Protein disulfide isomerase family proteins involved
in soybean protein biogenesis
Hiroyuki Wadahama
1,
*, Shinya Kamauchi
1,
*, 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
Many proteins that are synthesized in the endoplasmic
reticulum (ER) are folded with an accompanying forma-
tion of intramolecular disulfide bonds, aided by protein
disulfide isomerase (PDI) and related proteins, which
are characterized by thioredoxin motifs within their pri-
mary structure [1,2]. Both yeast and mammalian PDIs
are known to be multifunctional folding catalysts and
molecular chaperones, which catalyze the formation
and rearrangement of disulfide bonds between correct
pairs of cysteine residues in nascent polypeptide chains
within the ER [3]. Mammalian PDI functions not only
as a catalytic enzyme, but also as a subunit of both
microsomal triacylglycerol transfer protein [4] and
prolylhydroxylase [5]. The mammalian PDI family,
ER-60 ⁄ ERp57, which also has a protein oxidoreductase
activity, interacts and cooperates with calnexin and cal-
reticulin for oxidative folding of N-glycosylated proteins

designed from the expressed sequence tag clone sequences. The cDNA
encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or
GmPDIS-2, respectively. The nucleotide and amino acid sequence identities
of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both pro-
teins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL.
Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed
in Escherichia coli as soluble folded proteins that showed both an oxidative
refolding activity of denatured ribonuclease A and a chaperone activity.
Their domain structures were identified as containing two thioredoxin-like
domains, a and a¢, and an ERp29c domain by peptide mapping with either
trypsin or V8 protease. In cotyledon cells, both proteins were shown to dis-
tribute to the endoplasmic reticulum and protein storage vacuoles by con-
focal microscopy. Data from coimmunoprecipitation and crosslinking
experiments suggested that GmPDIS-1 associates with proglycinin, a pre-
cursor of the seed storage protein glycinin, in the cotyledon. Levels of
GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where
glycinin accumulates during seed development. GmPDIS-1, but not
GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.
Abbreviations
Ab, amyloid b-peptide; AZC,
L-azetidine-2-carboxylic acid; DSP, dithiobis(succinimidylpropionate); ER, endoplasmic reticulum; PDI, protein
disulfide isomerase; PSV, protein storage vacuole; UPR, unfolded protein response.
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 687
In plants, a genome-wide search of Arabidopsis thali-
ana identified a set of 22 orthologs of known PDI-like
proteins that was separated into 10 phylogenetic
groups [10]. Among these groups, five groups (I–V)
have two thioredoxin domains and show structural
similarities to PDI-like proteins in other higher eukary-
otes. The PDI family proteins that are categorized in

Research Soybean Index. As a result, two tentative
consensus sequences, TC176086 and TC176115, were
found. Using two primer sets designed from their nuc-
leotide sequences, we cloned two cDNAs from the
RNA extracted from young soybean leaves by RT-PCR.
These cDNAs encoded proteins, named GmPDIS-1
and GmPDIS-2, which consisted of 364 and 362 amino
acids, respectively (Fig. 1). The nucleotide and amino
acid sequence identities of GmPDIS-1 and GmPDIS-2
were 68% and 74%, respectively. Both proteins possess
a putative N-terminal secretory signal sequence and
two tandem thioredoxin-like motifs, with a CGHC
active site. Arginine residues (R122 and R241 of
GmPDIS-1, and R121 and R240 of GmPDIS-2), which
have been demonstrated to be involved in the regula-
tion of the active site redox potential in human PDI
[13,14], were conserved. In addition, glutamic acid resi-
dues (E51 and E170 of GmPDIS-1, and E50 and E169
of GmPDIS-2), which have been suggested to facilitate
the escape of the active site from a mixed disulfide
with the substrate [15], were also conserved. Most PDI
family proteins found in eukaryotic cells have C-ter-
minal, KDEL-related sequences that act as a signal for
retention in the ER [16,17]. However, GmPDIS-1 and
GmPDIS-2 lack this type of C-terminal signal. An
amino acid sequence similar to the C-terminal domain
of ERp29, an animal PDI-related protein [18,19], was
present in the C-terminal region of both GmPDIS-1
and GmPDIS-2.
The recombinant GmPDIS-1 and GmPDIS-2 pro-

sis for various time periods, the native recombinant
proteins were gradually degraded, resulting in the gen-
eration of smaller peptide fragments (data not
shown). The sites of proteolytic cleavage were deter-
mined to be Lys140 and Ile141 of GmPDIS-1 and
Lys139 and Ile140 of GmPDIS-2 by N-terminal
sequencing of the trypsin peptide fragments. The
N-terminal amino acid sequences of other peptide
fragments were AHHHHH, corresponding to the
N-terminal histidine tag of the recombinant proteins.
We then determined the C-terminal amino acid resi-
dues of the peptide fragments by measuring their
masses by MALDI-TOF MS. Most cleavage sites resi-
ded in two narrow regions, overlapping the putative
Soybean protein disulfide isomerase family H. Wadahama et al.
688 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
boundary regions in GmPDIS-1 and GmPDIS-2
between a and a¢ and a¢ and ERp29c, respectively
(Fig. 3). From these results, we concluded that both
GmPDIS-1 and GmPDIS-2 have a linear sequence of
three domains in an a–a¢–ERp29c pattern.
Several mammalian and yeast PDI family proteins
are known to function as molecular chaperones.
Therefore, we measured the molecular chaperone
activity, which prevents the aggregation of amyloid
b-peptide (Ab) (1–40) monomers. Such aggregation
can be initiated by the addition of ‘seed’, which was
obtained by sonication of Ab(1–40) aggregates into
50 lm Ab(1–40) monomers. This aggregation was
monitored as an increase in thioflavin T fluorescence.

or 38 kDa band in roots, stems, trifoliolate leaves, flow-
ers and cotyledons by western blotting (Fig. 5B). The
amounts of these proteins in leaves decreased during leaf
expansion.
GmPDIS-1 and GmPDIS-2 have an N-terminal signal
sequence for targeting to the ER, but lack a typical
ER-retention signal sequence, like the C-terminal
KDEL. We immunostained soybean cotyledons with
either rabbit anti-GmPDIS-1 serum or rabbit anti-GmP-
DIS-2 serum, and then clarified the subcellular localiza-
tion of GmPDIS-1 and GmPDIS-2 by confocal
microscopy. The specimens were double-stained with
guinea pig anti-BiP serum, as BiP is a well-known ER
resident protein [20–22]. To confirm the specificity of the
anti-BiP or anti-calreticulin serum, we performed west-
ern blotting analysis using soybean protein extracts.
Anti-BiP or anti-calreticulin serum immunoreacted with
a single 70 kDa or 54 kDa band corresponding to BiP
or calreticulin, respectively, in cotyledon extracts
(Fig. 6). In the immature cotyledon from an 80 mg bean
that was initiating the accumulation of seed storage pro-
teins, such as glycinin [23,24] and b-conglycinin [25,26],
in its protein storage vacuole (PSV), GmPDIS-1, Gm-
PDIS-2 and BiP were localized mainly to the ER
(Fig. 7A–D). Interestingly, the PSVs were also slightly
stained with anti-BiP serum. To confirm residence of
GmPDIS-1 and GmPDIS-2 in the lumen of the ER,
microsomes prepared from cotyledon cells were treated
with proteinase K in the absence or presence of Triton
AB

GmPDIS-1 associates with proglycinin in the
cotyledon cells
GmPDIS-1 and GmPDIS-2 were shown to have oxida-
tive folding activity in vitro and to be localized to the
ER of the cotyledon, suggesting that they may func-
tion in protein folding that is accompanied by the for-
mation of intramolecular disulfide bonds like those
of proglycinin [27]. We then attempted to detect an
association between GmPDIS-1 or GmPDIS-2 and
glycinin in the cotyledon cells by immunoprecipitation
with antibodies against GmPDIS-1, GmPDIS-2 and
glycinin after treatment with the protein crosslinker
dithiobis[succinimidylpropionate] (DSP). First, we
confirmed the immunoprecipitation of GmPDIS-1,
GmPDIS-2 and glycinin from the microsomal extract
of cotyledons from 150 mg beans by western blotting
analysis. The efficiencies of immunoprecipitation of
GmPDIS-1 and GmPDIS-2 were not influenced by
crosslinking treatment of the microsomes with DSP
prior to immunoprecipitation (Fig. 9A,B). Immunopre-
cipitation of glycinin acidic subunits was also con-
firmed (Fig. 9C). Second, the processing of proglycinin
to mature glycinin was monitored by pulse-chase
experiments in order to determine the labeling time of
glycinin with [
35
S]methionine and [
35
S]cysteine. Glyci-
nin molecules are synthesized as a single polypeptide

35
S]cysteine for 15 min and then chased in the
presence of cold methionine and cysteine. The labeled
glycinin was immunoprecipitated with anti-(glycinin
acidic subunit) serum. Immediately after pulse labeling
for 15 min, most of the label was in proglycinin
(Fig. 9D, lane 1). After a 6 h chase, the labeled pro-
glycinin decreased and the processed products, i.e. the
acidic and basic subunits of mature glycinin, appeared
(Fig. 9D, lane 4). On the basis of these results, we
labeled the cotyledons with [
35
S]methionine and
[
35
S]cysteine for 6 h to detect simultaneously proglyci-
nin and the acidic and basic subunits of mature glyci-
nin in the immunoprecipitation experiments. After
labeling, the microsomes from the cotyledons were
treated with the crosslinker DSP, solubilized, and
immunoprecipitated with nonimmune, rabbit anti-
GmPDIS-1 serum or rabbit anti-GmPDIS-2 serum.
The immunoprecipitants were treated with dithiothrei-
tol to reduce the disulfide bonds formed by crosslink-
ing with DSP, and then were subjected to a second
immunoprecipitation with anti-(glycinin acidic subunit)
serum. No band corresponding to glycinin was obser-
ved in immunoprecipitation experiment with the non-
immune serum (Fig. 9E, lane 2), whereas a 50–53 kDa
band corresponding to proglycinin was detected in

The synthesis of proglycinin was initiated when the
seeds achieved a mass of 50 mg, and increased gradu-
ally until they grew to 300 mg (Fig. 10G). On the
other hand, the synthesis of pro-b-conglycinin was ini-
tiated when the seeds achieved a mass of 40 mg. The
synthesis of pro-b-conglycinin increased until the
seeds grew to 70 mg and then decreased (Fig. 10E).
GmPDIS-1, GmPDIS-2, BiP and calreticulin were
expressed in the early stages of embryogenesis
(Fig. 10A–D). In the cotyledons of seeds with a mass
greater than 100 mg, the levels of GmPDIS-1 and BiP
increased proportionally to the synthesis of proglyci-
nin. Thus, GmPDIS-1 and BiP may be expressed to
enhance the machinery for the folding of seed storage
proteins such as proglycinin. However, this event
appeared to be independent of transcriptional regula-
tion, as the amounts of GmPDIS-1 and BiP mRNA
did not correlate with the levels of GmPDIS-1 and
BiP expression (Fig. 11A,C). The level of GmPDIS-2
did not correlate with the synthesis of proglycinin
(Fig. 10B,G). The amount of GmPDIS-2 mRNA cor-
related with the amount of protein (Fig. 11B). The
Fig. 6. Analysis of the specificities of anti-BiP serum and anti-
calreticulin serum. The purified recombinant BiP (lane 1), calreticu-
lin (lane 3) and soybean cotyledon extracts (20 lg of protein) (lanes
2 and 4) were subjected to 10% SDS ⁄ PAGE. Recombinant pro-
teins were stained with Coomassie Blue. Cotyledon proteins were
immunostained with anti-BiP serum (lane 2) or anti-calreticulin
serum (lane 4).
Soybean protein disulfide isomerase family H. Wadahama et al.

collected simultaneously are shown on the
right. Asterisks and arrows indicate PSVs
and ER networks, respectively. Bars:
10 lm.
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 693
stress) [34]. Thus, they are the products of UPR genes.
Both Arabidopsis PDI-like2-1 and alfalfa G1 expression
have been shown to be upregulated by tunicamycin
treatment [11,12]. In order to determine whether GmP-
DIS-1 and GmPDIS-2 respond to ER stress, we treated
soybean cotyledons with tunicamycin, dithiothreitol,
and l-azetidine 2-carboxylic acid (AZC), and measured
GmPDIS-1, GmPDIS-2, BiP and calreticulin mRNA
levels by real time RT-PCR. Both BiP and calreticulin
are encoded by well-known UPR genes [35,36]. GmP-
DIS-1, BiP and calreticulin expression were upregulat-
ed by all treatments with tunicamycin, dithiothreitol,
and AZC, suggesting that they are encoded by UPR
genes (Fig. 12). There were differences in the extent of
GmPDIS-1, BiP and calreticulin induction between the
stages of seed development. Under tunicamycin-
induced ER stress, GmPDIS-1, BiP and calreticulin
transcriptional induction were highest in the most
immature cotyledons, whereas under ER stress induced
with the proline analog AZC, transcriptional responses
were highest in the most mature cotyledons. On the
other hand, the expression of GmPDIS-2 was hardly
affected by treatment with tunicamycin, dithiothreitol,
or AZC.

C-terminal ER-retention signal. However, they are
colocalized with BiP to the ER. It is unclear whether
the existence of ER luminal proteins such as GmPDIS-1
and GmPDIS-2, which lack the KDEL sequence,
results from retention or retardation. The importance
of the ERp29c domain of Dictyostelium Dd-PDI for
ER retention was demonstrated by deletion mutation
experiments. In addition, it was demonstrated that the
ERp29c domain was sufficient to localize a green fluor-
escent protein chimera to the ER [39]. The C-terminal
ERp29c domains of both GmPDIS-1 and GmPDIS-2
may possibly play a similar role. Alternatively, these
proteins may be retained in the ER by association with
other ER-resident proteins, such as BiP. In addition to
the localization in the ER, localization of BiP, Gm-
PDIS-1 and GmPDIS-2 in the PSVs of the cotyledon
from the 220 mg bean was observed. Pimpl et al. indi-
cated that BiP was constitutively transported from the
ER to vacuoles via the Golgi [40]. The saturation of
the HDEL receptor with HDEL or KDEL proteins
was assumed to cause the BiP transport to vacuoles
via Golgi bodies. Tamura et al. reported that BiP and
the 62 kDa PDI that has a KDEL ER-retention signal
were constitutively transported to vacuoles in Arabid-
opsis cultured cells [41]. In this case, these proteins
were presumably transported to the vacuoles independ-
ently of the medial ⁄ trans-Golgi complex, as PDI that
Fig. 8. Localization of GmPDIS-1 and GmPDIS-2 in the lumen of
microsomes. Cytosol (lane 1) and microsomes (lanes 2–4) were iso-
lated from cotyledons (100 mg beans). Microsomes were treated

Fig. 9. Coimmunoprecipitation of GmPDIS-1 and proglycinin. Confirmation of immunoprecipitation of GmPDIS-1 (A), GmPDIS-2 (B) and glyci-
nin (C) with each specific antibody. Microsomes were isolated from cotyledons (150 mg beans) and treated with (+) or without (–) DSP. Pro-
teins were extracted and immunoprecipitated with anti-GmPDIS-1 serum (A), anti-GmPDIS-2 serum (B), or anti-(glycinin acidic subunit)
serum (C). The proteins extracted from the ER (lane 1) and the immunoprecipitants (lanes 2 and 3) were separated by SDS ⁄ PAGE and
immunoblotted with anti-GmPDIS-1 serum (A), anti-GmPDIS-2 serum (B), or anti-(glycinin acidic subunit) serum (C). Asterisks indicate rabbit
serum immunoglobulins recovered by the first immunoprecipitation in the immunoprecipitant. (D) Time-dependent processing of proglycinin
in the cotyledon. Cotyledons were labeled with Pro-mix L-[
35
S] in vitro labeling mix for 15 min (lane 1) and chased for 1 h (lane 2), 2 h
(lane 3) or 6 h (lane 4) at 25 °C. The extracts from the microsomes were subjected to immunoprecipitation with anti-(glycinin acidic subunit)
serum. The proteins in the precipitants were separated by SDS ⁄ PAGE and detected by fluorography. Pro 11S, proglycinin; 11S-A, glycinin
acidic subunits; 11S-B, glycinin basic subunits. (E) Coimmunoprecipitation experiments. Cotyledons were labeled with Pro-mix L-[
35
S] in vitro
labeling mix for 6 h. After labeling, microsomes were isolated and treated with (+) or without (–) DSP. The extracts from the microsomes
were subjected to immunoprecipitation with nonimmune serum (lanes 1 and 2), anti-GmPDIS-1 serum (lanes 3 and 4), or anti-GmPDIS-2
serum (lanes 5 and 6). The precipitants were treated with dithiothreitol and then subjected to a second immunoprecipitation with anti-(glyci-
nin acidic subunit) serum. The final precipitants were subjected to SDS ⁄ PAGE and analyzed by fluorography.
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 695
disulfide bonds in the ER of the cotyledon cells. In
addition, there is a possibility that GmPDIS-1 may
function as a molecular chaperone, as GmPDIS-1 had
molecular chaperone-like activity. In mammalian cells,
PDI family proteins were shown to be present in the
folding complex. For example, human PDI is a member
of the BiP system, composed of BiP, GRP94, ERdj3,
GRP170, etc. [46,47]. Another PDI family, ER-
60 ⁄ ERp57, forms a complex with either calnexin or cal-
reticulin to fold N-glycosylated proteins [6,48]. The for-

event. In both rice and maize, the presence of two
paralogs has been reported [10]. As GmPDIS-1 and
well-known UPR genes, such as BiP and calreticulin,
were induced in the cotyledons during seed maturation
after all treatments with tunicamycin, dithiothreitol, or
AZC, it is assumed that a mechanism that counters
ER stress exists in soybean cotyledons. The details of
such mechanisms in plants remain unknown. Arabidop-
sis and rice orthologs of Ire1, a sensor protein in sign-
aling pathways for transcriptional responses against
ER stress in yeast and mammals [50], have been identi-
fied and shown to be capable of acting as sensors of
ER stress in yeast cells [51,52]. In addition, an Arabid-
opsis transcription factor, AtbZIP60, has been found
to activate promoters through UPR cis -elements under
ER stress [53]. We found expressed sequence tag clones
that were predicted to be soybean orthologs of Ire1
(AW459105) and bZIP60 (TC226837). It is likely that
the pathways composed of these orthologs play main
roles in the induction of UPR genes in soybean cotyle-
dons, although such cis-elements have not yet been
identified. The extent of induction of GmPDIS-1, BiP
and calreticulin varied, and was dependent upon the
stage of bean development. It is likely that such differ-
ences in induction depend on the amount of misfolded
protein that accumulates after reagent treatment rather
A
B
C
D

DIS-1, were observed during the accumulation of the
storage proteins.
The primary structure and enzymatic functions of
GmPDIS-1 and GmPDIS-2 were very similar. How-
ever, their expression levels were regulated differently
in the cotyledon. This means that GmPDIS-2 plays
a physiologic role that is different from that of
GmPDIS-1. The levels of GmPDIS-2 were higher in
the immature cotyledon than in the mature cotyledon.
Thus, GmPDIS-2 may be important in early embryo-
genesis, whereas GmPDIS-1 may function in the fold-
ing of seed storage proteins and the alleviation of ER
stress. Because both GmPDIS-1 and GmPDIS-2 are
distributed ubiquitouly in other tissues, they might
assist folding of various proteins.
A
B
C
D
Fig. 11. Expression of GmPDIS-1 (A), GmPDIS-2 (B), BiP (C) and
calreticulin (D) mRNAs in soybean cotyledons during maturation.
Each mRNA was quantified by real time RT-PCR. Each value was
standardized by dividing by the value of actin mRNA. Values are
calculated as a percentage of the highest value obtained during
maturation.
Fig. 12. Responses of GmPDIS-1, GmPDIS-2, BiP and calreticulin
gene to ER stress induced by reagent treatment. Cotyledons from
68–88 mg (black bars), 137–142 mg (hatched bars) or 210–263 mg
(white bars) beans were divided into two halves and incubated in
the absence or presence of 250 lgÆmL

and calreticulin
The cloning of GmPDIS-1, GmPDIS-2 and BiP cDNAs was
performed by RT-PCR. Soybean trifoliolate center leaves
were frozen under liquid nitrogen and then ground into a
fine powder with a micropestle SK-100 (Tokken, Inc.,
Chiba, Japan). Total RNA was isolated using the RNeasy
Plant Mini kit (Qiagen Inc., Valencia, CA) according to the
manufacturer’s protocol. The amplification of cDNA from
total RNA was performed with a High Fidelity RNA PCR
kit (TaKaRa Bio Inc., Shiga, Japan) using the following
oligonucleotide primers: 5¢-GTCTGTGAATTCACGCGTC
CGGAAGAAGAAG-3¢ and 5¢-AAGTAAGAATTCACG
TTGAATATCTCCCAGC-3¢ for GmPDIS-1 (GenBank
accession number AB182630), 5¢-GAGAGACTCGAGTA
GGCGAGGATCGTTCAC and 5¢-AATC ATCTCGA G
CCGCTGAACTGAAAGAAA TGGC-3¢ for GmPDIS-2
(GenBank accession number AB182631), and 5¢-GGCAC
GAGATCGTCCATCGAGAAAGG-3¢ and 5¢-GTCCAAC
CGTCCTCCCTAGCATGAAG-3¢ for BiP (GenBank acces-
sion number AB210900). The amplified GmPDIS-1 and
GmPDIS-2 DNA fragments were digested with the restric-
tion enzymes EcoRI and XhoI, respectively, and subcloned
into the pUC118 vector (TaKaRa bio Inc.), which had been
cleaved with either EcoRI or XhoI, respectively. The ampli-
fied BiP DNA fragment was subcloned into the pT7Blue
vector (TaKaRa Bio Inc.).
Cloning of soybean calreticulin cDNA (GenBank acces-
sion number AB196794) was performed by the 5¢-RACE
method. Messenger RNA was isolated from total RNA with
the PolyATtract mRNA Isolation System (Promega

TACACCCTC-3¢ for calreticulin. The amplified DNA frag-
ment was subcloned into the ligation-independent cloning
site of the pET46Ek ⁄ LIC vector (EMD Biosciences, Inc.,
San Diego, CA). The recombinant proteins have the His-
tag linked to the N-terminus.
An expression plasmid encoding His-tagged mature b-con-
glycinin a¢ without the signal peptide (Met1–Lys30) or
prosequence (Gln31–Lys62) was constructed as follows.
b-Conglycinin a¢ cDNA (GenBank accession number
BAB64303.1) was amplified from total RNA by RT-PCR
using the following oligonucleotide primers: 5¢-TCCT
AGGTACCCGTATTAAGAATTTAAGATATACT-3¢ and
5¢-ATTACGTCGACCATTTAGTACTACATACTTATTC
AGTAAAAAGC-3¢. The amplified b-conglycinin a¢ DNA
fragment was digested with NdeI and XhoI and subcloned
into the pET31b(+) vector (EMD Biosciences, Inc.), which
had been cleaved with NdeI and XhoI. The recombinant pro-
teins have the His-tag linked to the C-terminus. The inserts
in the expression plasmid vectors were sequenced as des-
cribed above.
Expression and purification of recombinant
GmPDIS-1, GmPDIS-2, BiP, calreticulin and
b-conglycinin a¢
BL21(DE3) cells were transformed with the expression vec-
tors as described above. The expression of recombinant
Soybean protein disulfide isomerase family H. Wadahama et al.
698 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
proteins was induced by the addition of 0.4 mm isopropyl
thiogalactoside at 30 °C for 4 h. All recombinant proteins
expressed were soluble. The cells from 1 L of culture broth

)1
was used for recombinant GmP-
DIS-2.
Oxidative refolding assay with reduced RNaseA
PDI activity was assayed by the measurement of RNase
activity produced through the regeneration of the active
form from reduced RNaseA. Reduced RNaseA was
prepared as described previously by Creighton [56]. Each
reaction mixture comprised 200 mm [4-(2-hydroxyethyl)-
1-piperazinyl]ethanesulfonic acid (pH 7.5), 150 mm NaCl,
2mm CaCl
2
, 0.5 mm glutathione disulfide, 2 m m glutathi-
one, 1 mgÆmL
)1
reduced RNaseA, and 0.25 mgÆmL
)1
recombinant GmPDIS-1 or GmPDIS-2. The reaction mix-
ture was incubated at 25 °C. An aliquot (16 lL) of the
reaction mixture was removed, and RNaseA activity was
measured spectrophotometrically at 284 nm with cCMP as
the substrate [57]. Reactivation of reduced RNaseA in the
absence of recombinant protein was subtracted from reacti-
vation in the presence of either GmPDIS-1 or GmPDIS-2.
Limited proteolysis of GmPDIS-1 and GmPDIS-2
Purified recombinant GmPDIS-1 and GmPDIS-2 (50 lg)
were digested with either trypsin (1 lg) (Sigma-Aldrich
Inc.) in 100 mm Tris ⁄ HCl buffer (pH 8.0) at 25 °C for
60 min or V8 protease (2 lg) (Sigma-Aldrich Inc.) in
100 mm Tris ⁄ HCl buffer (pH 8.0) at 25 °C for 30 min

been mixed with a 200-fold volume of 50 mm gly-
cine ⁄ NaOH (pH 8.5) containing 5 lm thioflavin T (Wako
Pure Chemical Ind., Ltd, Osaka, Japan).
Antibodies
Antibodies were prepared by Operon Biotechnologies, K.K.
(Tokyo, Japan). Purified recombinant GmPDIS-1, GmP-
DIS-2, calreticulin and b-conglycinin a¢, and the soybean
glycinin acidic subunits (a gift from Fuji Oil Co., Osaka,
Japan), were emulsified with Freund’s complete adjuvant
and injected intradermally into a male rabbit. For prepar-
ation of antibody specific to the prosequence of b-conglyci-
nin a¢, a fragment of the propeptide (SEKDSYRNQAC)
was synthesized and injected into a male rabbit. For pre-
paration of antibody specific to BiP, purified recombinant
BiP was injected intradermally into a female guinea pig.
Western blotting analysis
Soybean trifoliolate center leaves, roots, flowers, stems and
cotyledons that had been frozen under liquid nitrogen were
ground into fine powders with a micropestle SK-100. Pro-
teins were extracted from 100 mg of tissue by boiling for
5 min in 200 lL of SDS ⁄ PAGE buffer [58] containing a
1% cocktail of protease inhibitors (Sigma-Aldrich Inc.).
The concentrations of proteins were measured with a pro-
tein assay kit (RC DC protein assay; Bio-Rad Laborator-
ies). Proteins were subjected to SDS ⁄ PAGE and blotted
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 699
onto a polyvinylidene difluoride membrane. The GmPDIS-1,
GmPDIS1-2, BiP, calreticulin, glycinin acidic subunit,
b-conglycinin a¢ and pro-b-conglycinin a¢ proteins were

into 3 · 3 · 1 mm cubes. The pieces of tissue were fixed
with 2% formaldehyde and 0.1% glutaraldehyde for 2 h at
room temperature. The fixed cotyledons were dehydrated
with a series of 50%, 50%, 70%, 70%, 80%, 90%, 95%
and 99.5% ethanol, 40 min each, at room temperature. The
dehydrated cotyledons were embedded in Historesin (Leica
Microsystems, Heidelberg, Germany) and sliced into sec-
tions. The sections of fixed cotyledon were mounted on a
glass slide and stained with primary antibodies, rabbit anti-
GmPDIS-1, anti-GmPDIS-2, anti-(glycinin acidic subunits),
or anti-b-conglycinin a¢ sera, and secondary antibodies,
goat anti-(rabbit IgG) serum (Cortex Biochem, San Lean-
dro, CA); this was followed by incubation with Cy5–strept-
avidin (GE Healthcare Bio-Sciences Corp., Piscataway,
NJ). For detection of BiP, specimens were stained with gui-
nea pig anti-BiP serum, and then with a Cy3-conjugated
goat anti-(guinea pig IgG) serum (Chemicon International,
Temecula, CA). The specimens were examined on an
MRC-1024 laser scanning confocal imaging system (Bio-
Rad Laboratories).
Proteinase K treatment of microsomes
Slices of cotyledons were homogenized by 20 strokes of a
Dounce homogenizer (Wheaton Science Products, Millville,
NJ) at 4 °Cin20mm sodium pyrophosphate buffer
(pH 7.5), containing 0.3 m mannitol (buffer A). The homo-
genate was placed into a cell strainer (BD Biosciences, San
Jose, CA) and centrifuged at 824 g for 40 min at 4 °Conan
RA200J rotor by a Kubota 1710. The filtered suspension
was centrifuged at 2770 g for 20 min at 4 °C on an RA200J
rotor by a Kubota 1710. The supernatant was centrifuged at

DSP. The homogenate
was placed on ice for 2 h. The crosslinking reaction was ter-
minated by the addition of 2 mm glycine for 30 min on ice.
The microsomes were prepared as described above.
Immunoprecipitation
The microsomal pellet was resuspended in 50 mm Tris ⁄ HCl
buffer (pH 7.5) containing 150 mm NaCl and 2% SDS, and
incubated at 37 °C for 2 h. The suspension was centrifuged
at 8000 g at 4 °C for 30 min on an RA50J rotor by a
Kubota 1710. The supernatant was diluted 50-fold with
50 mm Tris ⁄ HCl buffer (pH 7.5) containing 150 mm NaCl,
and precleared by the addition of 20 lL of protein A-conju-
gated Sepharose beads (50% slurry) (Sigma-Aldrich Inc.).
Immunoprecipitation was first carried out at 4 °C for 16 h
Soybean protein disulfide isomerase family H. Wadahama et al.
700 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
with nonimmmune serum, affinity-purified anti-GmPDIS-1,
anti-GmPDIS-2 or anti-(glycinin acidic subunit) sera. Anti-
gen–antibody complexes were absorbed onto protein
A-conjugated Sepharose beads at 4 °C for 1 h, and the
beads were washed five times with NaCl ⁄ P
i
containing
0.05% Tween-20. For the second immunoprecipitation, the
first immunoprecipitants were dissolved from the beads into
a 2% SDS ⁄ 0.4 m dithiothreitol solution at 100 °C for
5 min and diluted 50-fold with 20 mm borate buffer (pH 8)
containing 150 mm NaCl. The second immunoprecipitation
was carried out with anti-(glycinin acidic subunit) serum at
4 °C for 16 h. The antigen–antibody complexes were recov-

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