibodies, Alexa Fluor 594 Donkey anti-mouse antibody and Alexa Fluor 448 Donkey anti-rabbit antibody (Molecular Probes Inc.), diluted in PBS for 20 min, followed by four 5-min washes in PBS. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). All the incubations were carried out at room temperature. The coverslips were mounted onto slides with Mowiol (Calbiochem) containing 25 mg/ml DABCO (Sigma) as an anti-fade agent. Slides were viewed on a Leica confocal microscope, and images captured using a charge-coupled camera. Western Blotting—The proteins separated by SDS-PAGE were transferred electrophoretically from the gels onto a nitrocellulose membrane. Blots were blocked in TTBS (100 mm Tris-HCl, pH 7.5, 0.9% w/v NaCl, 0.1% v/v Tween 80) containing 3% (w/v) milk followed by incubation for 1 h in the primary antibody, rabbit anti-Myc polyclonal antibody (Santa Cruz Biotechnology) or anti-PDI antibody (31John D.C. Grant M.E. Bulleid N.J. EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (95) Google Scholar), diluted to 1:500 in TTBS buffer. Blots were then washed three times for 10 min each in TTBS and incubated with 1:1000 dilution of goat anti-rabbit IgG-horseradish peroxidase (DAKO), in TTBS, for 1 h. After washing three times for 10 min each in TTBS, the membranes were treated with Super Signal West Pico chemiluminescent substrate (Pierce) for 5 min. All incubations were carried out at room temperature, and the proteins were visualized by exposure to Fuji Medical x-ray film. Radiolabeling and Immunoisolation—For pulse-chase experiments, 2 × 106 cells per 6-cm dish were washed twice with prewarmed methionine- and cysteine-free Dulbecco's modified Eagle's medium (Sigma), and then preincubated in the same medium for 20 min at 37 °C. Each monolayer was pulse-labeled with 50 μCi of 35S-EXPRESS (Amersham Biosciences) for 10 min at 37 °C and then chased with excess of methionine and cysteine. DTT was added in the pulse-chase medium, where indicated. 0.5 mm cycloheximide (Sigma) was included in the chase medium to block completion of labeled nascent chains. At the end of the labeling period or chase times, the cells were transferred to ice and washed twice with ice-cold PBS containing 20 mmN-ethylmaleimide (NEM, Sigma) to minimize disulfide bond rearrangements. The cells were lysed in 1 ml of lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 20 mm NEM) for 10 min on ice. Lysates were centrifuged at 12,000 × g for 20 min to remove insoluble material and incubated overnight with goat anti-human tPA polyclonal antibody (Cambio) or mouse anti-human tPA monoclonal antibody (American Diagnostica Inc.) and protein GSepharose (Zymed Laboratories Inc.). To monitor secretion of tPA, chase media from the radiolabeled cells were collected at the indicated times and immunoisolated with monoclonal or polyclonal tPA antibodies. Immune complexes were washed in IP buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride) and solubilized in 50 μl of sample buffer (0.25 mm Tris-HCl, pH 6.8, 2% w/v SDS, 20% v/v glycerol, 0.004% w/v bromphenol blue). DTT (50 mm) was added to reduced samples where indicated. All samples, reduced and non-reduced, were boiled for 5 min prior to electrophoresis. Samples were resolved by SDS-PAGE and visualized by either autoradiography using Kodak Biomax MR film or phosphorimager analysis using a Fuji Film Bas 1800 phosphorimager. Glutathione Assays—Total glutathione levels were measured using the DTNB (5, 5′-dithiobis(2-nitrobenzoic acid), Sigma)-GSSG reductase recycling assay (32Anderson M.E. Methods Enzymol. 1985; 113: 548-555Crossref PubMed Scopus (2430) Google Scholar). CHO cells (2 × 106) were harvested by trypsinization, and the cell pellet was lysed in 100 μl of ice-cold 8 mm HCl/1.3% 5-sulfosalicylic acid (SSA, Sigma) by agitating with glass beads. The lysate was incubated on ice for 30 min. Glass beads, precipitated protein, and cell debris were removed by centrifugation at 12,000 × g for 5 min. Total glutathione was measured by adding 10 μl of the lysate to 1-ml assay mixture prewarmed to 30 °C in a spectrophotometer cuvette. GSSG reductase was then added with mixing to initiate the assay and change in absorbance at 405 nm was measured over 4 min. Standard curves were generated using 0.5-4 nmol of GSH solubilized in 8 mm HCl/1.3% SSA. PDI Redox State—The in vivo redox state of PDI was assayed by modification with the thiol-reactive reagent acetamido-maleimidylstilbene-disulfonic acid AMS (Molecular Probes). Cells were incubated for 10 min at 37 °C with or without DTT (5 mm) or 4,4′dithiodipyridine (0.5 mm). At the end of the incubation period, the cells were transferred to ice and washed twice with ice-cold PBS containing 20 mm NEM to minimize disulfide bond rearrangements. The cells were lysed in 1 ml of lysis buffer (without NEM) for 10 min on ice. Cell lysates were centrifuged at 12,000 × g for 20 min to remove insoluble material. The supernantant was transferred into a fresh tube and 1% SDS and 10 mm TCEP was added to all the samples. Samples were boiled for 2 min, cooled, and treated with 25 mm AMS for 60 min at room temperature, in the dark. Overexpression of Ero1-Lα Leads to Enhanced Oxidative Protein Folding—Our initial studies focused on the consequence of overexpressing Ero1 on the ability of tPA to form disulfide bonds, fold correctly and be secreted. Our approach was to generate stable cell lines expressing both t-PA and wild-type Ero1 or Ero1 containing single cysteine point mutations known to compromise function (18Frand A.R. Kaiser C.A. Mol. Biol. Cell. 2000; 11: 2833-2843Crossref PubMed Scopus (93) Google Scholar, 19Benham A.M. Cabibbo A. Fassio A. Bulleid N. Sitia R. Braakman I. EMBO J. 2000; 19: 4493-4502Crossref PubMed Google Scholar). A CHO cell line expressing tPA (CHO-tPA) was transfected with Myc-tagged Ero1-Lα and stable cell lines selected. Isolated cell lines were screened for expression of Ero1 by Western blot analysis (Fig. 1A, lanes 1-4). To assess the oxidation state of the transfected Ero1-Lα, cells were treated with the membrane permeable alkylating agent NEM, to block free thiol groups and prevent rearrangement of disulfide bonds. Analysis of the cell lysate by electrophoresis carried out under non-reducing conditions (Fig. 1A, lanes 5-8), revealed that the Myc-tagged exogenously expressed Ero1-Lα migrated with a greater mobility than when reduced prior to electrophoresis (Fig. 1A, lanes 1 and 5). The increased mobility indicates the formation of intrachain disulfide bonds as previously described for the endogenous protein (19Benham A.M. Cabibbo A. Fassio A. Bulleid N. Sitia R. Braakman I. EMBO J. 2000; 19: 4493-4502Crossref PubMed Google Scholar). Some higher molecular weight bands can also be seen when the cell lysate was separated under non-reducing conditions. These higher molecular weight bands are likely to be mixed disulfides between exogenously expressed Ero1-Lα and endogenous PDI, as characterized previously (12Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (216) Google Scholar). No such high molecular weight bands were seen with either cysteine mutants demonstrating their inability to form functional mixed disulfides with PDI (Fig. 1A, lanes 6 and 7). These results demonstrate that the wild-type Ero1-Lα expressed in the CHO cells has folded correctly and is functional as indicated by its ability to form intrachain disulfides and mixed disulfides with other known ER oxidoreductases. The intracellular location of exogenously expressed Ero1-Lα protein in the stably transfected CHO-tPA cells was examined by indirect immunofluorescence. Cells were stained with a mixture of anti-Myc antibody and antibody to an ER resident protein, calnexin. Secondary antibodies were conjugated to Alexa Fluor 594 (red) or Alexa Fluor 488 (green) dyes, to enable double labeling and to visualize Ero1-Lα and calnexin in the same cells. A characteristic ER reticular appearance for calnexin was visualized in cells. Overlay images of Myc with calnexin immunofluorescence showed co-localization of the protein with calnexin indicating that transfected Ero1-Lα is localized in the ER (Fig. 1B). In order to investigate the effect of overexpression of Ero1-Lα on the extent of glycosylation of tPA, untransfected (UT), and Ero1-Lα transfected (Ero1) stable CHO-tPA cells were radiolabeled for 10 min in the absence or presence of varying concentrations of DTT in the labeling medium. The cells were treated with NEM, the cells lysed and the tPA immunoisolated with a goat polyclonal antibody specific for tPA. The immunoisolated proteins were subjected to both reducing and non-reducing SDS-PAGE (Fig. 2, A and B). In the untransfected CHO-tPA cells, the tPA synthesized during the 10-min labeling period in the absence of DTT migrated as a doublet when reduced prior to electrophoresis (Fig. 2A, upper panel UT, lane 1) indicating that variable glycosylation had occurred. When subjected to non-reducing SDS-PAGE (Fig. 2B, upper panel UT, lane 1) tPA migrated with a greater mobility and with a more diffuse pattern than on reducing gels, indicating that during the 10 min labeling period in the absence of DTT, the tPA had formed intrachain disulfide bonds. As the concentration of DTT in the labeling medium was increased from 0 to 5 mm, the doublet progressively disappeared until only a single band of the over-glycosylated tPA was present at concentrations of DTT above 0.75 mm (Fig. 2A, lanes 2-10). This change in migration was accompanied by a progressive reduction in the formation of disulfide bonds as indicated by the slower migrating sharper bands under non-reducing conditions (Fig. 2B, lanes 2-10). Thus, at 1 mm DTT no disulfide bonds were formed as no faster migrating protein was seen when the sample was run under non-reducing conditions. In addition the reduction of proteins within the cell with higher concentrations of DTT also caused a slight decrease in mobility of the protein when run under reducing conditions (Fig. 2A, lanes 8-10). This is due to full alkylation by NEM of the 34 cysteine residues in tPA when the protein is reduced and is a further indication of the lack of disulfide bond formation at these concentrations of DTT. In the cell line overexpressing Ero1-Lα the tPA synthesized was more resistant to the effects of reduction with DTT. This resistance is apparent both by the presence of variably glycosylated tPA at DTT concentrations as high as 1 mm and a lack of full reduction of disulfide bonds at 2 mm DTT (Fig. 2B, middle panel). Hence overexpression of Ero1 leads to formation of disulfide bonds at higher DTT concentrations indicating that Ero1 can act as an oxidase and can function in the presence of reducing agents. Additionally the ability to form disulfide bonds corresponds to an ability to also fold into a conformation that restricts glycosylation at higher concentrations of DTT, again highlighting the intimate link between disulfide bond formation and protein folding. The ability of cysteine mutants of Ero1 to bring about a similar effect was also investigated. Mammalian Ero1 contains 14 cysteine residues of which three (Cys-391, -394, and -397) have been shown, by single point mutation, to be involved in either stabilization of structure or enzymatic function (12Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (216) Google Scholar, 19Benham A.M. Cabibbo A. Fassio A. Bulleid N. Sitia R. Braakman I. EMBO J. 2000; 19: 4493-4502Crossref PubMed Google Scholar). An analysis of single cysteine mutations has also been carried out with yeast Ero1 and four cysteine residues (Cys-100, Cys-105, Cys-352, and Cys-355) were found to be absolutely required for function (18Frand A.R. Kaiser C.A. Mol. Biol. Cell. 2000; 11: 2833-2843Crossref PubMed Scopus (93) Google Scholar). These correspond to Cys-99, Cys-104, Cys-394, and Cys-397 in the human protein. We created two Ero1 cysteine mutants with mutation in cys-99 or both cys-99 and cys-394 and prepared separate stable cell lines expressing these proteins (Fig. 1A). Both the cell line expressing Ero1-cys99 (Fig. 2, A and B, lower panel), and the cell line expressing Ero1-cys99/394 (data not shown) decreased the ability of the cells to resist the effect of DTT on disulfide bond formation. At 0.1 mm DTT there was no disulfide bond formation and the synthesized tPA was fully glycosylated (Fig. 2, A and B, lane 2). This result demonstrates that the effect on disulfide bond formation by reducing agent was a direct consequence of overexpression of Ero1 and not due to other pleiotropic effects such as the induction of another protein involved in disulfide bond formation. Ero1-Lα Accelerates Oxidative Folding of Correctly Folded tPA in the Mammalian ER—The previous experiments highlighted the role of Ero1 in facilitating disulfide bond formation in the presence of reducing agents. However these results do not address whether the overexpression of Ero1 actually accelerates the oxidative folding pathway. To address this point we studied the post-translational folding of tPA in cells following labeling in the presence of a concentration of DTT known to prevent disulfide bond formation. All previous studies using mild concentrations of DTT to inhibit co-translational disulfide bond formation in newly synthesized secretory and membrane proteins have shown that the effects of DTT are reversible, and that the proteins undergo post-translational disulfide bond formation when DTT was removed from the culture medium of the cells (20Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (332) Google Scholar, 21Valetti C. Sitia R. Mol. Biol. Cell. 1994; 5: 1311-1324Crossref PubMed Scopus (41) Google Scholar, 22Tatu U. Braakman I. Helenius A. EMBO J. 1993; 12: 2151-2157Crossref PubMed Scopus (113) Google Scholar, 23Tatu U. Hammond C. Helenius A. EMBO J. 1995; 14: 1340-1348Crossref PubMed Scopus (89) Google Scholar). These results demonstrated that oxidizing conditions within the ER necessary for disulfide bond formation could be rapidly restored when DTT was removed from the cells. To determine the time required to re-establish oxidizing conditions within the ER and to form the native complement of disulfide bonds in reduced tPA, CHO cells were pulsed in the presence of DTT and then chased with an excess of non-radiolabeled cysteine and methionine in the absence of DTT. Cycloheximide was added to the chase medium to inhibit protein synthesis. Cells were chased for up to 90 min, treated with NEM, lysed, and tPA immunoisolated using the goat anti-human tPA polyclonal antibody. Time courses for intracellular disulfide bond formation in the untransfected (UT) and Ero1-Lα transfected (Ero1) CHO-tPA cells were constructed by separating the immunoisolated proteins by SDS-PAGE under reducing and non-reducing conditions (Fig. 3). When untransfected CHO-tPA cells were labeled for 10 min in the presence of 5 mm DTT, non-reducing SDS-PAGE (Fig. 3B, UT) revealed a sharp band at 0 min into the chase, indicating that no disulfide bond formation had occurred. After 5 min of the chase the banding pattern became more diffuse and protein migrated with greater mobility indicating partial disulfide bond formation. By 10 min of the chase there was a further increase in mobility, which was also observed when the samples were reduced prior to electrophoresis (Fig. 3A, UT). This was due to differential alkylation of partially oxidized tPA. No further increase in mobility was observed after 45 min of chase indicating that in the untransfected CHO-tPA cells disulfide bond formation was complete between 30 and 45 min into the chase. In the Ero1-Lα-overexpressing cells, tPA migrated as a diffuse band of disulfide bonded protein within 5 min of the chase (Fig. 3B, Ero1) and there was no further increase in mobility after 20 min of chase, indicating that in these cells disulfide bond formation was complete between 10 and 20 min into the chase. Thus overexpression of Ero1-Lα accelerates the recovery from DTT treatment, allowing more rapid oxidative folding within mammalian cells. The acceleration of the oxidative pathway would suggest that the rate-limiting step in the pathway is the oxidation of PDI by Ero1 as any increase in Ero1 causes acceleration in the rate of oxidation of the substrate. An increase in the intensity of tPA immunoisolated during the chase period after labeling in the presence of DTT was observed at early ti
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