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Functional differences between human and yeast protein disulfide isomerase family proteins
BBRC Biochemical and Biophysical Research Communications 320 (2004) 359–365 www.elsevier.com/locate/ybbrc
Functional differences between human and yeast protein disulfide isomerase family proteinsq Taiji Kimura, Yasuhiro Hosoda, Yukiko Kitamura, Hideshi Nakamura,1 Tomohisa Horibe, and Masakazu Kikuchi* Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan Received 18 May 2004 Available online 15 June 2004
Abstract Previously, it has been reported that a mammalian protein disulfide isomerase (PDI), when expressed on a single copy number plasmid, can rescue growth of a PDI1-disrupted yeast. However, here, for the first time we demonstrated by tetrad analysis that human PDI (hPDI) is unable to replace yeast PDI (yPDI) when hPDI cDNA is integrated into the yeast chromosome. This observation indicates that hPDI is not functionally equivalent to yPDI. Estimation of the actual copy number of the plasmid, as well as comparison of isomerase and chaperone activities between human and yeast PDI homologues, indicates that one copy of hPDI cDNA is not sufficient to rescue the PDI1-disrupted strain. Notably, the isomerase activities of yPDI family proteins, Mpd1p, Mpd2p, and Eug1p, were extremely low, although yPDI itself exhibited twice as much isomerase activity as hPDI in vitro. Moreover, with the exception of Mpd1p, all hPDI and yPDI family proteins had chaperone activity, this being particularly strong in the case of yPDI and Mpd2p. These observations indicate that the growth of Saccharomyces cerevisiae is completely dependent on the isomerase activity of yPDI. Ó 2004 Published by Elsevier Inc. Keywords: Protein disulfide isomerase; Tetrad analysis; Isomerase activity; Chaperone activity; Yeast
Protein disulfide isomerase (PDI), which is one of the major enzymes assisting protein folding, catalyzes the formation, reduction, and isomerization of protein disulfide bonds in the endoplasmic reticulum (ER) [1]. PDI contains two thioredoxin-like motifs (CXXC) in two separate domains (a and a0 ) and an ER retention
q
Abbreviations: hPDI, human protein disulfide isomerase; hP5, human P5; hPDIR, human protein disulfide isomerase-related protein; yPDI, yeast PDI; ER, endoplasmic reticulum; IPTG, isopropyl-b-D thiogalactopyranoside; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. * Corresponding author. Present address: Department of Bioscience and Bioinformatics, College of Information Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan. Fax: +81-77-561-2659. E-mail address: [email protected] (M. Kikuchi). 1 Present address: Research and Development Division, Towa PHARMACEUTICAL CO., LTD, 26-7 Ichiban-Cho, Kadoma, Osaka 571-0033, Japan. 0006-291X/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.05.178
signal KDEL [2] at the C-terminus. Moreover, PDI has been reported to have chaperone and anti-chaperone activities [3] and may be involved in the quality control system, whereby misfolded proteins are destined for degradation in the cell [4] PDI is also known to facilitate the secretion of human lysozyme in Saccharomyces cerevisiae, reflecting its chaperone activity [5]. Recently, many PDI homologues have been identified, which are similar to PDI in structure. One of the characteristics of PDI homologues is that they have two or three CXXC motifs in their primary structures. Each motif functions as an active site for disulfide bond isomerization. Human P5 (hP5) [6] and human protein disulfide isomerase-related protein (hPDIR) [7] are members of human PDI family, and their functions in vitro were recently elucidated [8,9]. hP5 contains two CGHC motifs within two domains (a and a0 ) as well as an ER retention signal, KDEL, and it functions to catalyze the isomerization of insulin and prevent the
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aggregation of denatured proteins in vitro. However, these isomerase and chaperone activities of hP5 are less pronounced than those of hPDI [8]. On the contrary, hPDIR has three active sites (CSMC, CGHC, and CPHC) in three domains (a, a0 , and a0 ), and an ER retention signal, KEEL. hPDIR is different from previously identified PDI family members. hPDIR specifically binds to a1-antitrypsin and stimulates its oxidative refolding, however its isomerase and chaperone activities are lower than those of hPDI [9]. Recently, PDI and P5 have been identified from nucleoli isolated from cultured human cells [10], and it was demonstrated that expression of P5 in the embryonic midline is required for left/right asymmetries [11]. Despite these recent insights, the in vivo functions of hPDIR are still not clear. Saccharomyces cerevisiae PDI (yPDI or Pdi1p), which is encoded by the PDI1 gene, is essential for yeast cell growth [12]. Isomerization of nonnative disulfide bonds is suggested to be the essential function of Pdi1p in yeast [12], and its chaperone activity is considered to depend on its isomerase activity [13]. Moreover, S. cerevisiae contains three other nonessential genes with homology to PDI1 namely MPD1 [14], MPD2 [15], and EUG1 [16]. However, overexpression of these homologues in yPDI- and yPDI homologue-deleted strains indicated that Mpd1p is the only homologue capable of carrying out all the essential functions of Pdi1p [17]. Furthermore, Eug1p which contains two mutated CXXC active site motifs displays the equivalent function with Pdi1p [18]. Recently, it has been demonstrated that mammalian PDI family proteins can rescue the growth of a PDI1disrupted strain. In addition, overexpression of the a and a0 domains of rat PDI promoted the viability of a yeast strain in which PDI1 was disrupted by deletion [19]. Here, to understand the functional differences between hPDI, hP5, and hPDIR, human cDNAs encoding these proteins were inserted in a single copy number plasmid into PDI1-disrupted yeast, and we subsequently determined whether the viability of the yeast strain was rescued. We also inserted these cDNAs into the PDI1disrupted yeast chromosome to compare the functions of PDI and its homologues in vivo. Moreover, by assessing isomerase and chaperone activities in vitro, we also compared human and yeast PDI family proteins. Here, we detail the isomerase and chaperone activities of yPDI homologues and demonstrate that rescue of a PDI1-disrupted yeast strain depends on the level of expression of PDI and its homologues.
Materials and methods Strains, media, and yeast transformation. Saccharomyces cerevisiae strain trg1/TRG1 (MATa/MATa ura3/ura3 lys2/lys2 ade2/ade2 trp1/
trp1 his3/his3 leu2/leu2 TRG1/trg1::HIS3) was cultured in YPD medium for transformations, YPGal medium for expression, or SD medium for transformant selection at 30 °C. S. cerevisiae transformation was performed by electroporation using an electropulsator (Gene Transfer Equipment model GTE10 (Shimadzu)). Escherichia coli AD494 (DE3) {Dara , leu7967, DlacX74, DphoA, Pvu II, phoR, DmalF3, F 0 [lacþ , (lacIq ), pro], trxB::kan (DE3)} was used to express human and yeast PDI family proteins. Plasmid construction. XbaI and SalI sites were created in hPDI (cloned in our laboratory) and hPDIR cDNA [7] by site-directed mutagenesis using PCR. The hPDI XbaI site was introduced 1–6 nucleotides upstream of the 50 -terminus of the region encoding the secretory leader peptide and the hPDI SalI site was 4–9 nucleotides downstream of the coding region of the ER retention signal with 50 -TGA TCCGTGGGATCCATGCTGCGCCGCGCT-30 and 50 -CGGGTC TGGCTTGTCGACTTACAGTTCATC-30 as the upper and lower primers, respectively (mutated nucleotides are underlined). The PCR products were introduced between XbaI and SalI sites of the S. cerevisiae centromeric plasmid, pYEUra3 (Clontech). Similarly, amplified hP5 cDNA [6] and the yPDI gene (cloned in our laboratory) were inserted between XbaI and XhoI sites or BamHI and XhoI sites of pYEUra3, respectively. These plasmids were treated with NdeI to remove ARS1 and CEN4 coding regions, and the resultant plasmids were used for homologous recombination. To obtain pure PDI family proteins, we inserted the cDNAs of hPDI, hP5, and hPDIR, and the Mpd1p gene into the NdeI site of the pET15b plasmid, and the yPDI, Eug1p, and Mpd2p genes between the XhoI and BamHI sites of the pET15b plasmid. Expression and purification of His-tagged human and yeast PDI family proteins. E. coli AD494(DE3) was transformed with the plasmids of the pET15b-series and transformants were grown at 37 °C in LB medium containing 100 lg/ml ampicillin with shaking. When the OD at 600 nm reached 0.4–0.6, IPTG was added to a final concentration of 1 mM and incubation was continued at 30 °C for 6 h. Cells were collected by centrifugation, suspended in a 20 mM sodium phosphate buffer (pH 7.4), and disrupted with an ultrasonic cell disrupter. The supernatant was passed through a Minisart (Sartorius) (0.2 lm) and applied to a Ni2þ -chelating resin column (Amersham– Pharmacia Bioscience) for purification. Western blot analysis. Yeast transformants cultured in YPGal medium to the logarithmic phase were lysed using glass beads in a buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 5 mM MgCl2 , 50 mM KCl, 5% glycerol, 3 mM DTT, 1 mM PMSF, and 1 lg/ ml pepstatin A, and the lysate was centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and centrifuged again at 30,700 rpm at 4 °C for 90 min to obtain a microsomal pellet. The pellet was solubilized by heat treatment in the presence of Triton X-100. SDS–PAGE was carried out as described by Laemmli [20] using 12.5% (w/v) gels. After SDS–PAGE, the gels were electroblotted using a semidry method. The filters were blocked with 10% skim milk in PBS and reacted with anti-PDI, anti-P5, and anti-CGHC phage antibodies (5E) [21]. Anti-PDI was prepared in our laboratory, while anti-P5 and anti-yPDI were kind gifts of Dr. N. Takahashi and Dr. Y. Tsujimoto, and the anti-CGHC phage antibody (5E) was selected in our laboratory from the phage display library of Griffiths et al. [22] using hPDI and hPDIR as the ligands. Anti-PDI and anti-P5 were detected using HRP-conjugated goat anti-rabbit IgG and anti-CGHC phage antibody (5E) was detected using monoclonal mouse anti-M13. Tetrad analysis of strains which express the human PDI family proteins. Selected transformants were incubated for 1 or 2 days in YPD medium and then induced to sporulate in sporulation plates (10 g/L potassium acetate, 1 g/L bacto-yeast extract, 0.5 g/L glucose, and 20 g/ L bacto-agar) over an incubation period of 3–5 days at 28 °C. Spore formation was monitored microscopically. Once the cells had sporulated, the equivalent of 10 ll cells was resuspended in 200 ll distilled water containing 1.2 M sorbitol, 50 mM potassium phosphate buffer (pH 7.5), 14 mM 2-mercaptoethanol, and 0.2 mg/ml Zymolyase100T
T. Kimura et al. / Biochemical and Biophysical Research Communications 320 (2004) 359–365 (Seikagaku Kogyo). After 15–30 min incubation at 30 °C, a sample (20 ll) was spread on a thin layer of agar plate and the spores of each tetrad were separated using a micromanipulator (MMS-20, Shimadsu). These layers were then transferred to YPD and YPGal plates, and incubated at 30 °C. Analysis of isomerase and chaperone activities of human and yeast PDI family proteins. The isomerase activities of the PDI family proteins were determined by the method of Ibbetson and Freedman [23], in which the enzyme-catalyzed reduction of disulfide bonds of insulin by GSH is coupled to the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) by NADPH and glutathione reductase. Chaperone activity measured by the prevention of denatured rhodanese aggregation was analyzed using the method described by Martin et al. [24]. Briefly, bovine rhodanese was denatured in buffer A (6 M guanidium–HCl, 30 mM Tris–HCl, and 1 mM dithiothreitol [pH 7.2]) and the aggregation of denatured rhodanese was investigated by monitoring the increase in absorbance at 320 nm. Biomolecular interaction. Binding of mastoparan to immobilized yeast PDI family proteins was measured in a surface plasmon resonance apparatus, specifically a BIACORE 3000 instrument (Biacore). The yPDI, Eug1p, Mpd1p, and Mpd2p proteins were covalently coupled to the matrix of a CM5 sensor chip via amino groups, according to the manufacturer’s instructions. Mastoparan was then delivered to the protein-coated flow cell to observe binding. In the reverse experiment, Mpd2p was also used as the analyte. Data analysis was performed using BIACORE evaluation ver.3.1 software.
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Fig. 1. Structures of human and yeast PDI family proteins. An open box indicates the position of a thioredoxin-like domain. The CGHC, CSMC, CPHC, CLHC, CIHC, and CQHC motifs are known or putative active sites. The C-terminal KDEL, KEEL, and HDEL sequences are the estimated ER-retention signals.
Results One copy of human PDI cDNA fails to rescue a PDIdisrupted yeast strain Recently, it has been demonstrated that overexpression of mammalian PDI proteins can rescue the growth of a PDI1-disrupted yeast strain [25]. To compare the ability of human PDI family proteins, such as hPDI, hP5, and hPDIR, to overcome yeast PDI protein deficiency, we have fused their genes to the galactose-inducible and glucose-repressible GAL1 promoter immediately downstream of the translational start site. Yeast PDI shares several features with those of a group of mammalian proteins residing mainly in the lumen of the ER, as it has an amino-terminal secretary leader peptide, a carboxyl-terminal luminal retention signal, and two domains with the thioredoxin motif CGHC (Fig. 1). In contrast to its human relatives, which have no potential N-glycosylation sites, yeast PDI harbors five N-glycosylation sites, which are all modified in vivo [12]. The plasmids encoding the galactose-inducible PDI family proteins were introduced into a heterozygous S. cerevisiae TRG1/trg1::His3 diploid strain by transformation. The haploid progenies of these transformants were observed by tetrad analysis on solid media containing galactose (YPGal). The expression of PDI family proteins by these transformants was confirmed by immunoblot analysis using anti-bPDI, anti-hP5, anti-yPDI, and anti-CGHC phage antibodies (5E) (Fig. 2). Normally, yPDI is essential for yeast viability however hPDI centromeric
Fig. 2. Expression of PDI family proteins in the PDI1-disrupted yeast. PDI family proteins expressed in PDI1-disrupted yeast were extracted and detected by Western blotting as described in Materials and methods. Lane 1, marker proteins; lane 2, pYEUra3-hPDI; lane 3, pYEUra30 -hPDI; lane 4, pYEUra3-hP5; lane 5, pYEUra30 -hP5; lane 6, pYEUra3-hPDIR; lane 7, pYEUra30 -hPDIR; and lane 8, pYEUra30 yPDI.
plasmid, pYEUra3-hPDI, which is a single copy number plasmid, successfully replaced yPDI function. In contrast, the hPDI family proteins, hP5 and hPDIR, could not functionally replace yPDI (Table 1A). We also inserted the cDNAs of these hPDI family proteins into the yeast chromosome and performed tetrad analysis similar to that described above. This revealed that even hPDI could not replace yPDI using this homologous recombination approach (Table 1B). Theoretically, tetrad analysis with the YCp plasmid (pYEUra3-hPDI) should
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Table 1 Tetrad analysis of Saccharomyces cerevisiae PDI1/pdi1 D::HIS3 cells transformed with plasmids encoding hPDI family proteins Plasmid
Tetrads
Segregation of spores (viable:nonviable) 4:0
3:1
2:2
1:3
0:4
(A) pYEUra3-hPDI pYEUra3-hPDIR pYEUra3-hP5
20 20 20
15 0 0
1 0 0
2 17 16
2 2 4
0 1 0
(B) pYEUra30 -hPDI pYEUra30 -hPDIR pYEUra30 -hP5 pYEUra30 -yPDI
23 20 19 18
0 0 0 2
1 0 0 10
19 16 18 5
3 3 1 1
0 1 0 0
(A) and (B) indicate the experiments with yeast centromere (pYEUra3) and yeast integrating (pYEUra30 ) plasmids, respectively.
be fundamentally equivalent to that with the YIp plasmid (pYEUra30 -yPDI), however, different results were obtained with regard to PDI function. As indicated in Table 1A, most of the four spores were viable when YCp-hPDI (pYEUra3-hPDI) was used. In contrast, only one case that three spores were mainly viable was observed when YIp-yPDI (pYEUra30 -yPDI in Table 1B) was used. These results suggest that at least two or three copies of hPDI are necessary for yeast to be viable. For pYEUra3-hPDI, we calculated the plasmid copy number against the chromosome using Southern blotting, and found that yeast actually possess three copies of the plasmid (data not shown). It has already been reported that the actual copy number is more than two, even when a single copy number plasmid is used [26]. This report supports our results with regard to copy number of the YCp plasmid. Therefore, we confirmed that one copy of hPDI cDNA is not capable of rescuing the yPDI-deleted strain. Moreover, neither hP5 nor hPDIR was able to rescue the PDI1-disrupted strain, even when a YCp-type plasmid (pYEUra3-hP5 and pYEUra3-hPDIR) was used. Functional difference between human PDI family proteins and yeast PDI Although we found that only hPDI that differed from hP5 and hPDIR could replace yPDI function, it was notable that the strain containing a single copy of the hPDI gene did not grow. The isomerizing function of PDI was previously reported to be sufficient for yeast growth [13], therefore, we predicted that the yPDI isomerase activity is stronger than that of hPDI. To confirm this notion, we assessed the isomerase activity of hPDI, hPDIR, hP5, yPDI, and yPDI homologues. The isomerase activity of yPDI was twice that of hPDI (Fig. 3), while the activities of hP5 and hPDIR were less than 25% and approximately 1% that of yPDI, respectively. Given that all mammalian PDI family proteins were overexpressed in previous experiments when they rescued the PDI1-deleted strain, these observations
Fig. 3. Isomerase activities of the human and yeast PDI family proteins. The activity of wild-type hPDI was normalized to 100%. Each value is shown as the mean of two separate experiments.
suggest that their overexpression is necessary to mediate the rescue effect. In other words, yeast and mammalian PDIs differ concerning their maintenance of yeast viability. Recent studies on vertebrate PDI family proteins have produced several new observations and their functions are gradually being elucidated. However, the specific mechanisms of action of yeast PDI family proteins remain unknown. Although overexpressed Mpd1p rescued the PDI1-disrupted yeast strain, this was not observed for other yeast homologous proteins [17]. Thus, to elucidate the biochemical characteristics of yPDI family proteins, we expressed genes of yPDI family proteins in E. coli, purified the respective proteins, and measured their isomerase activities. As shown in Fig. 3, with the exception of yPDI itself, the yPDI family proteins had virtually no isomerase activity, with only Mpd1p displaying very weak activity. Based on these results (Fig. 3), we predicted that protein folding in S. cerevisiae is solely dependent on yPDI.
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Yeast PDI family proteins have significant chaperone activity As described above, yPDI family proteins generally possess extremely low isomerase activity, whereas it is known that mammalian PDI family proteins generally possess chaperone activity [3]. Previously, we demonstrated that the chaperone activities of hP5 and hPDIR were half that of hPDI [8,9]. Thus, here we examined the chaperone activity of yPDI protein homologues and compared them with the previously determined activities of hPDI family members. Mpd1p, which has comparatively weak isomerase activity, did not show any chaperone activity (Fig. 4). In contrast, Eug1p exhibited chaperone activity similar to those of hP5 and hPDIR. Surprisingly, Mpd2p had high chaperone activity (270% the level of hPDI). In general, the binding of mastoparan is an index of chaperone activity [27], so we also measured mastoparan binding to yeast PDI family proteins using the BIACORE system. The KD values of the mastoparan for yPDI, Eug1p, and Mpd2p were 3.25 106 , 4.21 104 , and 2.68 105 M, respectively, and mastoparan did not bind Mpd1p. These observa-
Fig. 4. Chaperone activities of the yeast and human PDI family proteins, examined using rhodanese as a substrate. The activity of wildtype hPDI was normalized to 100%. Each value is shown as the mean of two separate experiments.
Table 2 Dissociation constants of mastoparan and Mpd2p to yPDI family proteins Ligand
Dissociation constant (M) Analyte Mastoparan
yPDI Eug1p Mpd1p Mpd2p
6
3.25 10 4.21 104 ND 2.68 105
Mpd2p ND ND 5.97 106 8.95 106
The data analysis was performed using the BIA evaluation ver.3.1 software. ND, not detected.
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tions confirm that mastoparan binding correlates well with chaperone activity (Table 2).
Discussion Previously, the in vivo function of several mammalian PDI family proteins was studied by tetrad analysis using S. cerevisiae, and mouse PDI was found to rescue a PDI1-disrupted strain [25]. Here, we also employed tetrad analysis to assess the functional homology of hPDI family proteins as compared with yPDI and obtained different results. Specifically, in a homologous recombination experiment we found that hPDI proteins are unable to replace yPDI. Our results indicate, for the first time, that one copy of hPDI cDNA cannot rescue the PDI1-disrupted yeast strain, and that the ability of hPDI to promote yeast growth differs functionally from that of yPDI itself. Although yPDI is glycosylated in yeast, glycosylation per se does not affect the isomerase activity [28]. Here, to clearly distinguish yeast and mammalian PDIs, we have adopted several experimental approaches. The overall conclusions of these experiments are that in S. cerevisiae, the folding of proteins containing disulfide bonds may depend completely on yPDI and that strong isomerase activity is required for cell growth. With the exception of yPDI itself, all yPDI homologues had extremely weak or no isomerase activity. In contrast, they all possessed considerable chaperone activity, except for Mpd1p. This implies that the PDI1disrupted strain has very weak or no isomerase activity, but maintains tolerable chaperone activity. Furthermore, these results suggest that isomerase is a key enzyme for yeast cell growth. On the contrary, mammalian PDI family proteins generally exert both isomerase and chaperone activities, and share these functions to maintain cell growth. These observations apparently indicate that yeast completely rely on the isomerase function of yPDI to maintain growth, thus they suggest that the role of PDI fundamentally differs between yeast and mammal. Although the roles of Mpd1p, Mpd2p, and Eug1p are unknown in yeast, we have revealed their isomerase and chaperone activities in vitro. It was also previously reported that overexpressed Mpd1p rescues PDI1-disrupted yeast [14] and that overexpressed Mpd2p requires Mpd1p in the cell [18]. Here, we found that Mpd1p has extremely weak isomerase activity and that Mpd2p has extremely high chaperone activity. We examined whether or not Mpd2p interacts with other yeast PDI homologues using a BIACORE system, and found that it interacts with Mpd1p with a KD value of approximately 106 M (Table 2). However, interaction with Mpd2p did not increase the isomerase activity of Mpd1p. Thus, the interaction appears not to promote isomerase activity, but may alter or mediate other functions.
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hPDI and hER60 appear to correspond to yPDI and Eug1p within the yeast domain structure, respectively, but no yeast protein has been identified that corresponds to hP5 or hPDIR. Recently, the Eps1p homologue protein was identified in the human genome [29], however Mpd1p and Mpd2p human homologues have not yet been found. Thus, Mpd1p and Mpd2p appear to be really unique, in both structure and function, in different organisms. Mammalian PDI is widely known to be a multifunctional protein. To date, its isomerase and chaperone activities as well as its ability to function as a submit of some proteins have been described [30,31]. It was also reported that PDI accelerates infection by human immunodeficiency virus [32] and Toxoplasma gondii [33]. yPDI is an essential protein for yeast growth [12] and is more potent at maintaining yeast growth than hPDI family proteins (Table 1). Nevertheless, it was recently reported that hP5 is required for the establishment of left/right asymmetries during ontogeny [11] and hPDIR has the substrate specificity for oxidative refolding of a1-antitrypsin [9]. Here, we also reported that the functions of hPDI, hP5, and hPDIR differ from those of yPDI homologues. Moreover, recently, PDI and P5 were identified from nucleoli isolated from cultured human cells [10] presenting the possibility that PDI proteins have diverse functions beyond their isomerase and chaperone activities.
Acknowledgments We thank Dr. Greg. Winter for providing the human synthetic phage antibody library. We are also grateful to Drs. Nobuhiro Takahashi and Yoshiyuki Tsujimoto for providing the rabbit anti-human P5 antibody and anti-yeast PDI antibody.
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Functional differences between human and yeast protein disulfide isomerase family proteinsq Taiji Kimura, Yasuhiro Hosoda, Yukiko Kitamura, Hideshi Nakamura,1 Tomohisa Horibe, and Masakazu Kikuchi* Department of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan Received 18 May 2004 Available online 15 June 2004
Abstract Previously, it has been reported that a mammalian protein disulfide isomerase (PDI), when expressed on a single copy number plasmid, can rescue growth of a PDI1-disrupted yeast. However, here, for the first time we demonstrated by tetrad analysis that human PDI (hPDI) is unable to replace yeast PDI (yPDI) when hPDI cDNA is integrated into the yeast chromosome. This observation indicates that hPDI is not functionally equivalent to yPDI. Estimation of the actual copy number of the plasmid, as well as comparison of isomerase and chaperone activities between human and yeast PDI homologues, indicates that one copy of hPDI cDNA is not sufficient to rescue the PDI1-disrupted strain. Notably, the isomerase activities of yPDI family proteins, Mpd1p, Mpd2p, and Eug1p, were extremely low, although yPDI itself exhibited twice as much isomerase activity as hPDI in vitro. Moreover, with the exception of Mpd1p, all hPDI and yPDI family proteins had chaperone activity, this being particularly strong in the case of yPDI and Mpd2p. These observations indicate that the growth of Saccharomyces cerevisiae is completely dependent on the isomerase activity of yPDI. Ó 2004 Published by Elsevier Inc. Keywords: Protein disulfide isomerase; Tetrad analysis; Isomerase activity; Chaperone activity; Yeast
Protein disulfide isomerase (PDI), which is one of the major enzymes assisting protein folding, catalyzes the formation, reduction, and isomerization of protein disulfide bonds in the endoplasmic reticulum (ER) [1]. PDI contains two thioredoxin-like motifs (CXXC) in two separate domains (a and a0 ) and an ER retention
q
Abbreviations: hPDI, human protein disulfide isomerase; hP5, human P5; hPDIR, human protein disulfide isomerase-related protein; yPDI, yeast PDI; ER, endoplasmic reticulum; IPTG, isopropyl-b-D thiogalactopyranoside; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. * Corresponding author. Present address: Department of Bioscience and Bioinformatics, College of Information Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan. Fax: +81-77-561-2659. E-mail address: [email protected] (M. Kikuchi). 1 Present address: Research and Development Division, Towa PHARMACEUTICAL CO., LTD, 26-7 Ichiban-Cho, Kadoma, Osaka 571-0033, Japan. 0006-291X/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.05.178
signal KDEL [2] at the C-terminus. Moreover, PDI has been reported to have chaperone and anti-chaperone activities [3] and may be involved in the quality control system, whereby misfolded proteins are destined for degradation in the cell [4] PDI is also known to facilitate the secretion of human lysozyme in Saccharomyces cerevisiae, reflecting its chaperone activity [5]. Recently, many PDI homologues have been identified, which are similar to PDI in structure. One of the characteristics of PDI homologues is that they have two or three CXXC motifs in their primary structures. Each motif functions as an active site for disulfide bond isomerization. Human P5 (hP5) [6] and human protein disulfide isomerase-related protein (hPDIR) [7] are members of human PDI family, and their functions in vitro were recently elucidated [8,9]. hP5 contains two CGHC motifs within two domains (a and a0 ) as well as an ER retention signal, KDEL, and it functions to catalyze the isomerization of insulin and prevent the
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aggregation of denatured proteins in vitro. However, these isomerase and chaperone activities of hP5 are less pronounced than those of hPDI [8]. On the contrary, hPDIR has three active sites (CSMC, CGHC, and CPHC) in three domains (a, a0 , and a0 ), and an ER retention signal, KEEL. hPDIR is different from previously identified PDI family members. hPDIR specifically binds to a1-antitrypsin and stimulates its oxidative refolding, however its isomerase and chaperone activities are lower than those of hPDI [9]. Recently, PDI and P5 have been identified from nucleoli isolated from cultured human cells [10], and it was demonstrated that expression of P5 in the embryonic midline is required for left/right asymmetries [11]. Despite these recent insights, the in vivo functions of hPDIR are still not clear. Saccharomyces cerevisiae PDI (yPDI or Pdi1p), which is encoded by the PDI1 gene, is essential for yeast cell growth [12]. Isomerization of nonnative disulfide bonds is suggested to be the essential function of Pdi1p in yeast [12], and its chaperone activity is considered to depend on its isomerase activity [13]. Moreover, S. cerevisiae contains three other nonessential genes with homology to PDI1 namely MPD1 [14], MPD2 [15], and EUG1 [16]. However, overexpression of these homologues in yPDI- and yPDI homologue-deleted strains indicated that Mpd1p is the only homologue capable of carrying out all the essential functions of Pdi1p [17]. Furthermore, Eug1p which contains two mutated CXXC active site motifs displays the equivalent function with Pdi1p [18]. Recently, it has been demonstrated that mammalian PDI family proteins can rescue the growth of a PDI1disrupted strain. In addition, overexpression of the a and a0 domains of rat PDI promoted the viability of a yeast strain in which PDI1 was disrupted by deletion [19]. Here, to understand the functional differences between hPDI, hP5, and hPDIR, human cDNAs encoding these proteins were inserted in a single copy number plasmid into PDI1-disrupted yeast, and we subsequently determined whether the viability of the yeast strain was rescued. We also inserted these cDNAs into the PDI1disrupted yeast chromosome to compare the functions of PDI and its homologues in vivo. Moreover, by assessing isomerase and chaperone activities in vitro, we also compared human and yeast PDI family proteins. Here, we detail the isomerase and chaperone activities of yPDI homologues and demonstrate that rescue of a PDI1-disrupted yeast strain depends on the level of expression of PDI and its homologues.
Materials and methods Strains, media, and yeast transformation. Saccharomyces cerevisiae strain trg1/TRG1 (MATa/MATa ura3/ura3 lys2/lys2 ade2/ade2 trp1/
trp1 his3/his3 leu2/leu2 TRG1/trg1::HIS3) was cultured in YPD medium for transformations, YPGal medium for expression, or SD medium for transformant selection at 30 °C. S. cerevisiae transformation was performed by electroporation using an electropulsator (Gene Transfer Equipment model GTE10 (Shimadzu)). Escherichia coli AD494 (DE3) {Dara , leu7967, DlacX74, DphoA, Pvu II, phoR, DmalF3, F 0 [lacþ , (lacIq ), pro], trxB::kan (DE3)} was used to express human and yeast PDI family proteins. Plasmid construction. XbaI and SalI sites were created in hPDI (cloned in our laboratory) and hPDIR cDNA [7] by site-directed mutagenesis using PCR. The hPDI XbaI site was introduced 1–6 nucleotides upstream of the 50 -terminus of the region encoding the secretory leader peptide and the hPDI SalI site was 4–9 nucleotides downstream of the coding region of the ER retention signal with 50 -TGA TCCGTGGGATCCATGCTGCGCCGCGCT-30 and 50 -CGGGTC TGGCTTGTCGACTTACAGTTCATC-30 as the upper and lower primers, respectively (mutated nucleotides are underlined). The PCR products were introduced between XbaI and SalI sites of the S. cerevisiae centromeric plasmid, pYEUra3 (Clontech). Similarly, amplified hP5 cDNA [6] and the yPDI gene (cloned in our laboratory) were inserted between XbaI and XhoI sites or BamHI and XhoI sites of pYEUra3, respectively. These plasmids were treated with NdeI to remove ARS1 and CEN4 coding regions, and the resultant plasmids were used for homologous recombination. To obtain pure PDI family proteins, we inserted the cDNAs of hPDI, hP5, and hPDIR, and the Mpd1p gene into the NdeI site of the pET15b plasmid, and the yPDI, Eug1p, and Mpd2p genes between the XhoI and BamHI sites of the pET15b plasmid. Expression and purification of His-tagged human and yeast PDI family proteins. E. coli AD494(DE3) was transformed with the plasmids of the pET15b-series and transformants were grown at 37 °C in LB medium containing 100 lg/ml ampicillin with shaking. When the OD at 600 nm reached 0.4–0.6, IPTG was added to a final concentration of 1 mM and incubation was continued at 30 °C for 6 h. Cells were collected by centrifugation, suspended in a 20 mM sodium phosphate buffer (pH 7.4), and disrupted with an ultrasonic cell disrupter. The supernatant was passed through a Minisart (Sartorius) (0.2 lm) and applied to a Ni2þ -chelating resin column (Amersham– Pharmacia Bioscience) for purification. Western blot analysis. Yeast transformants cultured in YPGal medium to the logarithmic phase were lysed using glass beads in a buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 5 mM MgCl2 , 50 mM KCl, 5% glycerol, 3 mM DTT, 1 mM PMSF, and 1 lg/ ml pepstatin A, and the lysate was centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and centrifuged again at 30,700 rpm at 4 °C for 90 min to obtain a microsomal pellet. The pellet was solubilized by heat treatment in the presence of Triton X-100. SDS–PAGE was carried out as described by Laemmli [20] using 12.5% (w/v) gels. After SDS–PAGE, the gels were electroblotted using a semidry method. The filters were blocked with 10% skim milk in PBS and reacted with anti-PDI, anti-P5, and anti-CGHC phage antibodies (5E) [21]. Anti-PDI was prepared in our laboratory, while anti-P5 and anti-yPDI were kind gifts of Dr. N. Takahashi and Dr. Y. Tsujimoto, and the anti-CGHC phage antibody (5E) was selected in our laboratory from the phage display library of Griffiths et al. [22] using hPDI and hPDIR as the ligands. Anti-PDI and anti-P5 were detected using HRP-conjugated goat anti-rabbit IgG and anti-CGHC phage antibody (5E) was detected using monoclonal mouse anti-M13. Tetrad analysis of strains which express the human PDI family proteins. Selected transformants were incubated for 1 or 2 days in YPD medium and then induced to sporulate in sporulation plates (10 g/L potassium acetate, 1 g/L bacto-yeast extract, 0.5 g/L glucose, and 20 g/ L bacto-agar) over an incubation period of 3–5 days at 28 °C. Spore formation was monitored microscopically. Once the cells had sporulated, the equivalent of 10 ll cells was resuspended in 200 ll distilled water containing 1.2 M sorbitol, 50 mM potassium phosphate buffer (pH 7.5), 14 mM 2-mercaptoethanol, and 0.2 mg/ml Zymolyase100T
T. Kimura et al. / Biochemical and Biophysical Research Communications 320 (2004) 359–365 (Seikagaku Kogyo). After 15–30 min incubation at 30 °C, a sample (20 ll) was spread on a thin layer of agar plate and the spores of each tetrad were separated using a micromanipulator (MMS-20, Shimadsu). These layers were then transferred to YPD and YPGal plates, and incubated at 30 °C. Analysis of isomerase and chaperone activities of human and yeast PDI family proteins. The isomerase activities of the PDI family proteins were determined by the method of Ibbetson and Freedman [23], in which the enzyme-catalyzed reduction of disulfide bonds of insulin by GSH is coupled to the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) by NADPH and glutathione reductase. Chaperone activity measured by the prevention of denatured rhodanese aggregation was analyzed using the method described by Martin et al. [24]. Briefly, bovine rhodanese was denatured in buffer A (6 M guanidium–HCl, 30 mM Tris–HCl, and 1 mM dithiothreitol [pH 7.2]) and the aggregation of denatured rhodanese was investigated by monitoring the increase in absorbance at 320 nm. Biomolecular interaction. Binding of mastoparan to immobilized yeast PDI family proteins was measured in a surface plasmon resonance apparatus, specifically a BIACORE 3000 instrument (Biacore). The yPDI, Eug1p, Mpd1p, and Mpd2p proteins were covalently coupled to the matrix of a CM5 sensor chip via amino groups, according to the manufacturer’s instructions. Mastoparan was then delivered to the protein-coated flow cell to observe binding. In the reverse experiment, Mpd2p was also used as the analyte. Data analysis was performed using BIACORE evaluation ver.3.1 software.
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Fig. 1. Structures of human and yeast PDI family proteins. An open box indicates the position of a thioredoxin-like domain. The CGHC, CSMC, CPHC, CLHC, CIHC, and CQHC motifs are known or putative active sites. The C-terminal KDEL, KEEL, and HDEL sequences are the estimated ER-retention signals.
Results One copy of human PDI cDNA fails to rescue a PDIdisrupted yeast strain Recently, it has been demonstrated that overexpression of mammalian PDI proteins can rescue the growth of a PDI1-disrupted yeast strain [25]. To compare the ability of human PDI family proteins, such as hPDI, hP5, and hPDIR, to overcome yeast PDI protein deficiency, we have fused their genes to the galactose-inducible and glucose-repressible GAL1 promoter immediately downstream of the translational start site. Yeast PDI shares several features with those of a group of mammalian proteins residing mainly in the lumen of the ER, as it has an amino-terminal secretary leader peptide, a carboxyl-terminal luminal retention signal, and two domains with the thioredoxin motif CGHC (Fig. 1). In contrast to its human relatives, which have no potential N-glycosylation sites, yeast PDI harbors five N-glycosylation sites, which are all modified in vivo [12]. The plasmids encoding the galactose-inducible PDI family proteins were introduced into a heterozygous S. cerevisiae TRG1/trg1::His3 diploid strain by transformation. The haploid progenies of these transformants were observed by tetrad analysis on solid media containing galactose (YPGal). The expression of PDI family proteins by these transformants was confirmed by immunoblot analysis using anti-bPDI, anti-hP5, anti-yPDI, and anti-CGHC phage antibodies (5E) (Fig. 2). Normally, yPDI is essential for yeast viability however hPDI centromeric
Fig. 2. Expression of PDI family proteins in the PDI1-disrupted yeast. PDI family proteins expressed in PDI1-disrupted yeast were extracted and detected by Western blotting as described in Materials and methods. Lane 1, marker proteins; lane 2, pYEUra3-hPDI; lane 3, pYEUra30 -hPDI; lane 4, pYEUra3-hP5; lane 5, pYEUra30 -hP5; lane 6, pYEUra3-hPDIR; lane 7, pYEUra30 -hPDIR; and lane 8, pYEUra30 yPDI.
plasmid, pYEUra3-hPDI, which is a single copy number plasmid, successfully replaced yPDI function. In contrast, the hPDI family proteins, hP5 and hPDIR, could not functionally replace yPDI (Table 1A). We also inserted the cDNAs of these hPDI family proteins into the yeast chromosome and performed tetrad analysis similar to that described above. This revealed that even hPDI could not replace yPDI using this homologous recombination approach (Table 1B). Theoretically, tetrad analysis with the YCp plasmid (pYEUra3-hPDI) should
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Table 1 Tetrad analysis of Saccharomyces cerevisiae PDI1/pdi1 D::HIS3 cells transformed with plasmids encoding hPDI family proteins Plasmid
Tetrads
Segregation of spores (viable:nonviable) 4:0
3:1
2:2
1:3
0:4
(A) pYEUra3-hPDI pYEUra3-hPDIR pYEUra3-hP5
20 20 20
15 0 0
1 0 0
2 17 16
2 2 4
0 1 0
(B) pYEUra30 -hPDI pYEUra30 -hPDIR pYEUra30 -hP5 pYEUra30 -yPDI
23 20 19 18
0 0 0 2
1 0 0 10
19 16 18 5
3 3 1 1
0 1 0 0
(A) and (B) indicate the experiments with yeast centromere (pYEUra3) and yeast integrating (pYEUra30 ) plasmids, respectively.
be fundamentally equivalent to that with the YIp plasmid (pYEUra30 -yPDI), however, different results were obtained with regard to PDI function. As indicated in Table 1A, most of the four spores were viable when YCp-hPDI (pYEUra3-hPDI) was used. In contrast, only one case that three spores were mainly viable was observed when YIp-yPDI (pYEUra30 -yPDI in Table 1B) was used. These results suggest that at least two or three copies of hPDI are necessary for yeast to be viable. For pYEUra3-hPDI, we calculated the plasmid copy number against the chromosome using Southern blotting, and found that yeast actually possess three copies of the plasmid (data not shown). It has already been reported that the actual copy number is more than two, even when a single copy number plasmid is used [26]. This report supports our results with regard to copy number of the YCp plasmid. Therefore, we confirmed that one copy of hPDI cDNA is not capable of rescuing the yPDI-deleted strain. Moreover, neither hP5 nor hPDIR was able to rescue the PDI1-disrupted strain, even when a YCp-type plasmid (pYEUra3-hP5 and pYEUra3-hPDIR) was used. Functional difference between human PDI family proteins and yeast PDI Although we found that only hPDI that differed from hP5 and hPDIR could replace yPDI function, it was notable that the strain containing a single copy of the hPDI gene did not grow. The isomerizing function of PDI was previously reported to be sufficient for yeast growth [13], therefore, we predicted that the yPDI isomerase activity is stronger than that of hPDI. To confirm this notion, we assessed the isomerase activity of hPDI, hPDIR, hP5, yPDI, and yPDI homologues. The isomerase activity of yPDI was twice that of hPDI (Fig. 3), while the activities of hP5 and hPDIR were less than 25% and approximately 1% that of yPDI, respectively. Given that all mammalian PDI family proteins were overexpressed in previous experiments when they rescued the PDI1-deleted strain, these observations
Fig. 3. Isomerase activities of the human and yeast PDI family proteins. The activity of wild-type hPDI was normalized to 100%. Each value is shown as the mean of two separate experiments.
suggest that their overexpression is necessary to mediate the rescue effect. In other words, yeast and mammalian PDIs differ concerning their maintenance of yeast viability. Recent studies on vertebrate PDI family proteins have produced several new observations and their functions are gradually being elucidated. However, the specific mechanisms of action of yeast PDI family proteins remain unknown. Although overexpressed Mpd1p rescued the PDI1-disrupted yeast strain, this was not observed for other yeast homologous proteins [17]. Thus, to elucidate the biochemical characteristics of yPDI family proteins, we expressed genes of yPDI family proteins in E. coli, purified the respective proteins, and measured their isomerase activities. As shown in Fig. 3, with the exception of yPDI itself, the yPDI family proteins had virtually no isomerase activity, with only Mpd1p displaying very weak activity. Based on these results (Fig. 3), we predicted that protein folding in S. cerevisiae is solely dependent on yPDI.
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Yeast PDI family proteins have significant chaperone activity As described above, yPDI family proteins generally possess extremely low isomerase activity, whereas it is known that mammalian PDI family proteins generally possess chaperone activity [3]. Previously, we demonstrated that the chaperone activities of hP5 and hPDIR were half that of hPDI [8,9]. Thus, here we examined the chaperone activity of yPDI protein homologues and compared them with the previously determined activities of hPDI family members. Mpd1p, which has comparatively weak isomerase activity, did not show any chaperone activity (Fig. 4). In contrast, Eug1p exhibited chaperone activity similar to those of hP5 and hPDIR. Surprisingly, Mpd2p had high chaperone activity (270% the level of hPDI). In general, the binding of mastoparan is an index of chaperone activity [27], so we also measured mastoparan binding to yeast PDI family proteins using the BIACORE system. The KD values of the mastoparan for yPDI, Eug1p, and Mpd2p were 3.25 106 , 4.21 104 , and 2.68 105 M, respectively, and mastoparan did not bind Mpd1p. These observa-
Fig. 4. Chaperone activities of the yeast and human PDI family proteins, examined using rhodanese as a substrate. The activity of wildtype hPDI was normalized to 100%. Each value is shown as the mean of two separate experiments.
Table 2 Dissociation constants of mastoparan and Mpd2p to yPDI family proteins Ligand
Dissociation constant (M) Analyte Mastoparan
yPDI Eug1p Mpd1p Mpd2p
6
3.25 10 4.21 104 ND 2.68 105
Mpd2p ND ND 5.97 106 8.95 106
The data analysis was performed using the BIA evaluation ver.3.1 software. ND, not detected.
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tions confirm that mastoparan binding correlates well with chaperone activity (Table 2).
Discussion Previously, the in vivo function of several mammalian PDI family proteins was studied by tetrad analysis using S. cerevisiae, and mouse PDI was found to rescue a PDI1-disrupted strain [25]. Here, we also employed tetrad analysis to assess the functional homology of hPDI family proteins as compared with yPDI and obtained different results. Specifically, in a homologous recombination experiment we found that hPDI proteins are unable to replace yPDI. Our results indicate, for the first time, that one copy of hPDI cDNA cannot rescue the PDI1-disrupted yeast strain, and that the ability of hPDI to promote yeast growth differs functionally from that of yPDI itself. Although yPDI is glycosylated in yeast, glycosylation per se does not affect the isomerase activity [28]. Here, to clearly distinguish yeast and mammalian PDIs, we have adopted several experimental approaches. The overall conclusions of these experiments are that in S. cerevisiae, the folding of proteins containing disulfide bonds may depend completely on yPDI and that strong isomerase activity is required for cell growth. With the exception of yPDI itself, all yPDI homologues had extremely weak or no isomerase activity. In contrast, they all possessed considerable chaperone activity, except for Mpd1p. This implies that the PDI1disrupted strain has very weak or no isomerase activity, but maintains tolerable chaperone activity. Furthermore, these results suggest that isomerase is a key enzyme for yeast cell growth. On the contrary, mammalian PDI family proteins generally exert both isomerase and chaperone activities, and share these functions to maintain cell growth. These observations apparently indicate that yeast completely rely on the isomerase function of yPDI to maintain growth, thus they suggest that the role of PDI fundamentally differs between yeast and mammal. Although the roles of Mpd1p, Mpd2p, and Eug1p are unknown in yeast, we have revealed their isomerase and chaperone activities in vitro. It was also previously reported that overexpressed Mpd1p rescues PDI1-disrupted yeast [14] and that overexpressed Mpd2p requires Mpd1p in the cell [18]. Here, we found that Mpd1p has extremely weak isomerase activity and that Mpd2p has extremely high chaperone activity. We examined whether or not Mpd2p interacts with other yeast PDI homologues using a BIACORE system, and found that it interacts with Mpd1p with a KD value of approximately 106 M (Table 2). However, interaction with Mpd2p did not increase the isomerase activity of Mpd1p. Thus, the interaction appears not to promote isomerase activity, but may alter or mediate other functions.
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hPDI and hER60 appear to correspond to yPDI and Eug1p within the yeast domain structure, respectively, but no yeast protein has been identified that corresponds to hP5 or hPDIR. Recently, the Eps1p homologue protein was identified in the human genome [29], however Mpd1p and Mpd2p human homologues have not yet been found. Thus, Mpd1p and Mpd2p appear to be really unique, in both structure and function, in different organisms. Mammalian PDI is widely known to be a multifunctional protein. To date, its isomerase and chaperone activities as well as its ability to function as a submit of some proteins have been described [30,31]. It was also reported that PDI accelerates infection by human immunodeficiency virus [32] and Toxoplasma gondii [33]. yPDI is an essential protein for yeast growth [12] and is more potent at maintaining yeast growth than hPDI family proteins (Table 1). Nevertheless, it was recently reported that hP5 is required for the establishment of left/right asymmetries during ontogeny [11] and hPDIR has the substrate specificity for oxidative refolding of a1-antitrypsin [9]. Here, we also reported that the functions of hPDI, hP5, and hPDIR differ from those of yPDI homologues. Moreover, recently, PDI and P5 were identified from nucleoli isolated from cultured human cells [10] presenting the possibility that PDI proteins have diverse functions beyond their isomerase and chaperone activities.
Acknowledgments We thank Dr. Greg. Winter for providing the human synthetic phage antibody library. We are also grateful to Drs. Nobuhiro Takahashi and Yoshiyuki Tsujimoto for providing the rabbit anti-human P5 antibody and anti-yeast PDI antibody.
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