Title: The Contributions of Protein Disulfide Isomerase and Its Homologues to Oxidative Protein Folding in the Yeast Endoplasmic Reticulum
Abstract: In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 ± 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization. In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 ± 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization. Disulfide bonds provide added stability to extracellular proteins by covalently cross-linking two cysteines. Disulfide formation is often error-prone, particularly in the early stages of folding (1Gilbert H.F. J. Biol. Chem. 1997; 272: 29399-29402Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), and pairing the correct cysteines into disulfides requires that any mispaired disulfides must be broken and reformed in a different configuration to reach the native structure. In bacteria, disulfides are formed in the periplasm by an elaborate system of oxidases and isomerases that assure the correct cysteines are connected (2Collet J.F. Bardwell J.C. Mol. Microbiol. 2002; 44: 1-8Crossref PubMed Scopus (176) Google Scholar, 3Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (446) Google Scholar). In eukaryotes this post-translational modification occurs in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CPY, carboxypeptidase Y; Mal-PEG, maleimide-conjugated polyethylene glycol polymer; PDI, protein disulfide isomerase; DTT, dithiothreitol.1The abbreviations used are: ER, endoplasmic reticulum; CPY, carboxypeptidase Y; Mal-PEG, maleimide-conjugated polyethylene glycol polymer; PDI, protein disulfide isomerase; DTT, dithiothreitol. where a complex set of enzyme catalysts promotes correct disulfide formation. In yeast (4Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 5Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) and mammalian cells (6Cabibbo A. Pagani M. Fabbri M. Rocchi M. Farmery M.R. Bulleid N.J. Sitia R. J. Biol. Chem. 2000; 275: 4827-4833Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) the oxidizing equivalents for disulfide formation are generated principally by Ero1p. These disulfides in turn are delivered to protein disulfide isomerase (Pdi1p), an essential folding catalyst of the endoplasmic reticulum (7Farquhar R. Honey N. Murant S.J. Bossier P. Schultz L. Montgomery D. Ellis R.W. Freedman R.B. Tuite M.F. Gene (Amst.). 1991; 108: 81-89Crossref PubMed Scopus (107) Google Scholar). Both yeast and mammalian protein disulfide isomerase (PDI) are composed of four domains (termed a, b, b′, and a′) and an anionic tail (c) (8Edman J.C. Ellis L. Blacher R.W. Roth R.A. Rutter W.J. Nature. 1985; 317: 267-270Crossref PubMed Scopus (475) Google Scholar). The two catalytic domains (a and a′) are located at the ends of the molecule, and each contains an active site with the sequence CGHC. The catalytic thioredoxin domains are separated by two non-catalytic thioredoxin domains (b and b′) (9Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) in a multidomain structure (abb′a′c). When the active-site cysteines of PDI are in a disulfide (oxidized) form, the enzyme can introduce disulfides into proteins (oxidase activity) through thiol/disulfide exchange. However, when the active-site cysteines of PDI are present in a dithiol (reduced) form, the active site is able to catalyze the reduction or isomerization of substrate disulfides (10Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 619-625Crossref PubMed Scopus (118) Google Scholar, 11Walker K.W. Lyles M.M. Gilbert H.F. Biochemistry. 1996; 35: 1972-1980Crossref PubMed Scopus (142) Google Scholar). Although PDI exhibits both its oxidase and isomerase activities in vitro, and is by far the most active disulfide isomerase known, some uncertainty exists about its function in vivo. The PDI1 gene is essential in yeast (7Farquhar R. Honey N. Murant S.J. Bossier P. Schultz L. Montgomery D. Ellis R.W. Freedman R.B. Tuite M.F. Gene (Amst.). 1991; 108: 81-89Crossref PubMed Scopus (107) Google Scholar). A Pdi1p mutant with no active-site cysteines and no redox-related activity will not complement the lethal deletion of the PDI1 gene (12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Clearly, a catalytic redox function of Pdi1p, either its oxidase or isomerase activity, is essential for yeast growth and viability. Surprisingly, mutant forms of Pdi1p that are significantly defective in either the oxidase or isomerase activities will rescue the deletion mutant of PDI1. Mutating a single cysteine in both active sites (CGHS:CGHS) impairs the oxidase activity of PDI, but these mutants retain significant isomerase activity in vitro (11Walker K.W. Lyles M.M. Gilbert H.F. Biochemistry. 1996; 35: 1972-1980Crossref PubMed Scopus (142) Google Scholar, 12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). These oxidase-deficient mutants (12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 13Holst B. Tachibana C. Winther J.R. J. Cell Biol. 1997; 138: 1229-1238Crossref PubMed Scopus (71) Google Scholar) restore viability to cells lacking Pdi1p, leading to the suggestion that the oxidase activity is not its essential function (12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). However, other evidence implies that the primary function of PDI is as an oxidase. Mutants in the CXXC active site of Pdi1p showed increased sensitivity to DTT that correlated to a decreased rate of folding of a secretory protein (13Holst B. Tachibana C. Winther J.R. J. Cell Biol. 1997; 138: 1229-1238Crossref PubMed Scopus (71) Google Scholar). Pdi1p also accepts oxidizing equivalents from the ER oxidase, Erolp, and transfers them to substrate proteins (14Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Consistent with this view, Frand and Kaiser (14Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar), using a gel-shift assay that modifies each free cysteine of Pdi1p with a 0.5-kDa mass label, showed that most of the cysteines of yeast PDI appear to be oxidized in the yeast ER. In addition, a single catalytic domain of PDI (a or a′), which is grossly deficient in isomerase activity (5% of wild type) but has 50% of the oxidase activity of PDI in vitro, will replace yeast PDI and maintain normal growth and viability (15Xiao R. Solovyov A. Gilbert H.F. Holmgren A. Lundstrom-Ljung J. J. Biol. Chem. 2001; 276: 27975-27980Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Titration of the expression levels of the isomerase-deficient PDIa′ domain shows that yeast requires no more than 6% of PDI isomerase activity but needs 60% or more of its oxidase activity (16Solovyov A. Xiao R. Gilbert H.F. J. Biol. Chem. 2004; 279: 34095-34100Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The imbalanced need for PDI fundamental activities raises several important questions about how disulfide formation and isomerization occur in the ER and whether or not PDI, the most active disulfide isomerase in vitro, even displays this activity in vivo. Redundancies in ER disulfide-forming pathways or compensating changes in the expression level or the oxidation state of PDI might confound the apparent requirements for PDI catalytic activities. There are four PDI1 homologues in the yeast ER (MPD1, MPD2, EUG1, and EPS1) (17Cherry J.M. Ball C. Weng S. Juvik G. Schmidt R. Adler C. Dunn B. Dwight S. Riles L. Mortimer R.K. Botstein D. Nature. 1997; 387: 67-73Crossref PubMed Google Scholar). None is essential, and they are all normally expressed at low levels (18Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar). However, all of them will rescue the Δpdi1 mutation when overexpressed (19Tachibana C. Stevens T.H. Mol. Cell. Biol. 1992; 12: 4601-4611Crossref PubMed Scopus (130) Google Scholar, 20Tachikawa H. Takeuchi Y. Funahashi W. Miura T. Gao X.D. Fujimoto D. Mizunaga T. Onodera K. FEBS Lett. 1995; 369: 212-216Crossref PubMed Scopus (51) Google Scholar, 21Tachikawa H. Funahashi W. Takeuchi Y. Nakanishi H. Nishihara R. Katoh S. Gao X.D. Mizunaga T. Fujimoto D. Biochem. Biophys. Res. Commun. 1997; 239: 710-714Crossref PubMed Scopus (36) Google Scholar, 22Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). One of these homologues, Mpd2p, becomes essential when an oxidase-deficient PDI mutant (CGHS: CGHS) replaces wild-type PDI (18Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar). The availability of an isomerase defective form of yeast Pdi1p (the a′ domain) that provides the essential function has allowed us to determine how PDI and its ER homologues contribute to disulfide isomerization in the yeast ER. In the experiments described below we use oxidase and isomerase-defective variants of PDI to show that limiting the oxidase activity limits cell growth even in the presence of compensatory mechanisms. Despite the importance of its oxidase activity, assays based on the maturation of yeast carboxypeptidase Y (CPY) suggest that PDI does display its isomerase activity in vivo. Using a more sensitive gel-shift assay to detect reduced PDI active sites, we also find that a significant fraction of the PDI active sites are in the reduced state, capable of catalyzing disulfide isomerization. ER homologues of PDI also provide oxidase and isomerase activity to the yeast ER, but surprisingly, yeast strains that have all the homologues of Pdi1p deleted still show wild-type growth rates even when isomerase-deficient Pdi1p provides the only source of PDI function. High levels of disulfide isomerization are not essential to the survival and growth of yeast, suggesting an evolutionary process in yeast that may select against essential proteins that require disulfide isomerization. Strains and Plasmids—Saccharomyces cerevisiae strains with complete deletions of PDI1 were obtained from Ron Raines (University of Wisconsin, Madison, WI) (12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and from Norgaard et al. (18Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar). Strains with null mutations of PDI1, EUG1, MPD1, MPD2, and EPS1 alone and in combination are described in Norgaard et al. (18Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar). A wild-type strain (CRY1) was obtained from Steve Elledge (Baylor College of Medicine). The yeast expression vector YPP414-YPS is derived from plasmid pRS414 (23Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), which is a centromere-based, yeast-bacterial shuttle vector with a TRP1 yeast selectable marker. The pRS414 was digested with KpnI and EcoRI, and an 885-bp fragment encoding PDI1 promoter was inserted. This was followed by digestion with EcoRI and NotI and insertion of a 129-bp expression cassette fragment. This expression cassette contains BglII/BamHI sites that are in-frame with an N-terminal yeast PDI signal sequence to direct the expressed protein to the ER and a C-terminal yeast ER-retention sequence (HDEL). The coding sequences of rat PDI, the a and a′ domains of yeast PDI (yeast PDIa, yeast PDIa′) were PCR-amplified to contain a 5′-BamHI site and a 3′-BglII site. Yeast PDI was PCR-amplified to contain a BamHI site at both the 5′ and 3′ terminus. The yeast PDIa′ was amplified using the oligonucleotides 5′-GGG TCC CAG ATC TGA TTC CTC TGT CTT CCA ATT GGT C and 3′-AGA TTT AGG ATC CGA CGT CGA AGT GAC C GT TTT CCT TG as primers. Yeast PDIa was amplified using the oligonucleotides 5′-AAA TTT AGA TCT CCT GAA GAC TCC GCT G and 3′-GCG GAG CGG ATC CTT GCT TGA TCA TGA ATT GGA CAA TG as primers. After digestion with BglII and BamHI (rat PDI and yeast PDIa′) or BamHI (yeast PDI), the PCR-amplified fragments were inserted into the corresponding site of YPP414-YPS. The yeast a domain includes amino acids from Pro-Glu-Asp-Ser to Met-IIe-Lys-Gln (amino acids 30–139) of the yeast PDI sequence, and the yeast a′ domain includes amino acids from Glu-Asn-Gln-Asp to Phe-Asp-Val-Asp (amino acids 263–377). Growth Phenotypes—Overnight cultures grown in synthetic complete media with 2% glucose (SC medium) were diluted to ∼10,000 cells/ml, and aliquots of 30 μl were placed onto freshly made SC plates. The plates were incubated for 2 days at 30 °C before being photographed. The ability of yeast strains to grow in the presence of different concentrations of DTT was assayed by applying cells in the same way on freshly prepared SC plates containing 0–3 mm DTT. The plates were photographed after incubation at 30 °C for 2 days. Determination of the Oxidation State of PDI—Maleimide-conjugated polyethylene glycol (Mal-PEG) was obtained from Nektar Therapeutics (San Carlos, CA) and was purified by gel filtration on a PD-10 column (Amersham Biosciences) to remove low molecular weight maleimides. These lower molecular weight maleimides react with free sulfhydryl groups but do not shift the molecular weight, leading to incomplete shifts of the molecular weight on SDS-PAGE in fully reduced controls. Yeast cells complemented by yeast PDI, yeast PDIa′, or rat PDI were grown to mid-log phase in yeast extract/peptone/dextrose. Trichloroacetic acid was added to a final concentration of 20% to intact cells that had not been pelleted by centrifugation to avoid potential changes in the redox state. After centrifugation, the insoluble material was resuspended in ice-cold 40% trichloroacetic acid, and cells were lysed by vortexing with glass beads (Sigma). Lysates were removed from the glass beads and centrifuged, and the precipitated protein was washed with ice-cold acetone. The protein was resuspended in 5 mm gel-filtered Mal-PEG in non-reducing SDS sample buffer (3% SDS, 0.2 m Tris-HCl, pH 8, glycerol, bromphenol blue) and incubated for 30 min at room temperature then quenched by the addition of DTT to a final concentration of 50 mm. Reduced and oxidized controls were prepared by resuspending the proteins in either 10 mm DTT or 1 mm 5,5′-dithiobis(2-nitrobenzoic acid) in non-reducing 2× SDS sample buffer, incubating for 30 min at room temperature, acid-precipitating the protein with trichloroacetic acid (20% final concentration), resuspending in Mal-PEG, and then quenching by the addition of DTT to a final concentration of 50 mm. Samples were resolved by SDS-PAGE on precast 4–20% Tris-HCl gels (Bio-Rad) and transferred to nitrocellulose. Yeast PDI and yeast PDIa′ were visualized by probing with a polyclonal anti-yeast PDI antibody (from Jakob Winther) followed by a secondary antibody conjugated to horseradish peroxidase. Rat PDI was visualized by probing with a polyclonal anti-rat PDI antibody followed by a secondary antibody conjugated to horseradish peroxidase. Enhanced chemiluminescence (Amersham Biosciences) was used to detect the PDI species. Band intensities were determined using Scion Image software with film exposures in the linear range of intensities. The number of cysteines present as sulfhydryl groups was determined by multiplying the intensity of each band times the number of modified sulfhydryl groups represented by the position of the band. These intensities were summed and divided by the total intensity to estimate the average number of available sulfhydryl groups in the original protein. Protein Expression and Purification from Escherichia coli—Yeast PDI and yeast PDIa′ were PCR-amplified to contain a 5′-NdeI site and a 3′-XhoI site. After digestion with NdeI and XhoI, the PCR products were inserted between the corresponding sites of pET23a (Novagen, Madison, WI). E. coli strain BL21 (DE3) (Invitrogen) transformed with the appropriate pET23a vector was grown at 37 °C in LB media supplemented with 100 μg/ml ampicillin to an absorbance of 1.0 at 600 nm and induced by adding isopropyl β-d-thiogalactoside to a final concentration of 1 mm. After 4 h the cells were harvested by centrifugation, suspended in ice-cold loading buffer (pH 7.5, 20 mm phosphate, 0.5 mm NaCl, and 100 mm imidazole), and disrupted by sonication. After centrifugation at 12,000 × g for 15 min at 4 °C, the supernatant was applied to a HiTrap chelating column (Amersham Biosciences) pre-charged with nickel and equilibrated with loading buffer. The column was washed with 10 ml of loading buffer and then with 5 ml of elution buffer containing 300 mm imidazole. Eluate fractions containing PDI were pooled and dialyzed against 50 mm Tris-HCl, pH 8.0, and 1 mm EDTA to remove the imidazole. The purity of proteins was analyzed by SDS/PAGE and estimated to be >90%. Ribonuclease Refolding Assays—Renaturation of reduced RNase was followed in a continuous assay as described previously (24Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 613-619Crossref PubMed Scopus (351) Google Scholar). The formation of active RNase was measured spectrophotometrically by monitoring hydrolysis of the RNase substrate, cCMP, at 296 nm. Each sample contained 4.5 mm cCMP, 1 mm GSH, 0.2 mm GSSG, 100 mm Tris-HCl, pH 8.0, 1 mm EDTA, 8 μm reduced RNase, and 0–9 μm yeast PDI or yeast PDIa′. The assay was performed at 25 °C and initiated by the addition of reduced RNase. Carboxypeptidase Y Radiolabeling and Immunoprecipitations— Strains were grown in SC medium lacking methionine and cysteine, pelleted, and resuspended at 1 × 108 cells/ml in the same medium. Cells were labeled with 40 μCi of [35S]methionine and [35S]cysteine per A600 unit for 15 min. An excess of methionine and cysteine was introduced, and samples of 5 × 107 cells were collected at 0, 10, 20, and 30 min in 10 mm NaN3 and 10 mm NaF and lysed by resuspension in 100 μl of 20% trichloroacetic acid and agitation with glass beads. After centrifugation, the insoluble material was washed with ice-cold acetone four times, and the pellets were resuspended in IP buffer (50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1% Triton, 0.2% SDS, and 150 mm NaCl), boiled for 5 min, and incubated with Pansorbin (50 μl) (Calbiochem) for 20 min. After centrifugation, dilution buffer (60 mm Tris-HCl, pH 7.4, 1.25% Triton, 190 mm NaCl, 6 mm EDTA) was added to the supernatant before incubation with 6 μl of anti-CPY antibody (Jakob Winther) overnight at 4 °C. Immune complexes were collected with protein A-Sepharose (Amersham Biosciences), washed with IP buffer, 1 ml of urea buffer (50 mm Tris-HCl, pH 7.4, 0.2% SDS, 2 m urea, 250 mm NaCl, 5 mm EDTA, 1% Triton), 1 ml of low salt buffer (50 mm Tris-HCl, pH 7.4, 0.2% SDS, 5 mm EDTA, 1% Triton, 50 mm NaCl), and 1 ml of detergent-free buffer (50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 150 mm NaCl) and solubilized in 60 μl of sample loading buffer (80 mm Tris-HCl, pH 6.8, 10% 2-Mercaptoethanol, 2% SDS, 10% glycerol, 0.1% bromphenol blue). Samples were resolved by SDS-PAGE and transferred to nitrocellulose. The immunoprecipitated protein was exposed to a phosphor screen from Amersham Biosciences for 24–36 h. Screens were scanned using StormScan scanners to detect radiolabeled CPY bands. Complementation of the Δpdi1 Deletion with a Single Catalytic Domain of PDI—Isomerase-deficient mutants of PDI support growth when expressed from the inducible-repressible GAL1–10 promoter (16Solovyov A. Xiao R. Gilbert H.F. J. Biol. Chem. 2004; 279: 34095-34100Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To compare the abilities of individual yeast Pdi1p catalytic domains to support growth when expressed from the endogenous PDI1 promoter on a low copy (cen) plasmid, genes encoding the a (yeast PDIa) and a′ (yeast PDIa′) domains of yeast PDI, full-length yeast PDI and rat PDI were individually introduced into Δpdi1 S. cerevisiae using a plasmid shuffling method (12Laboissiere M.C. Sturley S.L. Raines R.T. J. Biol. Chem. 1995; 270: 28006-28009Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Expression of yeast PDIa′ complemented the Δpdi1 deletion with only a slightly reduced growth rate compared with the wild-type yeast PDI (Fig. 1). Rat PDI, despite the fact that its oxidase and isomerase activities are similar to those of yeast PDI in vitro (25Zhan X. Schwaller M. Gilbert H.F. Georgiou G. Biotechnol. Prog. 1999; 15: 1033-1038Crossref PubMed Scopus (25) Google Scholar), supported significantly slower growth (Fig. 1). The yeast PDIa domain and the individual catalytic domains of rat PDI were not able to support growth when expressed from the PDI1 promoter; however, they all rescued the lethal phenotype when overexpressed from the PDI1 promoter on a multicopy plasmid (2 μm) (data not shown). Activities of yeast PDIa′ in Vitro—The individual catalytic domains (a and a′) of mammalian PDI are active oxidases but have little isomerase activity (15Xiao R. Solovyov A. Gilbert H.F. Holmgren A. Lundstrom-Ljung J. J. Biol. Chem. 2001; 276: 27975-27980Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 26Darby N.J. Penka E. Vincentelli R. J. Mol. Biol. 1998; 276: 239-247Crossref PubMed Scopus (152) Google Scholar). When overexpressed, these mammalian catalytic domains will rescue the lethal deletion of PDI1. Although the yeast protein is predicted to have a domain structure that is similar to the mammalian protein, the catalytic domains of the yeast protein have not been characterized. Full-length yeast PDI and yeast PDIa′ were expressed in E. coli and purified through a C-terminal His6 tag. Domain boundaries were predicted by homology modeling of the yeast PDIa′ domain using 3D-PSSM (27Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar). As with the mammalian catalytic domains, the oxidase activity of yeast PDIa′ with reduced ribonuclease A as substrate is 50% that of an equivalent molar concentration of wild-type yeast PDI (based on moles of protein rather than active sites), but the isomerase activity is quite low, only 5% of wild type (Fig. 2). The Role of ER Homologues of PDI—All known disulfide isomerases are members of the thioredoxin family, and MPD1, MPD2, EUG1, and EPS1 are the only four thioredoxin family homologous of PDI1 thought to be expressed in the ER of yeast (17Cherry J.M. Ball C. Weng S. Juvik G. Schmidt R. Adler C. Dunn B. Dwight S. Riles L. Mortimer R.K. Botstein D. Nature. 1997; 387: 67-73Crossref PubMed Google Scholar). None of these homologues is essential, but they might provide isomerase and/or oxidase activity that becomes essential when it is not provided by yeast PDI1. If so, the ability of yeast PDIa′ or rat PDI to support viability may depend on chromosomal copies of other ER homologues. To test this we shuffled the various plasmids containing yeast PDIa′, yeast PDI, and rat PDI into strains carrying a pdi1 null mutation in combination with deletions of the homologues. Surprisingly, yeast PDIa′ behaves exactly like full-length yeast Pdi1p, supporting near wild-type growth in all strains, including the strain in which all homologues are absent (Table I). Rat PDI, however, is able to rescue the Δpdi1 deletion only if the homologue, MPD2, is present. All strains rescued by rat PDI display slow growth rates, a result similar to that reported previously with an oxidase-deficient yeast PDI mutant in which both active sites were mutated to CGHS (18Norgaard P. Westphal V. Tachibana C. Alsoe L. Holst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (106) Google Scholar)Table IRescue of PDI deficiencies by expression of various PDIsIntroduced geneYeast strainΔpdi1Δpdi1Δeug1Δpdi1Δmpd1Δpdi1Δmpd2Δpdi1Δeug1Δmpd1Δpdi1Δeug1Δmpd2Δpdi1Δmpd1Δmpd2Δpdi1Δeug1Δmpd1Δmpd2Δeps1Yeast PDI++++++++++++++++++++++++++++++++Yeast PDIa′++++++++++++++++++++++++Rat PDI+++-+--- Open table in a new tab Redox state of PDI in Vivo—The in vivo redox states of yeast PDI, yeast PDIa′ and rat PDI were examined using a gel-shift assay based on mobility shifts caused by the reaction of a free sulfhydryl group with a Mal-PEG (28Wu H.H. Thomas J.A. Momand J. Biochem. J. 2000; 351: 87-93Crossref PubMed Scopus (63) Google Scholar). To preserve the oxidation state of these proteins, trichloroacetic acid was directly added to intact cells to quench any thiol-disulfide exchange followed by disruption and treatment with Mal-PEG in the presence of SDS. Mal-PEG alkylation of a single sulfhydryl results in an apparent molecular mass shift of ∼15 kDa as observed by SDS-PAGE (Fig. 3). The Mal-PEG mass-shift on SDS-PAGE indicates the number of cysteines that are present as sulfhydryl groups. Those that are in disulfide oxidation states will not shift with Mal-PEG treatment. Because yeast PDI has six total sulfhydryl groups, the unshifted band (Fig. 3) represents PDI molecules in which all the cysteines are in disulfides. When Pdi1p is trichloroacetic acid-extracted from a Δpdi1 deletion strain complemented with yeast PDI and treated with Mal-PEG, bands corresponding to zero, and two Mal-PEG additions predominate along with a small amount of PDI shifted by the addition of four Mal-PEG, suggesting that wild-type PDI is partially reduced in vivo (Fig. 3). A similar result is observed in a wild-type yeast strain (CRY1) with only a chromosomal copy of wild-type yeast PDI1 (Fig. 3). In the CRY1 strain, there are 1.7 ± 0.4 (n = 5) free sulfhydryl groups. The predominant bands represent fully oxidized Pdi1p and a species shifted by two Mal-PEG modifications. The band representing four sulfhydryl modifications is somewhat more prominent in the CRY1 strain and represents 16 ± 6% (n = 5) of the total intensity compared with 8 ± 6% (n = 11) in the strain complemented with yeast Pdi1p expressed from a plasmid under control of the PDI1 promoter. However, the difference is not statistically significant (p > 0.05). Quantitation of the band intensities can be used to determine how many of the sulfhydryl groups are in each oxidation state. Because a population of active sites may be partially reduced/oxidized, the number of sulfhydryl groups that are available represents an average across all the sites. Approximately 1.3 ± 0.3 (n = 11) sulfhydryl groups (of six total) are found in yeast Pdi1p present in the yeast ER. However, rat PDI is much more reduc