Correspondence to: Jakob R. Winther, Carlsberg Laboratory, Department of Yeast Genetics, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Tel:45 3327 5282 Fax:45 3327 4766 E-mail:jrw{at}crc.dk.
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Abstract |
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PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.
Key Words: protein disulfide isomerase, ER, protein folding, carboxypeptidase Y, Saccharomyces cerevisiae
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Introduction |
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An important aspect of folding of secretory proteins is the formation of native disulfide bonds, a process catalyzed by protein disulfide isomerase (PDI)1 (
In most organisms, a number of genes encoding PDI-like proteins are found. In Escherichia coli, at least four proteins with thioredoxin-like domains have convincingly been shown to be involved in various aspects of disulfide bond formation and shuffling in the periplasm (
In Saccharomyces cerevisiae, the complete genome sequence shows that the number of putative PDIs is most likely limited to five, of which the PDI1 gene is the only essential gene (40% to Pdi1p and contains two thioredoxin-like domains (Fig 1). Unlike all known PDIs, the active sites of Eug1p have a CXXS motif (
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Recently, a novel PDI-like protein named Eps1p was characterized. It contains a single thioredoxin-like domain and is localized to the ER membrane (Fig 1). It has been proposed to be involved in ER quality control, as deletion of the gene suppresses a dominant-negative pma1 mutant. PMA1 encodes a plasma membrane proton ATPase, which is essential for growth. The mutant in question prevents wild-type Pma1p from being transported to the plasma membrane. In a strain deleted for eps1, both mutant and wild-type Pma1p are transported to the plasma membrane, thereby allowing growth (
In previous genetic analyses of pdi1 complementation, be it heterologous or homologous, the potential contributions from the Pdi1p homologues have been disregarded. With the present work, we wish to determine the role of the homologues in thiol oxidation and, in particular, to determine how important these enzymes are when PDI activity is compromised. To do this, we have constructed yeast strains simultaneously deleted for two or more of these genes. The success of this approach is illustrated by the observation that a mutant form of Pdi1p, Pdi1pCGHS-CGHS, (this nomenclature is adopted for describing the sequence of the two active sites of the protein; the sequence of the more NH2-terminal active site is written first), which is defective in oxidation, is dependent on an endogenous homologue for its ability to complement the pdi1 deletion. We have also revealed differences in the functionality between the PDI-like proteins, as MPD1 is the only PDI1 homologue that, when overexpressed, is able to suppress a strain deleted for all genes of the PDI1 family.
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Materials and Methods |
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Media and Materials
Yeast cells were grown in standard YPD and SC media (
Strains and Plasmids
All plasmids used in this study are listed in Table 1. The yeast strains used in this study are listed in Table 2.
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Deletion of the PDI1 gene was described in 330 bp.
The plasmid pPN196 is derived from pFA6-kanMX3 (700 bp.
The vector pFA6-kanMX3 (1,480 bp. The mating type of the MPD2 deleted strain was changed from
to a by crossing to an isogenic strain. This strain was subsequently crossed with a
pdi1
eug1 [PDI1] strain (
pdi1
eug1 [PDI1] denotes a yeast strain deleted for PDI1 and EUG1 and complemented by PDI1 on a plasmid).
The EPS1 gene was amplified by PCR using the primers oBH61 (5' CGC GGA TCC GCG CAA GAA ATT CTA TCC AGG 3') and oBH62 (5' TCC CCG CGG GGA TTT TTA TGG TAG GCG TGC 3'), and the PCR product was cloned into the EcoRV site of pBCSK+ (Stratagene). The SalI-XhoI and EcoRV-EcoRV fragments of a plasmid containing the EPS1 ORF were inserted into the SalI and SmaI sites, respectively, of YDp-L (800 bp.
Of the plasmids listed in Table 1, pBH1464, pBH1711, pBH1857, pBH1966, and pCT58 have been described elsewhere (
MPD1 and MPD2 used to construct the plasmids for complementation were amplified by PCR from yeast chromosomal DNA. MPD1 was amplified using the oligonucleotides oBH57 (5' CGC GGA TCC GCG TCC ACT TAA CAC AAT TAG G 3') and oBH58 (5' TCC CCG CGG GGA CTT ATT CTT ATG CCC C 3') as primers, which introduced a BamHI site upstream of the ORF and a SacII site downstream of MPD1, respectively. After digestion with BamHI and SacII, the PCR-amplified fragment was inserted between the corresponding sites of pBH1692, resulting in plasmid pBH1800. The sequence of the cloned ORF was found to be identical to the published sequence, except for a silent C to T mutation at nucleotide position +168 relative to the ATG codon. MPD2 was amplified using the oligonucleotides oBH59 (5' CGC GGA TCC GCG GCG AGT CTA GTG CAA GTA CG 3') and oBH60 (5' TCC CCG CGG GGA CTT ATA TTG CGG CTA ACG 3'). The use of oBH59 and oBH60 also introduced a BamHI site upstream and a SacII site downstream, respectively, of the ORF. After digestion with BamHI and SacII, the PCR-amplified fragment was inserted between the corresponding sites of pBH1692, giving pBH1806. The sequence of the cloned ORF was found to be identical to the published sequence.
The genes encoding the Pdi1p homologues were introduced via a plasmid shuffle procedure (
pPN379 contains 1,500 base pairs of the MPD1 promoter fused to lacZ. A PCR product, made using the primers oPN25 (5' ACG CGC GGA TCC ACA GTC TTA GGG AAG TAA CC 3') and oPN26 (5' ACG ACG GGA TCC ATT ATA TGT CAA ATT TTG TCT CTC C 3'), was digested with BamHI and inserted into the corresponding site of pFN8 (
Pulse Labeling and Immunoprecipitation
The pulse-chase and immunoprecipitation experiments were carried out essentially as described by prc1 were made PRC1 by integration of the wild-type allele of PRC1 using the plasmid pIPRC. This plasmid is an EcoRI shrink of pTSY3 (
prc1::HIS3 using the plasmid described in
For Western blots, the cells (4 OD600 of cells per sample) were collected by centrifugation and resuspended in 5% (wt/vol) TCA. After centrifugation, the pellet was washed twice in 100% acetone and subsequently resuspended in 100 µl of lysis buffer (100 mM MOPS, pH 7.0, 2% SDS) containing 20 mM AMS to modify free thiols. Also, a protease inhibitor cocktail (Boehringer) was included, according to the supplier's instructions. Approximately 50 µl of acid-washed glass beads (212300 µm; Sigma-Aldrich) were added to each sample. The samples were then vortexed for 30 min in an Eppendorf Mixer 5432 at room temperature, and incubated for a further 30 min at room temperature to complete the sulfhydryl modification. CPY was subsequently immunoprecipitated as described by
Growth Phenotypes
Cultures grown overnight in SC + 5xLeu + 5xAde were diluted to a concentration of 5,000 cells/ml (5xLeu or 5xAde denotes five times of the normal SC concentration of the compound). 20 µl were applied on SC + 5xLeu + 5xAde plates. The plates were incubated for 2 d at 30°C before being photographed. The ability of yeast strains to grow in the presence of DTT was determined by spotting cells in the same way onto freshly prepared SC + 5xLeu + 5xAde plates buffered to pH 5 and containing 02.5 mM DTT. The plates were subsequently incubated in a CO2 atmosphere to minimize air oxidation of DTT. After 2 d at 30°C, the plates were photographed.
Measurement of ß-Galactosidase Activity
ß-galactosidase assays were performed using a protocol modified from 0.1 in a total volume of 25 ml in 100-ml flasks. The culture was grown at 30°C until an OD600
0.3. An appropriate amount of culture was harvested by centrifugation and the pellet was resuspended in 450 µl buffer Z (
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Results |
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MPD1 Is the Only PDI1 Homologue that, on Its Own, Is Able to Ensure Cell Viability
From combined in vivo and in silico analysis, five genes can be identified as being members of or obvious candidates for the PDI family: PDI1, EUG1, MPD1, MPD2, and EPS1 (
We were interested in studying why yeast and other eukaryotes are equipped with such an array of PDI-like proteins. More specifically, we wished to uncover putative synthetic phenotypes by systematic disruption of several PDI1 homologues in the same cell, and to investigate each homologue's effectiveness in suppression of a pdi1 deletion when expressed at roughly the same levels as Pdi1p. To do so, we constructed null mutations of PDI1 EUG1, MPD1, MPD2, and EPS1, alone and in combinations. The first important result of this experiment is that simultaneous deletion of all four Pdi1p homologues causes no obvious growth defect on synthetic complete medium under normal laboratory conditions. Thus, no synthetic phenotypes are displayed as long as PDI1 is present. We then placed the MPD1, MPD2, EUG1, and EPS1 open reading frames under the control of the PDI1 promoter on a low-copy TRP1-based plasmid. These plasmids were introduced into a pdi1 strain carrying PDI1 on a URA3-based plasmid. Growth on 5-FOA, which selects for loss of the PDI1-containing URA3-based plasmid, was used as a measure of the ability of a gene to suppress the PDI1 deletion. The result is that Eug1p, Mpd1p, and Mpd2p are all able to replace Pdi1p when produced to roughly the same level. However, their effectiveness at doing so varies greatly. A
pdi1 strain complemented by MPD1 under control of the PDI1 promoter (
pdi1 [MPD1]) showed a small reduction in growth rate compared with the wild type, while the
pdi1 [MPD2] and
pdi1 [EUG1] strains showed larger reductions in growth rates (Fig 2). We next wanted to see whether the ability of these plasmids to complement the pdi1 deletion was dependent on chromosomal copies of the other PDI1 homologues, or whether synthetic effects could be revealed. This was accomplished by shuffling the various plasmids containing the homologues into strains containing a pdi1 deletion in combination with deletions of the homologues. The pattern of viability in this array of strains did reveal functional differences between the MPD1, MPD2, and EUG1 gene products (Table 3). Deletion of EUG1 affected neither viability nor growth rate in any of the yeast strains investigated. Overexpression of EUG1, on the other hand, was able to complement the pdi1 deletion only if MPD1 and MPD2 were both present. Differences in the functionality of Mpd1p and Mpd2p were seen, as overexpression of MPD2 was no longer able to rescue a pdi1 deletion if MPD1 was also deleted, while overexpression of MPD1 rescued all strains. Not even expression of MPD2 from a high-copy plasmid rescued a pdi1 deletion in the absence of MPD1. The difference could, in principle, reflect very low steady state levels of Mpd2p compared with Mpd1p, caused by differences in protein stability. To test this, we constructed plasmids containing MPD1 and MPD2, in which the sequence encoding a COOH-terminal myc-tag followed by the ER retention signal HDEL were added. Both constructs were found to phenocopy the wild-type genes in their ability to complement a pdi1-deleted strain. A Western blot of extracts from these strains probed with myc-antibodies shows that Mpd2p-myc is present at roughly the same level as Mpd1p-myc when both tagged genes are expressed under the control of the PDI1-promoter (data not shown). This indicates that in a strain completely depleted of other Pdi1p-like proteins, Mpd1p is the only homologue able to provide all Pdi1p functions required for survival.
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EPS1 was not able to rescue a pdi1-deleted strain when expressed under control of the PDI1-promoter. It has previously been shown, however, that it can complement, when expressed from a 2 µm plasmid (
The importance of the oxidizing CXXC motif was shown with specific EUG1 and PDI1 mutants. Mutation of the active sites of Eug1p to CXXC converts Eug1p to a more Pdi1p-like enzyme as judged by its improved ability to rescue a pdi1 strain (
The protein Ero1p is required for ER oxidation of protein thiols (
The PDI1 Homologues Are Expressed at a Much Lower Level than PDI1
Since the PDI1 homologues are able to complement the pdi1 deletion only when expressed under control of the PDI1 promoter, their normal levels are likely to be substantially lower than that of the PDI1 gene. To estimate expression levels, we measured the ß-galactosidase activity from reporter constructs in which the promoter regions of all the genes in question were fused to lacZ. In an otherwise wild-type yeast, the ß-galactosidase activity obtained from the MPD1 and EUG1 reporters were 45% of the activity from the PDI1-lacZ reporter, while that of the MPD2-lacZ reporter was only 0.2% (Table 4). This indicates that the homologous proteins are present at a much lower concentration than Pdi1p.
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The question was posed as to whether the inability of MPD1 to complement a pdi1 deletion is due only to the normally low abundance of the gene product or whether it also is a consequence of reduced catalytic potency compared with Pdi1p. To test this, we placed PDI1 under control of the weak MPD1 promoter. Interestingly, such a plasmid was able to rescue a strain simultaneously deleted for pdi1, eug1, mpd1, mpd2, and eps1 (data not shown). This shows that it is not only the lower expression levels of the PDI1 homologues that make them incapable of fulfilling PDI function.
The Effects of Mutations in PDI1 Are Enhanced in Strains Lacking Pdi1p Homologues
DTT sensitivity is closely linked to the effectiveness of the oxidizing apparatus of the ER (
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The rate of intracellular folding of proCPY can be monitored, as only folded proteins are allowed to exit the ER (
We followed the maturation of proCPY to CPY in strains depleted for PDIs. Cells were labeled with [35S]methionine for 15 min and chased for 0, 15, and 60 min. After immunoprecipitation with CPY antibody, the samples were subjected to SDS-PAGE and analyzed using a PhosphorImager. As seen in Fig 4 A, the rate of proCPY folding was strongly dependent on the gene that was used to rescue the pdi1 deletion. The half-time of proCPY maturation in the PDI1-complemented strain is 510 min, whereas the half time increased to 30 min in strains rescued by overexpression of MPD1. When the pdi1 deletion is rescued by overexpression of EUG1, proCPY maturation is almost arrested, accompanied by accumulation of the p1 ER form of proCPY. This effect is even more pronounced in the strain rescued by overexpression of MPD2. Deletion of the genes encoding the various homologues had no, or very little, effect on the rate of proCPY maturation as long as the strains were complemented by PDI1 (Fig 4 B), indicating that the Pdi1p homologues do not normally make a significant contribution to proCPY folding. However, the homologues do recognize proCPY as a substrate in the absence of Pdi1p, because maturation of proCPY takes place, although at a reduced rate.
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As Eug1p is not on its own able to carry out oxidation reactions, oxidation should be less efficient in a pdi1 strain complemented by EUG1. We tested this assumption by looking at the redox state of proCPY under steady state. Extracts were made in the presence or absence of the thiol modifying agent AMS. AMS modifies free sulfhydryl groups only, and the modification causes a mobility shift on SDS-PAGE due to the bulkiness of the AMS group. The cell extracts were separated on an SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with CPY antibodies. As seen in Fig 5 A, proCPY in a wild-type strain is not modified by AMS, while treatment of the cells with DTT before the AMS modification causes a large mobility shift on the gel. This corresponds to modification of all eleven cysteines in the fully reduced proCPY. proCPY in the
pdi1 [EUG1] strain is partially AMS modifiable, suggesting that a significant portion of the proCPY is not completely oxidized at steady state. This indicates that oxidation is in fact compromised in the EUG1 complemented strain.
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Pdi1p Depletion Affects Protein Glycosylation
In the pdi1 [EUG1], the
pdi1 [MPD2], and the
pdi1
eug1
mpd1
mpd2
eps1 [MPD1] strains, the p1 and the p2 forms of proCPY are not fully separated (Fig 4A and Fig B). The p1 form in these strains appears to migrate slower than wild type. Also, the mature form of CPY shows a faster mobility than in the wild-type strain. That these differences in mobility originate from glycosylation defects is confirmed by their disappearance upon treatment of the samples with endoglycosidase H (data not shown). Since the p1 form migrates slower (most clearly seen in Fig 5 B), this defect must originate in the ER.
The compound calcofluor white interferes with cell wall assembly, and hypersensitivity towards this compound has therefore been used to indicate defects in cell-wall biogenesis. Several mutants showing hypersensitivity towards calcofluor white are affected in enzymes involved in the glycosylation apparatus (pdi1 [EUG1], the
pdi1 [MPD2], and the
pdi1
eug1
mpd1
mpd2
eps1 [MPD1] strains all were extremely sensitive towards calcofluor white (data not shown).
Pdi1p Homologues Play No Significant Role in ER-associated Degradation
PDI-like proteins have been implicated in quality control and ER-associated degradation of misfolded ER proteins (-factor and CPY with a single amino acid mutation, CPY* (
mpd1
mpd2
eug1
eps1 strain (Fig 6 A). Surprisingly, however, substitution of PDI1 with MPD1, MPD2, or EUG1 also had no significant effect on CPY* degradation (Fig 6 B). To rule out any possible inconsistencies as compared with the experimental procedures used by
pdi1 [PDI1SGHS-CGHC] strain used by
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Discussion |
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When cysteine residues enter the lumen of the ER as part of a translocated polypeptide chain, they encounter a change in the redox environment that ultimately promotes formation of intrachain and/or interchain disulfide bonds. In the generally accepted view, PDI is involved in two reactions during the oxidative folding of a protein: (a) the introduction of disulfide bonds in a polypeptide substrate by transfer of a disulfide bond between the two PDI active site cysteines (i.e., in the oxidized state) to the substrate, and (b) rearrangement of the incorrect disulfide bonds that most likely form during complex folding reactions.
The discovery of Ero1p as important for oxidative folding suggests a pathway for the flow of oxidizing equivalents from yet unknown donors to Ero1p, to the CXXC-motif of Pdi1p and further to the polypeptide substrate (pdi1 [EUG1] strain is partially reduced (Fig 5 A) clearly demonstrates the decreased oxidative capacity of this strain.
pdi1 ero1-1 strain is not rescued by any of the homologues, they must also be dependent on the Ero1p pathway. A weak allele of ERO1 would not affect the ability of the homologues to complement had they used a different pathway. This is consistent with the model that Ero1p not only transfers oxidizing equivalents to Pdi1p and Mpd2p but also to Mpd1p.
As pdi1 deletion is lethal, the question arises as to what essential cellular process or processes are compromised by Pdi1p depletion, or, in this case, substitution of Pdi1p with one of the homologues. We find that mature CPY in some of our strains migrates differently than CPY in a wild-type strain, indicating a changed outer chain glycosylation (Fig 4 and Fig 5 B). Defects in the ER folding apparatus could give rise to diminished steady state levels of the enzymes involved in glycosylation. Furthermore, these cells show hypersensitivity to calcofluor white, which is probably due to cell wall defects. Consistent with this, the morphology of the cells is changed as a consequence of Pdi1p depletion. It is most prominent in the pdi1 [EUG1] strain, where odd-shaped cells often are encountered.
There are several indications from both yeast and higher eukaryotes that PDI may be involved in quality control and degradation of misfolded secretory proteins. However, we find no effect of deleting the homologues in the presence of a wild-type copy of PDI1. Likewise, there is no reduction in the rate of CPY* degradation on substitution of Pdi1p with any of the homologues Mpd1p, Mpd2p, or Eug1p (Fig 6). It therefore appears that neither Pdi1p nor the homologues play a unique, irreplaceable role in degradation of CPY*. This is surprising in view of the previous observation that substitution of Pdi1p with a Pdi1pSGHS-CGHC mutant form results in significant stabilization of CPY* (
Mpd1p ranks as the most Pdi1p-like of the homologues, as it can act as the sole PDI in a pdi1
eug1
mpd1
mpd2
eps1 strain and, under these conditions, support an almost normal growth. However, the observation that expression of PDI1 from the weak MPD1 promoter is sufficient for survival, in conjunction with the multiple defects seen in the pdi1-deleted strains rescued by overexpressed homologues, demonstrates the superior catalytic potency of Pdi1p. In the absence of Pdi1p, both Mpd1p and/or Mpd2p appear to be important contributors to protein oxidation in the ER. However, MPD1 and MPD2 do not seem equally well suited to carry out protein oxidation, since MPD2 requires MPD1 to be present to restore viability of a pdi1-deleted strain. Also, a pdi1-deleted strain rescued by MPD2 is severely affected in proCPY folding. The redox potential of the PDI-like protein from E. coli, DsbA, is strongly dependent on the nature of the two amino acid residues between the active site cysteines (
pdi1 [MPD2CGHC], the mutation did not enable the mutant to complement a
pdi1
eug1
mpd1
mpd2
eps1 strain (data not shown).
In this paper, we have addressed the functionality of the Pdi1p homologues using an approach that focuses on the ability of the homologues to replace Pdi1p in various strain backgrounds. We have seen that there are clear differences in the ability of the Pdi1p homologues to ensure growth when Pdi1p function is compromised, suggesting different in vivo responsibilities. Some of the genes are targets of the unfolded protein response (
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Footnotes |
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Dr. Westphal's present address is The Burnham Institute, La Jolla, CA 92037.
Dr. Tachibana's present address is University of Washington, Department of Genetics, Seattle, WA 98195-7360.
Dr. Alsøe's present address is The Norwegian Radium Hospital, Department of Immunology, Montebello, 0310 Oslo, Norway.
Dr. Holst's present address is Danish Veterinary and Food Administration, Department of Food Safety and Toxicology, DK-2860 Søborg, Denmark.
1 Abbreviations used in this paper: 5-FOA, 5-fluoro-orotic acid; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; CPY, carboxypeptidase Y; ONPG, o-nitrophenyl-ß-D-galactoside; PDI, protein disulfide isomerase.
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Acknowledgements |
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We thank Morten C. Kielland-Brandt and Søren W. Rasmussen for critical reading of the manuscript. We are grateful to Chris Kaiser and Alison Frand for providing the ero1-1 strain, and Reiner Hitt and Dieter Wolf for providing the prc1-1 plasmid.
Submitted: 22 May 2000
Revised: 22 December 2000
Accepted: 25 December 2000
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References |
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