Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins Shinya Kamauchi 1, * , †, Hiroyuki Wadahama 1, *, Kensuke Iwasaki 1 , Yumi Nakamoto 2 , Keito Nishizawa 2 , Masao Ishimoto 2 , Teruo Kawada 1 and Reiko Urade 1 1 Graduate School of Agriculture, Kyoto University, Uji, Japan 2 National Agricultural Research Center for Hokkaido Region, Sapporo, Japan Secretory, organelle and membrane proteins are folded with the assistance of molecular chaperones and other folding factors in the endoplasmic reticulum (ER). In many cases, protein folding in the ER is accompanied by N-glycosylation and the formation of disulfide bonds [1]. The directed formation of disulfide bonds in a nas- cent polypeptide chain is thought to be catalyzed by protein disulfide isomerase (PDI; EC 5.3.4.1) and PDI- related family proteins that belong to the thioredoxin superfamily [2–4]. Animal PDI has been shown to act not only as a thiol-oxidoreductase enzyme, but also as a molecular chaperone [5]. PDI is thought to bind poly- peptides through its hydrophobic region and to form, break and isomerize disulfide bonds in these polypep- Keywords endoplasmic reticulum; molecular chaperone; protein disulfide isomerase; soybean; storage protein Correspondence R. Urade, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Fax: +81 774 38 3758 Tel: +81 774 38 3757 E-mail: urade@kais.kyoto-u.ac.jp †Present address Osaka Bioscience Institute, Suita, Japan *These authors contributed equally to this work Database The nucleotide sequence data for the cDNA of GmPDIL-1 and GmPDIL-2 and genomic GmPDIL-1 and GmPDIL-2 are available in the DDBJ ⁄ EMBL ⁄ GenBank databases under accession numbers AB182628, AB185851, AB300660 and AB300661, respectively (Received 26 December 2007, revised 22 February 2008, accepted 18 March 2008) doi:10.1111/j.1742-4658.2008.06412.x Protein disulfide isomerase family proteins play important roles in the fold- ing of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disul- fide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmP- DIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon–intron structure across plant spe- cies. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmP- DIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with pro- glycinin, a precursor of the seed storage protein glycinin, and the a¢-subunit of b-conglycinin, a seed storage protein found in cotyledon cells under con- ditions that disrupt the folding of glycinin or b-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones. Abbreviations AZC, L-azetidine-2-carboxylic acid; DSP, dithiobis(succinimidylpropionate); ER, endoplasmic reticulum; ERSE, endoplasmic reticulum stress- responsive element; PDI, protein disulfide isomerase; PVDF, poly(vinylidene difluoride). 2644 FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS tides [6]. In plants, a genome-wide search of Arabidop- sis thaliana identified a set of 22 PDI orthologs sepa- rated into 10 phylogenetic groups [7]. Groups I and II have two thioredoxin domains and show structural simi- larities to PDI of other eukaryotes. Genes encoding group I or II PDIs are present in several plants [8–16]. Among them, group I PDIs of rice and Rubiaceae were shown to affect folding of proglutelin in vivo and cystine knot defense proteins in vitro [15,17]. In soybean cotyledon cells, large quantities of stor- age protein are synthesized in the ER during seed development to reserve carbon and nitrogen for germi- nation and early growth [18]. Primary soybean seed storage proteins are globulins called glycinin and b-conglycinin. They are folded and assemble into tri- mers in the ER, and are then transported to, and deposited in, protein storage vacuoles [19]. Glycinin is synthesized as a precursor subunit that undergoes two proteolytic processing steps; the first is a removal of an N-terminal signal peptide in the ER, and the second is fragmentation of the precursor into 40 kDa acidic and 20 kDa basic subunits in protein storage vacuoles [20,21]. The second processing step is required for assembly into hexamers [22]. A major glycinin, A1aB1b, possess two intramolecular disulfide bonds, Cys12–Cys45 and Cys88–Cys298, which are required for hexamer assembly and structural stability [23–25]. Folding and the formation of the disulfide bonds of glycinin are predicted to be facilitated by some PDI family members. Previously, we identified novel PDI family proteins belonging to group IV (GmPDIS-1 and GmPDIS-2) and group V (GmPDIM), and showed that GmPDIS-1 and GmPDIM associated with proglycinin in the ER [26,27]. However, involvement of other PDI family proteins in the folding of the stor- age proteins remains a topic of investigation. In this study, we isolated cDNA clones and genomic sequences encoding soybean group I and II PDI family members. We present the tissue distribution, cellular localization and modulation of expression of the proteins encoded by genes from each of these two groups during soybean seed development. We provide evidence of an association between GmPDIL-1 or GmPDIL-2 and proglycinin or b-conglycinin during the course of the folding process of these proteins. Results Cloning and expression of GmPDIL-1 and GmPDIL-2 To clone the soybean ortholog of Arabidopsis PDI-like 1-1 and PDI-like 1-3 categorized in groups I and II [7], a blast search was performed using the nucleotide sequence of PDI-like 1-1 or PDI-like 1-3 cDNA from the Institute for Genomic Research Soy- bean Index. As a result, tentative consensus sequences, TC188262 from PDI-like 1-1 and TC176115 from PDI-like 1-3, were found. Using primer sets designed from their nucleotide sequences, we cloned cDNAs derived from young soybean leaves by RT-PCR. These cDNAs encoded proteins, named GmPDIL-1 and GmPDIL-2, which were 525 and 551 amino acids long, respectively (supplementary Figs S1 and S2). Both pro- teins possess a putative N-terminal secretory signal sequence and two thioredoxin-like motifs with a CGHC active site. Arginines (Arg128 and Arg482 of GmPDIL-1 and Arg163 and Arg505 of GmPDIL-2) known to be involved in the regulation of the active site redox potential in human PDI [28,29] were con- served. In addition, glutamic acid residues (Glu67 and Glu412 of GmPDIL-1, and Glu95 and Glu434 of GmPDIL-2), suggested to facilitate the release of the active site from a mixed disulfide with substrate [30], were also conserved. GmPDIL-1 and GmPDIL-2 pos- sessed C-terminal, KDEL-related sequences that func- tion in ER retention [31,32]. The amino acid sequence identity of GmPDIL-1 and GmPDIL-2 to each other, minus the signal peptides, was 30%. Recombinant GmPDIL-1 and GmPDIL-2 proteins were expressed in Escherichia coli and purified (Fig. 1A,B). Both recombinant proteins were soluble and eluted in a monomeric form from a gel filtration column (data not shown). To examine the helical con- tent, far-UV CD was performed. Both GmPDIL-1 and GmPDIL-2 yielded CD spectra that reflected folded globular protein, and the calculated a-helical content was 34% and 28% for PDIL-1 and PDIL-2, respec- tively (data not shown). The domain structures of GmPDIL-1 and GmPDIL-2 were predicted to form a linear sequence of four domains in an a–b–b¢–a¢ orien- tation beginning at the region of conserved domain sequence homology. We subjected the recombinant GmPDIL-1 and GmPDIL-2 proteins to limited prote- olysis with either trypsin or V8 protease to determine their domain boundaries. After proteolysis for various time periods, the native recombinant proteins were gradually degraded, resulting in the generation of smaller-sized peptide fragments (data not shown). The sites of proteolytic cleavage were determined to be Lys77, Lys152, Lys162 and Glu39 of GmPDIL-1 by N-terminal sequencing of the trypsin peptide fragments and the V8 protease peptide fragments, respectively. In addition, four cleavage sites were identified by measur- ing the masses of the peptide fragments by MALDI- TOF MS. Among the cleavage sites, four resided in S. Kamauchi et al. Two soybean protein disulfide isomerases FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS 2645 two narrow regions, overlapping the putative bound- ary regions in GmPDIL-1 between a and b and b¢ and a¢ (Fig. 1C). In the case of GmPDIL-2, Lys175, Glu43, Glu62, Glu68 and Glu135 were identified by N-termi- nal sequencing of the enzymatic digest fragments, respectively, and Glu541 was identified by MALDI- TOF MS. These cleavage sites were located in regions of N-terminal extension, in the C-terminal tail, and in the putative boundary regions between a and b (Fig. 1D). As lysine, arginine or glutamic acid residues are present in the putative boundary region between b and b¢ in GmPDIL-1, and between b and b¢ and b¢ and a¢ in GmPDIL-2, the structure of these regions may be protease resistant. The activity of recombinant GmPDIL-1 and GmP- DIL-2, which catalyze oxidative refolding of reduced and denatured RNaseA, was examined. The specific activities of GmPDIL-1 and GmPDIL-2 were 472 and 300 mmol RNaseAÆmin )1 Æmol )1 , respectively (Fig. 2A). Several mammalian and yeast PDI family proteins are known to function as molecular chaperones [5]. We measured the molecular chaperone activity, which pre- vents the aggregation of unfolded rhodanese. Aggrega- tion occurred over 14 min without PDI, but was inhibited by GmPDIL-2 in a concentration-dependent manner (Fig. 2C). In the presence of 2.4 lm GmPDIL- 2 (molar ratio of 6 : 1 to rhodanese), 30% of the rhodanese aggregation was inhibited for at least 14 min. GmPDIL-1 exhibited slight, but significant, chaperone activity at a molar ratio of 6 : 1 to rhoda- nese (Fig. 2B). Cloning of GmPDIL-1 and GmPDIL-2 genomic sequences Genomic sequences encoding GmPDIL-1 or GmPDIL- 2 were cloned and sequenced. The alignment and comparison with the corresponding cDNA showed that GmPDIL-1 and GmPDIL-2 were composed of 10 and 12 exons, respectively (supplementary Fig. S3). A C D B Fig. 1. Prediction of the GmPDIL-1 and GmPDIL-2 domain structures. Recombinant GmPDIL-1 (A) and GmPDIL-2 (B) in E. coli (lane 1) were purified by His-tag column chromatography (lane 2), followed by gel filtration chromatography (lane 3). Proteins in each effluent were sepa- rated by 10% SDS ⁄ PAGE and stained with Coomassie Blue. (C,D) Schematic representation of cleavage sites in recombinant GmPDIL-1 (C) and GmPDIL-2 (D) by limited proteolysis with trypsin and V8 protease. The upper line represents recombinant protein. The boxes below indi- cate the domain boundaries predicted by an NCBI conserved domain search. The arrows indicate the determined cleavage sites. Black boxes in domains a and a¢ represent the CGHC motif. A closed circle with a bar represents an N-glycosylation consensus site. SP, signal peptide. Two soybean protein disulfide isomerases S. Kamauchi et al. 2646 FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS Nucleotide sequences of the ORF of both PDI genes were exactly the same as those of cDNAs cloned in this study. Comparisons of soybean genomic sequences of GmPDIL-1 or GmPDIL-2 with those of Arabidopsis (AGI number At1g77510), rice (MOsDb number Os04g35600) and wheat (accession number AJ277377) [33] or Arabidopsis (AGI number At3g54960) and rice (MOsDb number Os02g01010) showed a significant conservation of the exon–intron structure across these species. The exon–intron structure across GmPDIL-1 and GmPDIL-2 was considerably different. All introns of GmPDIL-1 and GmPDIL-2 matched the branchpoint consensus sequence in plants (YTNAN) upstream of the 3¢-site. The second, third, fifth, sixth and seventh introns of GmPDIL-1 had the plant branchpoint consensus sequence TTNAN, whereas the first, fourth, eighth and ninth introns of GmPDIL-1 had the consensus sequence CTNAN [34]. In the case of GmPDIL-2, 10 of the 11 introns had TTNAN, and the eighth intron possessed CTNAN. Promoter regions of around 1 kb and 1.5 kb upstream of each start codon of GmPDIL-1 and GmP- DIL-2 were analyzed. A search for elements upstream of the coding region of GmPDIL-1 in the database of plant promoters (PLACE: http://www.dna.affrc.go.jp/ PLACE/) detected ER stress-responsive element (ER- SEs), CCAAT-N 9 -CCACG [35], and a number of cis-acting regulatory elements involved in endosperm- specific gene regulation, including G-box, DPBF core Dc3, E-box, SEF 1 motif and SEF 4 motifs (Table 1). In the promoter region of GmPDIL-2, cis-acting regu- latory elements involved in the regulation of endo- sperm-specific genes, AACA motif, DPBF core Dc3, E-box, RY-repeat and SEF 1 motif, were found (Table 2). However, no ER stress regulatory element was found. GmPDIL-1 mRNA, but not GmPDIL-2 mRNA, is upregulated by ER stress Expression of genes encoding ER-resident proteins is known to be upregulated by the accumulation of unfolded protein in the ER (i.e. ER stress) in plant cells [36,37]. As the consensus sequence of the ERSE was found in the promoter region of GmPDIL-1, the potential for GmPDIL-1 response to ER stress was Fig. 2. Activity of recombinant GmPDIL-1 and GmPDIL-2. (A) Oxi- dative refolding activity of the recombinant GmPDIL-1, GmPDIL-2, GmPDIS-1, GmPDIS-2 and GmPDIM. Activity was assayed by the measurement of RNase activity produced through the regeneration of the active form of reduced RNaseA. Data represent the mean ± SD for four experiments. The data for GmPDIS-1, GmP- DIS-2 and GmPDIM are from Wadahama et al. [26,27]. (B,C) Chap- erone activity of GmPDIL-1 (B) and GmPDIL-2 (C). The aggregation of rhodanese (0.4 l M) was measured without (solid circles) or with 0.8 l M GmPDIL-2 (open triangles), 2.4 lM GmPDIL-1 (open squares) or 2.4 l M GmPDIL-2 (open squares). Each value repre- sents the mean of three experiments. Bars represent SD. The statistical significance of difference was determined between aggregations in the reaction with and without GmPDIL1 or GmPDIL-2 by the unpaired Student t -test. *P < 0.01. S. Kamauchi et al. Two soybean protein disulfide isomerases FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS 2647 examined. First, we performed microarray analysis of transcripts from soybean cotyledons treated with or without an ER-stress inducing reagent, tunicamycin, on a GeneChip (Affymetrix, Santa Clara, CA, USA) designed from soybean-expressed sequence tags. Many (488) probe sets showed a mean variation of ‡ 4-fold under tunicamycin treatment (supplementary Table S1). In total, 178 genes, including some encoding PDI family members, GmPDIL-1, GmPDIS-1 [26] and GmPDIM [27], were identified by homology search of these probe sets. Probes sets for GmPDIL-2 exhibited little variation (data not shown). To confirm the induc- tion of GmPDIL-1 mRNA by ER stress, the cotyledon was treated with or without tunicamycin or l-azeti- dine-2-carboxylic acid (AZC), and mRNA levels of GmPDIL-1 and GmPDIL-2 were measured by real- time RT-PCR. Expression of GmPDIL-1 was upregu- lated by treatments with tunicamycin and AZC in a similar manner to expression of the well-known unfolded protein response genes BiP and calreticulin (Fig. 3). On the other hand, the expression of GmP- DIL-2 was not affected by treatment with either tunicamycin or AZC. Tissue distribution and cellular localization of GmPDIL-1 and GmPDIL-2 Anti-GmPDIL-1 and anti-GmPDIL-2 sera specifically recognized recombinant GmPDIL-1 and GmPDIL-2, respectively (Fig. 4A, lanes 1 and 4). Anti-GmPDIL-1 and anti-GmPDIL-2 sera did not immunoreact with recombinant GmPDIL-2 and GmPDIL-1, respectively (data not shown). Anti-GmPDIL-1 serum immunore- acted with bands of 60 and 63 kDa (Fig. 4A, lane 2), whereas anti-GmPDIL-2 serum immunoreacted with a single 72 kDa band in western analysis of cotyledon proteins (Fig. 4A, lane 5). These bands were not detected with anti-GmPDIL-1 and anti-GmPDIL-2 sera pretreated with purified recombinant GmPDIL-1 and GmPDIL-2, respectively (Fig. 4A, lanes 3 and 6), suggesting that such antibodies specifically immunore- acted with GmPDIL-1 and GmPDIL-2 in the cotyle- don. GmPDIL-1 and GmPDIL-2 have two and five consensus sequences for N-glycosylation, respectively (Fig. 1C,D). When cotyledon proteins were digested with glycosidase F, the mass of bands that immuno- reacted with GmPDIL-1 or GmPDIL-2 sera became Table 1. Putative regulatory motifs found within the promoter sequences of GmPDIL-1. Base substitutions are in lower-case letters. Motif Consensus sequence Function Strand Distance from ATG Sequence ERSE CCAAT-N9-CCACG Putative cis-acting element involved in unfolded protein response + 150–168 CgAAT-gatatttcg-CCACG ) 115–133 CgAAT-ctcatgtcc-CCACG CACGTG motif (G-box) CACGTG Essential for expression of b-phaseolin gene during embryogenesis in bean, tobacco and Arabidopsis + 68–73 CACGTG ) 68–73 CACGTG DPBFcore Dc3 ACACNNG bZIP transcription factors, Dc3 promoter-binding factor-1 and fator-2 binding core sequence; found in the carrot Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by abscisic acid ) 68–73 ACACgtG + 350–356 ACACagG E-box CANNTG E-box of napA storage protein gene of Brassica napus. Sequence is also known as RRE (R response element). Conserved in many storage protein gene promoters + 68–73 CAcgTG + 442–447 CAaaTG + 515–520 CAacTG ) 68–73 CAcgTG ) 459–464 CAttTG ) 795–798 CAaaTG ) 948–953 CAacTG SEF 1 motif ATATTTAWW Sequence found in 5¢-upstream region of soybean b-conglicinin gene ) 649–657 ATATTTAat ) 701–709 ATATTTAta SEF 4 motif RTTTTTR Sequence found in the 5¢-upstream region of soybean b-conglycinin gene + 812–818 aTTTTTa + 860–866 aTTTTTa ) 290–296 aTTTTTa ) 724–730 aTTTTTa ) 801–807 aTTTTTg ) 812–818 aTTTTTa Two soybean protein disulfide isomerases S. Kamauchi et al. 2648 FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS smaller, shifting from 60 and 63 kDa to 59 and 62 kDa, or from 72 kDa to 63 kDa (Fig. 4B), suggest- ing N-glycosylation of these proteins in soybean. Isoelectric points (pI) of GmPDIL-1 and GmPDIL-2 deglycosylated with glycosidase F were determined by two-dimensional electrophoresis (Fig. 4C). Two spots of 60 and 63 kDa with similar pI values (5.0 and 5.1), corresponding to bands detected on SDS ⁄ PAGE, were detected with anti-GmPDIL-1 serum in cotyledon pro- teins. These pI values are almost consistent with that of the recombinant GmPDIL-1. A single spot was detected with anti-GmPDIL-2 serum. The pI of the spot (4.9) was consistent with that of the recombinant GmPDIL-2. GmPDIL-1 and GmPDIL-2 were found Table 2. Putative regulatory motifs found within the promoter sequences of GmPDIL-2. Base substitutions are in lower-case letters. Motif Consensus sequence Function Strand Distance from ATG Sequence AACA motif AACAAAC Core of AACA motifs found in rice glutelin genes, involved in controlling endosperm-specific expression + 870–876 AACAAAC ) 1521–1527 AACAAAC DPBFcore Dc3 ACACNNG bZIP transcription factors, Dc3 promoter-binding factor-1 and factor-2 binding core sequence; found in the carrot Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by abscisic acid + 591–597 ACACgtG ) 170–176 ACACttG ) 756–762 ACACaaG ) 838–844 ACACagG E-box CANNTG E-box of napA storage protein gene of Brassica napus. Sequence is also known as RRE (R response element). Conserved in many storage protein gene promoters + 81–86 CAacTG + 592–597 CAcgTG ) 90–95 CAacTG ) 171–176 CActTG ) 529–534 CAttTG ) 564–569 CAatTG ) 793–798 CAatTG ) 1458–1463 CAtaTG RY repeat CATGCAY RY repeat in seed storage protein genes in legumes such as soybean + 728–733 CATGCA SEF 1 motif RTTTTTR Sequence found in the 5¢-upstream region of soybean b-conglycinin gene + 421–427 aTTTTTa + 1185–1191 gTTTTTa + 1206–1212 aTTTTTa ) 288–294 aTTTTTa ) 622–628 aTTTTTg ) 642–648 aTTTTTa ) 784–790 aTTTTTa ) 1185–1191 aTTTTTa Fig. 3. Response of GmPDIL-1 and GmP- DIL-2 gene expression to ER stress. Cotyle- dons from 137–142 mg or 210–263 mg beans were divided into two halves and incubated in the absence or presence of tunicamycin (TM) for 24 h (A) or AZC for 18 h (B), respectively. The mRNAs of GmP- DIL-1, GmPDIL-2, BiP or calreticulin (CRT) were quantified by real-time RT-PCR. Each value was standardized by correcting for actin mRNA. Fold expression change was calculated as the ratio of mRNA in the sam- ples treated with the stress reagent to that in the untreated sample. Data represent the mean ± SD for three experiments. Data for *BiP and *CRT are from Wadahama et al. [26]. S. Kamauchi et al. Two soybean protein disulfide isomerases FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS 2649 to be expressed in roots, stems, trifoliolate leaves, flow- ers and cotyledons by immunodetection (Fig. 4D). Expression in leaves decreased during leaf expansion. Levels of GmPDIL-1 increased until the seeds grew to 70 mg (Fig. 5B). Thereafter, the level remained almost constant. Thus, GmPDIL-1 may be expressed to enhance the machinery for the folding of seed stor- age proteins. However, this event appeared to be inde- pendent of transcriptional regulation, as the amounts of GmPDIL-1 mRNA did not correlate with the levels of GmPDIL-1 protein expression (Fig. 5A). Levels of GmPDIL-2 and GmPDIL-2 mRNA did not correlate with the synthesis of storage proteins (Fig. 5C,D). GmPDIL-1 and GmPDIL-2 have both an N-termi- nal signal sequence for targeting to the ER and a C-terminal ER retention sequence (KDEL). To confirm localization of GmPDIL-1 and GmPDIL-2 to the ER, microsomes prepared from cotyledon cells were sepa- rated by sucrose gradient in the presence of MgCl 2 or EDTA and analyzed by western blotting (Fig. 6A). Peaks for GmPDIL-1, GmPDIL-2 and BiP, well known as an ER lumen protein, were detected at a density of 1.21 gÆmL )1 in the presence of MgCl 2 .In the presence of EDTA, which causes release of ribo- somes from the rough ER, the peaks of GmPDIL-1, GmPDIL-2 and BiP displayed a similar shift to the lighter sucrose fractions (density of 1.16 gÆmL )1 ). This suggests localization of GmPDIL-1 and GmPDIL-2 to the rough ER. Then, to confirm residence of GmP- DIL-1 and GmPDIL-2 in the ER lumen, microsomes prepared from cotyledon cells were treated with pro- teinase K in the absence or presence of Triton X-100. A B D C Fig. 4. Expression of GmPDIL-1 and GmPDIL-2 in soybean tissues. (A) Cross-reactivity of antiserum prepared against recombinant GmPDIL- 1 or GmPDIL-2 with recombinant GmPDIL-1 (20 ng) (lane 1) and GmPDIL-2 (20 ng) (lane 4) and the cotyledon proteins (10 lg) (lanes 2, 3, 5 and 6). Anti-GmPDIL-1* and anti-GmPDIL-2* represent anti-GmPDIL-1 serum (1 lL) and anti-GmPDIL-2 serum (1 lL) treated with the recom- binant GmPDIL-1 (4 lg) and the recombinant GmPDIL-2 (10 lg), respectively. (B) GmPDIL-1 and GmPDIL-2 were N-glycosylated proteins in soybean. The proteins extracted from the cotyledon were treated without (lane 1) or with (lane 2) glycosidase F. The cotyledon proteins (10 lg) and recombinant GmPDIL-1 (20 ng) or GmPDIL-2 (20 ng) (lane 3) were separated by SDS ⁄ PAGE and immunostained with anti-GmP- DIL-1 or anti-GmPDIL-2 serum. (C) Separation of recombinant GmPDIL-1 and GmPDIL-2 and GmPDIL-1 and GmPDIL-2 expressed in the soy- bean cotyledon by two-dimensional electrophoresis. (D) Detection of GmPDIL-1 and GmPDIL-2 in soybean tissues. Thirty micrograms of protein extracted from the cotyledon (80 mg bean), root, stem, 3 cm leaf, 6 cm leaf, 9 cm leaf and flower were separated by 10% SDS ⁄ PAGE and immunostained with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum. Two soybean protein disulfide isomerases S. Kamauchi et al. 2650 FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS Both GmPDIL-1 and GmPDIL-2 were resistant to protease treatment in the absence of detergent, and were degraded in the presence of Triton X-100 (Fig. 6B), suggesting that GmPDIL-1 and GmPDIL-2 are luminal proteins. Association of GmPDIL-1 and GmPDIL-2 with proglycinin and b-conglycinin a¢-subunit in cotyledon cells GmPDIL-1 and GmPDIL-2 were shown to have oxi- dative folding activity in vitro and to localize to the cotyledon ER, suggesting that they may function in proglycinin folding that is accompanied by the forma- tion of intramolecular disulfide bonds. We sought to detect an association between GmPDIL-1 or GmP- DIL-2 and glycinin in cotyledon cells by immunopre- cipitation from a cotyledon microsomal extract pretreated with the protein crosslinker dithiobis(succin- imidylpropionate) (DSP). First, we confirmed the immunoprecipitation of GmPDIL-1 and GmPDIL-2 from the microsomal extract with anti-GmPDIL-1 serum and anti-GmPDIL-2 serum, respectively (Fig. 7A,B). Each immunoprecipitant was analyzed by western blotting with anti-GmPDIL-1 or anti-GmP- DIL-2 serum. GmPDIL-1 and GmPDIL-2 were immu- noprecipitated irrespective of crosslinking treatment. Following metabolic labeling of nascent proteins with [ 35 S]methionine and [ 35 S]cysteine, glycinin was immu- noprecipitated with anti-(glycinin acidic subunit) serum and detected by fluorography (Fig. 7C, lanes 3 and 4). Most of the label was found in proglycinin. After labeling, microsomes from the cotyledons were crosslinked, solubilized, and immunoprecipitated with preimmune serum, anti-GmPDIL-1 serum or anti- GmPDIL-2 serum. The immunoprecipitants were trea- ted with dithiothreitol to reduce the disulfide bonds formed by crosslinking, and subjected to a second immunoprecipitation with anti-(glycinin acidic subunit) serum. No band was observed in the preimmune serum sample (Fig. 7C, lanes 1 and 2). Little proglycinin was detected in the immunoprecipitant with anti-GmPDIL-1 A C D B Fig. 5. Expression of GmPDIL-1 and GmP- DIL-2 in soybean cotyledons during matura- tion. mRNA of GmPDIL-1 (A) and GmPDIL-2 (C) was quantified by real-time RT-PCR. Each value was standardized with actin mRNA. Values are calculated as a percent- age of the highest value obtained during maturation. Bars represent SD of four experiments. Thirty micrograms of proteins extracted from the cotyledons was sepa- rated by 10% SDS ⁄ PAGE and immuno- stained with anti-GmPDIL-1 serum (B) and anti-GmPDIL-2 serum (D). A B Fig. 6. Localization of GmPDIL-1 and GmPDIL-2 in the ER lumen. (A) Microsomes were isolated from cotyledons (100 mg beans) and fractionated on isopicnic linear sucrose gradients in the presence of MgCl 2 or EDTA. Proteins from each gradient fraction were analyzed by western blotting with anti-GmPDIL-1 serum, anti-GmPDIL-2 serum, and anti-BiP serum, respectively. The top of the gradient is on the left. Density (gÆmL )1 ) is indicated at the top. (B) Microsomes were treated without (lanes 1 and 2) or with (lanes 3 and 4) pro- teinase K in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Triton X-100. Micosomal proteins (10 lg) were analyzed by western blotting with anti-GmPDIL-1 serum and anti-GmPDIL-2 serum, respectively. S. Kamauchi et al. Two soybean protein disulfide isomerases FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS 2651 serum or anti-GmPDIL-2 serum (data not shown). Sufficient detection of proglycinin associated with GmPDIL-1 and GmPDIL-2 was difficult, as only a few proglycinin molecules may associate transiently with PDI proteins in the ER. Then, similar experi- ments were performed with dithiothreitol-treated coty- ledons. Unfolded proglycinin may increase in the ER in the presence of dithiothreitol. Some proglycinin was detected in the immunoprecipitant from the cotyledon untreated with DSP, but much more proglycinin was detected in the immunoprecipitant with anti-GmPDIL-1 and anti-GmPDIL-2 sera from the cotyledon treated with DSP (Fig. 7C, lanes 5–8). These results suggest that GmPDIL-1 and GmPDIL-2 molecules associate with unfolded proglycinin in the lumen of the ER in the presence of dithiothreitol. Recombinant proteins, especially GmPDIL-2, exhib- ited chaperone activity in vitro, raising the question of whether they act as chaperones for proteins such as b-conglycinin, which have no intramolecular disulfide bonds. We then looked for an association between GmPDIL-1 or GmPDIL-2 and b-conglycinin a¢-sub- unit. [ 35 S]b-conglycinin a¢-subunit was confirmed to be immunoprecipitated with anti-(b-conglycinin a¢-sub- unit) serum (Fig. 7D, lanes 3 and 4). b-Conglycinin a¢-subunit was hardly detected in the immunoprecipi- tant with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum (data not shown). Then, the immunoprecipita- tion was performed with the cotyledons treated with tunicamycin. Tunicamycin may increase unfolded b-conglycinin a¢-subunit in the ER, as the folding effi- ciency of glycoproteins such as b-conglycinin a¢-sub- unit may be lowered by inhibition of N-glycosylation. b-Conglycinin a¢-subunit was detected in the immuno- precipitants with anti-GmPDIL-1 serum only from the cotyledons treated with DSP (Fig. 7D, lane 6). Some b-conglycinin a¢-subunit was detected in the immuno- precipitants with anti-GmPDIL-2 serum from cotyle- dons untreated with DSP (Fig. 7D, lane 7). Much more b-conglycinin a¢-subunit was detected in the immunoprecipitants with anti-GmPDIL-2 serum from the cotyledons treated with DSP (Fig. 7D, lane 8). These results suggest that GmPDIL-1 and GmPDIL-2 associate with b-conglycinin a¢-subunit in the lumen of the ER in the presence of tunicamycin. A C B D Fig. 7. Coimmunoprecipitation of GmPDIL-1 or GmPDIL-2 and pro- glycinin or b-conglycinin a¢-subunit. Confirmation of immunoprecipi- tation of GmPDIL-1 and GmPDIL-2 with each specific antibody. Microsomes were isolated from cotyledons (150 mg beans) and treated with (+) or without ()) DSP. Proteins were extracted and immunoprecipitated with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum. The proteins extracted from the ER (lane 1) and the immu- noprecipitants (lanes 2 and 3) were analyzed by western blotting with anti-GmPDIL-1 serum (A) or anti-GmPDIL-2 serum (B). Aster- isks indicate rabbit serum immunoglobulins recovered by the first immunoprecipitation in the immunoprecipitant. (C,D) Coimmunopre- cipitation experiments. Cotyledons were pretreated with dithiothrei- tol (C) or tunicamycin (D) and labeled with Pro-mix L-[ 35 S] in vitro labeling mix for 1 h. After labeling, microsomes were isolated and treated with (+) or without ()) DSP. The extracts from the micro- somes were subjected to immunoprecipitation with preimmune serum (lanes 1 and 2), anti-(glycinin acidic subunit) serum (C, lanes 3 and 4) anti-(b-conglycinin a¢-subunit) serum (D, lanes 3 and 4), anti-GmPDIL-1 serum (lanes 5 and 6), or anti-GmPDIL-2 serum (lanes 7 and 8). The precipitants were subjected to a second immu- noprecipitation with anti-(glycinin acidic subunit) serum (C) or anti- (b-conglycinin a¢-subunit) serum (D). The final precipitants were subjected to SDS ⁄ PAGE and analyzed by fluorography. The position of proglycinins (pro11S) or b-conglycinin a¢-subunit (7S-a¢) is indi- cated on the right. Two soybean protein disulfide isomerases S. Kamauchi et al. 2652 FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS Discussion In this study, we cloned and characterized the cDNAs of GmPDIL-1 and GmPDIL-2 as members of the PDI family. The amino acid sequences and putative domain structures of GmPDIL-1 and GmPDIL-2 were similar to each other, and both recombinant proteins exhibited thiol-oxidoreductase and chaperone activities. Antise- rum against recombinant GmPDIL-1 immunoreacted with two soybean proteins of comparable masses that had similar pIs. It is unclear whether either protein was the product of the GmPDIL-1 gene or of different genes. As the masses of both bands appeared to be smaller to similar extents after treatment with glycosi- dase F, it is unlikely that the differences in size were due to differences in glycosylation. In other plant spe- cies, gene duplications are found at high frequencies. Previously, we found other soybean PDI family genes, those encoding GmPDIS-1 and GmPDIS-2, which might have arisen by gene duplication [26]. Kainuma et al. purified a 63 kDa soybean protein from cotyle- dons and characterized it as a PDI family protein [38]. N-terminal amino acid sequences of the peptide frag- ments from the purified 63 kDa protein were analyzed, and the sequence of a 63 amino acid fragment was determined. Within the sequence, 58 amino acids were identical to the amino acid sequence of GmPDIL-1, suggesting that PDIL-1 and the 63 kDa PDI are homologous proteins encoded by different genes. Therefore, it seems likely that the doublet band may be the 63 kDa PDI and GmPDIL-1. GmPDIL-1, GmPDIS-1 and GmPDIM mRNAs, but not GmPDIL-2 and GmPDIS-2 mRNAs, were ele- vated after ER stress [26,27]. Expression of the Arabid- opsis orthologs of GmPDIL-1, GmPDIS-1 and GmPDIM has been revealed to be induced by ER stress by DNA microarray analysis [36,37,39]. In the promoter regions of GmPDIL-1, GmPDIM [27], and their Arabidopsis orthologs, consensus sequences of ERSE were found. Consensus sequences of ERSE were frequently found in the promoter region of other Ara- bidopsis genes responsive to ER stress [36,37]. In addi- tion, a novel Arabidopsis transcription factor, AtbZIP60, has been shown to activate transcription from ERSE [40]. Therefore, genes of these PDI family members may be unfolded protein-responsive genes that play important roles in maintaining homeostasis of the ER under conditions of stress. The consensus sequences for seed-specific expression were found in the promoter regions of both GmPDIL-1 and GmPDIL-2. However, the mRNA expression patterns of GmPDIL-1 and GmPDIL-2 were different, suggesting that the expression of these genes in cotyle- dons is regulated differently and varies from that observed for storage proteins [18]. A large amount of soybean storage proteins is synthesized in cotyledon cells during seed maturation [18], suggesting that abun- dant, nascent, unfolded proteins are translocated to the ER lumen. A rapid increase in the workload of the ER, as a result of the synthesis of storage proteins, may elicit an ER stress response. However, it seems unlikely that ER stress arises during the normal matu- ration process of soybean seeds as decreases in the mRNA levels of GmPDIL-1, GmPDIS-1 [26] and GmPDIM [27] were observed during the accumulation of the storage proteins. Expression of certain seed stor- age proteins changed as a function of sulfur supply. Under conditions of no sulfur, expression of glycinin, a sulfur-rich storage protein, was decreased. In contrast, expression of the b-conglycinin b-subunit, a sulfur-poor storage protein, was elevated [41]. Sulfur regulation by such proteins is mediated by O-acetyl- l-serine levels [42]. On the other hand, the levels of GmPDIL-1 and GmPDIL-2 mRNAs were not affected by the level of sulfur (supplementary Fig. S4), suggest- ing that the levels of these mRNAs are not regulated in a manner responsive to the expression levels of stor- age proteins. The protein levels of both GmPDIL-1 and GmPDIL-2 were also differentially regulated in cotyledons during seed development. Protein levels of GmPDIL-1, GmPDIS-1 and GmPDIM dramatically increased during seed maturation, but GmPDIL-2 and GmPDIS-2 were expressed at low levels during the same stage. These results may reflect the importance of GmPDIL-1 in seed maturation. In general, the PDI family proteins catalyze the for- mation of disulfide bonds on nascent polypeptide chains in the ER. Hence, GmPDIL-1 and GmPDIL-2 may support proglycinin folding that accompanies the formation of disulfide bonds in the ER of cotyledon cells. However, the association of GmPDIL-1 or GmP- DIL-2 and proglycinin was barely detectable under normal conditions, whereas this association was detected in the presence of dithiothreitol, which inhib- its disulfide bond formation in the ER and may cause the accumulation of unfolded proglycinin. As the active sites of the PDI family proteins are reduced in the ER in the presence of dithiothreitol, neither GmP- DIL-1 nor GmPDIL-2 forms a mixed disulfide bond with the cysteine residues of proglycinin. Therefore, GmPDIL-1 and GmPDIL-2 could noncovalently asso- ciate with proglycinin in the presence of dithiothreitol, suggesting that GmPDIL-1 and GmPDIL-2 may func- tion as molecular chaperones for proglycinin rather than thiol-oxidoreductases. The chaperone activity of GmP- DIL-1 for rhodanese was low (Fig. 2B). GmPDIL-1 S. Kamauchi et al. Two soybean protein disulfide isomerases FEBS Journal 275 (2008) 2644–2658 ª 2008 The Authors Journal compilation ª 2008 FEBS 2653 [...]... (2007) Protein disulfide isomerase family proteins involved in soybean protein biogenesis FEBS J 274, 687–703 27 Wadahama H, Kamauchi S, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T & Urade R (2008) A novel plant protein disulfide isomerase family homologous to animal P5: molecular cloning and characterization as a functional protein for folding of soybean seed storage proteins FEBS J 275, 399–410 28... et al Expression and purification of recombinant GmPDIL-1 and GmPDIL-2 E coli BL21(DE3) cells were transformed with the expression vectors described above The expression of recombinant proteins was induced by the addition of 0.4 mm isopropyl thiogalactoside at 30 °C for 4 h; expressed recombinant proteins were soluble Extraction and purification of recombinant proteins was performed as described previously... determined by MALDI-TOF MS on an AXIMA-CFR MALDI-TOF MS plus (Shimadzu Biotech, Kyoto, Japan) RNaseA refolding assay PDI activity was assayed by the measurement of RNase activity produced through the regeneration of the active form from reduced RNaseA as described previously [26] Chaperone activity assessment Chaperone activity was assayed as described previously [49] Aggregation of rhodanese (0.4 lm,... chaperones appear to collaborate in response to substrate proteins Mapping of association sites of chaperones and PDI family proteins along individual substrate polypeptides will be necessary for clarification of the mechanism of action of this protein family Experimental procedures Plants Soybean (Glycine max L Merrill cv Jack) seeds were planted in 5 L pots and grown in a controlled environmental chamber at... blot analysis was performed essentially as described previously [26] Briefly, proteins were extracted from the frozen tissues by boiling in SDS ⁄ PAGE buffer To cleave N-glycans of the proteins, proteins were extracted from the cotyledons in 0.2% SDS ⁄ 0.1 m Tris ⁄ HCl (pH 8.6) ⁄ 1% Nonidet P-40 Proteins (0.4 mg) were treated with 10 mU of glycosidase F (Sigma-Aldrich Inc.) at 37 °C for 16 h Proteins were... Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains Plant Physiol 108, 327–335 13 Coughlan SJ, Hastings C & Winfrey RJ Jr (1996) Molecular characterisation of plant endoplasmic reticulum Identification of protein disulfide- isomerase as the major reticuloplasmin Eur J Biochem 235, 215–224 14 Huang D-J, Chen H-J & Lin Y-H (2005) Isolation and. .. cotyledon These proteins may play different physiological roles in soybean embryogenesis An association of GmPDIS-1 and GmPDIM, but not of GmPDIS-2, GmPDIL-1, or GmPDIL-2, with either proglycinin or b-conglycinin was detected under normal conditions [26,27] Among soybean PDI family members, GmPDIS-1 and GmPDIM may primarily function in the folding of seed storage proteins In the ER, molecular chaperones. .. collected and assayed by western blotting Labeling of cotyledons We thank Ms Masatoshi Izumo for technical support in identifying the C-terminal amino acid of the peptide produced by protease digestion We thank Ms Akie Ko for assaying the oxidative refolding activity This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences and a Grant-in-Aid for. .. structure of yeast protein disulfide isomerase suggests cooperativity between its active sites Cell 124, 1085–1088 7 Houston NL, Fan C, Xiang QY, Schulze JM, Jung R & Boston RS (2005) Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins Plant Physiol 137, 762–778 8 Shorrosh BS & Dixon RA (1991) Molecular. .. specific for individual proteins will clarify these results An association of GmPDIL-1 or GmPDIL-2 with b-conglycinin a¢-subunit was also detected in the presence of tunicamycin, which inhibits N-glycosylation and may cause the accumulation of unfolded b-conglycinin a¢-subunit in the ER As mature b-conglycinin a¢-subunit possesses no disulfide bonds, GmPDIL-1 and GmPDIL-2 may act as molecular chaperones for . Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins Shinya. protein disulfide isomerase family homologous to animal P5: molecular cloning and characterization as a functional protein for folding of soybean seed storage proteins.