Báo cáo khoa học: Protein disulfide isomerase family proteins involved in soybean protein biogenesis docx

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

[6–8]. The genes of these PDI families are unfolded pro-
tein response (UPR) genes, which are induced by the
accumulation of unfolded proteins in the ER [9].
Keywords
endoplasmic reticulum; protein disulfide
isomerase; soybean; storage protein;
unfolded protein response
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:
Database
The nucleotide sequence data for GmPDIS-1,
GmPDIS-2, BiP, calreticulin and
b-conglycinin a¢ are available in the
DDBJ ⁄ EMBL ⁄ GenBank databases under
accession numbers AB182630, AB182631,
AB210900, AB196794 and AB113351
*These authors contributed equally to this
work
(Received 23 July 2006, revised 5 October
2006, accepted 22 November 2006)
doi:10.1111/j.1742-4658.2006.05613.x
Protein disulfide isomerase family proteins are known to play important
roles in the folding of nascent polypeptides and the formation of disulfide
bonds in the endoplasmic reticulum. In this study, we cloned two similar
protein disulfide isomerase family genes from soybean leaf (Glycine max L.
Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers

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

group IV are the smallest molecules (approximately
360 amino acids) in groups I–V of these proteins. The
amino acid sequences of the group IV proteins, other
than the sequence of the thioredoxin domain, were dif-
ferent from those of groups I–III and group V. Group
IV proteins lack a KDEL-like ER retrieval signal. The
genes of group IV have been identified only in plant
and Dictyostelium genomes. Previously, it was shown
that mRNA of the Arabidopsis group IV ortholog was
induced by ER stress [11,12]. This finding implies that
group IV proteins play an important role in quality
control of proteins in the ER; however, their cellular
localization, function and physiologic roles remain
unclear.
In this study, we report the isolation of cDNA
clones that encode two soybean group IV PDI ortho-
logs, GmPDIS-1 and GmPDIS-2. The identification of
their domain structures, tissue distribution, cellular
localization and changes in expression during soybean
seed embryogenesis are described. In addition, we pro-
vide evidence to suggest an association between
GmPDIS-1 and proglycinin, a seed storage protein, in
the course of the folding process.
Results
cDNA cloning and expression of GmPDIS-1 and
GmPDIS-2
To clone the soybean ortholog of Arabidopsis PDI-
like2-1 categorized in group IV [10], a blast search
was performed using the nucleotide sequence of PDI-
like2-1 cDNA from The Institute for Genomic

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-

teins were expressed in Escherichia coli and purified
(Fig. 2A,B). Both recombinant proteins were soluble
and eluted in a monomeric form from a gel-filtration
column (data not shown). To determine whether both
recombinant proteins were folded, far-UV CD was
performed. Both GmPDIS-1 and GmPDIS-2 yielded
CD spectra typical of a folded protein (data not
shown). The activity of recombinant GmPDIS-1 and
GmPDIS-2 (i.e. the catalysis of oxidative refolding of
the reduced, denatured RNaseA) was measured. The
specific activities of GmPDIS-1 and GmPDIS-2 were
66 and 43 mmol RNaseAÆmin
)1
Æmol
)1
, respectively
(Fig. 2C). The specific activity of bovine PDI was
431 mmol RNaseAÆmin
)1
Æmol
)1
.
The domain structures of GmPDIS-1 and Gm-
PDIS-2 were predicted to be a linear sequence of three
domains in an a–a¢–ERp29 C-terminal-like domain
(ERp29c) from the region of sequence homology to
the conserved domains. Hence, we subjected the
recombinant GmPDIS-1 and GmPDIS-2 proteins to
limited proteolysis with either trypsin or V8 protease
to determine their domain boundaries. After proteoly-

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.

The intensity of the fluorescence increased almost lin-
early over 60 min. The seed-dependent aggregation of
Ab(1–40) monomers was inhibited by b oth GmPDIS-1
and GmPDIS-2 in a concentration-dependent manner
(Fig. 4). In the presence of 2 lm GmPDIS-1 or
0.5 lm GmPDIS-2 (molar ratio of 1 : 25 or 1 : 100 to
Ab), almost all Ab aggregation was inhibited for at
least 40 min.
Tissue distribution and cellular localization of
GmPDIS-1 and GmPDIS-2
We prepared antibodies against recombinant GmPDIS-1
and GmPDIS-2. Anti-GmPDIS-1 serum specifically
Fig. 1. Multiple amino acid sequence alignment of GmPDIS-1, GmPDIS-2, Arabidopsis PDI-like2-1 (AtPDIL2-1), and alfalfa G1 (MsG1) [64]. A
multiple alignment of the polypeptides was generated using
CLUSTAL W. Numbers refer to the amino acid number, asterisks indicate amino
acid matches, and dashes represent gaps between the sequences. The putative signal sequence (underlined), active site CGHC motifs (sha-
ded in black), conserved arginine (shaded in gray) and conserved glutamic acid (boxes) are indicated.
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 689
immunoreacted to recombinant GmPDIS-1, but not
to recombinant GmPDIS-2, whereas anti-GmPDIS-2
serum immunoreacted strongly to recombinant GmPDIS-2
and weakly to recombinant GmPDIS-1 (Fig. 5A, lanes
1–4). Immunoglobulin molecules that immunoreact to
GmPDIS-1 in anti-GmPDIS-2 serum were eliminated
by pretreatment of the serum with purified recombinant
GmPDIS-1 (Fig. 5A, lanes 5 and 6). Hence, we used
pretreated anti-GmPDIS-2 serum for the experiments
described below. Anti-GmPDIS-1 serum or anti-Gm-
PDIS-2 serum was immunoreacted with a single 40 kDa

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

C
Fig. 2. Activity of the recombinant GmPDIS-1 and GmPDIS-2. The
recombinant GmPDIS-1 (A) and GmPDIS-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 separated by 10% SDS ⁄ PAGE and stained with Coomassie
Blue. (C) PDI activity of the recombinant GmPDIS-1 (left bar) and
GmPDIS-2 (right bar). The activity was assayed by the measure-
ment of RNase activity produced through the regeneration of the
active form from reduced RNaseA. Each value represents the mean
of six (GmPDIS-1) or eight experiments (GmPDIS-2).
A
B
Fig. 3. Schematic representation of cleavage sites in GmPDIS-1 (A) and GmPDIS-2 (B) by limited proteolysis. The upper line represents
recombinant protein. The boxes below indicate the domain boundaries predicted by an NCBI conserved domain search. The arrows indicate
the determined cleavage sites. Black boxes in domain a and a¢ represent the CGHC motif.
Soybean protein disulfide isomerase family H. Wadahama et al.
690 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
X-100. Both GmPDIS-1 and GmPDIS-2 were resistant
to protease treatment in the absence of detergent.
Because of disruption of microsome membranes in the
presence of Triton X-100, GmPDIS-1 and GmPDIS-2
were degraded by the protease treatment (Fig. 8). Sim-
ilar phenomena were observed in the case of BiP and
calreticulin, which are well known as luminal proteins of
the ER. In the cotyledon from the 220 mg bean that
heavily accumulated seed storage proteins in its PSVs,
BiP was visualized in both the PSV and the ER
(Fig. 7E,F). Images of GmPDIS-1 and GmPDIS-2 over-
lapped with those of BiP (Fig. 7G,H).

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

chain and associate as trimers in the ER [28,29]. These
trimers move to the PSV, where a processing enzyme
cleaves them into acidic and basic polypeptide chains
[26,30]. Nascent proteins in the isolated cotyledons
Fig. 4. Inhibition of Ab aggregation by
GmPDIS-1 and GmPDIS-2. Seed-dependent
aggregation of 50 l
M Ab(1–40) in the pres-
ence of GmPDIS-1 (A) or GmPDIS-2 (B)
(closed circle, 0 l
M; closed square, 0.2 lM;
open circle, 0.5 l
M; closed triangle, 1.0 lM;
open triangle, 2.0 l
M). Each value repre-
sents the mean of two experiments.
AB
Fig. 5. Expression of GmPDIS-1 and GmPDIS-2 in soybean tissues. (A) Crossreactivity of the antibody prepared against recombinant
GmPDIS-1 or GmPDIS-2 with recombinant GmPDIS-1 (2.4 lg) (lanes 1, 3 and 5) and GmPDIS-2 (2.4 lg) (lanes 2, 4 and 6). Anti-GmPDIS-2*
represents anti-GmPDIS-2 serum (1 lL) treated with purified recombinant GmPDIS-1 (5 lg) to eliminate the antibodies that crossreact with
GmPDIS-1. (B) Detection of GmPDIS-1 and GmPDIS-2 in soybean tissues. Thirty-microgram samples of protein extracted from the cotyledon
(80 mg bean), root, stem, 3 cm length leaf (Leaf-3), 6 cm leaf (Leaf-6), 10 cm leaf (Leaf-10) and flower were separated by 10% SDS ⁄ PAGE
and immunostained with anti-GmPDIS-1 serum (lanes 1–7) or anti-GmPDIS-2 (lanes 8–14) serum pretreated with recombinant GmPDIS-1 as
described under (A).
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 691
were metabolically pulse-labeled with [
35
S]methionine
and [

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 immunoprecipitant with anti-GmPDIS-1 serum
(Fig. 9E, lane 4). These results suggest that GmPDIS-1
molecules associate with proglycinin in the lumen of
the ER. As coimmunoprecipitation of proglycinin
with GmPDIS-1 was dependent on DSP treatment,
GmPDIS-1 may noncovalently associate with proglyci-
nin in the ER. On the other hand, slight coimmuno-
precipitation of proglycinin with GmPDIS-2 was
detected by DSP treatment (Fig. 9E, lane 6).
Changes in the levels of GmPDIS-1 and GmPDIS-2
during seed development
Very large amounts of seed storage proteins are syn-
thesized and translocated to the ER during the matur-
ation stage of embryogenesis. Under such conditions,
the folding machinery, composed of molecular chaper-
ones and foldases, may be strengthened for folding of
de novo synthesized seed storage proteins. Therefore,
we determined the relationships between changes in
expression levels of both GmPDIS-1 and GmPDIS-2
and the synthesis of storage proteins during develop-
ment of soybeans by western blotting. In addition, the
expression levels of both BiP, a universal ER chaper-
one [31], and calreticulin, known as a chaperone for
glycoprotein folding [32,33], were determined. Pro-
b-conglycinin, possessing an N-terminal prosequence,
and proglycinin are transient protein forms that are
present in the ER prior to processing in the PSV
[28,29]. Hence, the amounts of pro-b-conglycinin and
proglycinin are considered to be nearly equivalent to
the synthesis levels of both b-conglycinin and glycinin.

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.

692 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
levels of calreticulin (Fig. 10D) and its mRNA
(Fig. 11D) were slightly higher in the cotyledons of
70 mg seeds, in which the synthesis of the pro-b-con-
glycinin glycoprotein had reached the most advanced
stage (Fig. 10E).
GmPDIS-1, but not GmPDIS-2, is induced
by ER stress
Many ER-resident proteins are upregulated by the
accumulation of unfolded protein in the ER (i.e. ER
A
B
C
D
E
F
G
H
Fig. 7. Localization of GmPDIS-1 and GmP-
DIS-2 in soybean cotyledons. Cotyledons at
early (80 mg bean, A–D) or late (220 mg
bean, E–H) stages of seed development
were immunostained with a combination of
anti-b-conglycinin a¢ serum (green) and anti-
BiP serum (red) (A, E), anti-(glycinin acidic
subunit) serum (green) and anti-BiP serum
(red) (B, F), anti-GmPDIS-1 serum (green)
and anti-BiP serum (red) (C, G), or anti-
GmPDIS-2 serum (green) and anti-BiP
serum (red) (D, H). Visible light images

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.

Discussion
In this study, we cloned and characterized the cDNAs
of GmPDIS-1 and GmPDIS-2 as members of the PDI
family. The amino acid sequences of GmPDIS-1 and
GmPDIS-2 were similar to each other. Both had two
tandem thioredoxin-like domains, a and a¢. Their thio-
redoxin-like domain organization occurred in tandem
at the N-terminus and was the same as that of mam-
malian P5 [37]. However, the C-terminal regions of
both GmPDIS-1 and GmPDIS-2 had no sequence
similarity to P5. Their C-terminal domains were similar
to the C-terminal domain of mammalian ERp29
[16,17]. Recombinant GmPDIS-1 and GmPDIS-2
showed molecular chaperone-like activity that inhibited
the aggregation of Ab(1–40). We detected oxidative
refolding of the unfolded RNaseA by both recombin-
ant GmPDIS-1 and GmPDIS-2. This refolding corres-
ponded to 15% and 10% of bovine PDI activity,
respectively. The amino acid sequences of the two thio-
redoxin domains of GmPDIS-1 and GmPDIS-2 were
similar to those of the other PDI family proteins that
exhibited an oxidative refolding activity for RNase
[38]. In addition, a group of amino acids that are
essential for oxidative refolding activity was con-
served between GmPDIS-1 and GmPDIS-2. Therefore,
it seems possible that the low activities of both
GmPDIS-1 and GmPDIS-2 may be due to their low
affinity for unfolded RNaseA.
Most of the identified PDI family proteins are resi-
dents of the ER. GmPDIS-1 and GmPDIS-2 lack the

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

with proreinase K (lanes 3 and 4) in the absence (lane 3) or pres-
ence (lane 4) of Triton X-100. Proteins (10 lg) were separated by
10% SDS ⁄ PAGE, blotted on a poly(vinylidene difluoride) membrane
and immunostained with specific antibodies against GmPDIS-1,
GmPDIS-2, BiP and calreticulin, respectively. The asterisk indicates
a band of degraded calreticulin.
Soybean protein disulfide isomerase family H. Wadahama et al.
694 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
was located in the vacuoles had high-mannose glycans
but not Golgi-processed complex glycans. In soybean
cotyledon, two routes were identified for transporting
proglycinin from the ER directly to the PSV and from
the ER to the PSV via the Golgi [42,43]. It remains
unclear, however, how GmPDIS-1, GmPDIS-2 and
BiP are transported from the ER to the PSV and what
role these proteins play in the PSV.
In this study, the association of GmPDIS-1 with pro-
glycinin molecules was demonstrated. The proglycinin
protomer has two disulfide bonds, an intrachain bond
and an interchain bond between the acidic and basic
subunits [27]. The importance of the disulfide bonds
has been demonstrated for the assembly of glycinins
[44]. The structural stability of the mature glycinin
molecule required both disulfide bridges to be intact
[45]. In general, PDI family proteins catalyze the for-
mation of disulfide bonds on nascent polypeptide
chains in the ER. Hence, GmPDIS-1 may support
proglycinin folding that accompanies the formation of
AB C
ED

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-

mation of such complexes is thought to facilitate the
attachment of the PDI family to substrates, resulting in
an increase in the folding rate of the substrate [7,49].
GmPDIS-1 may form a complex with other ER chaper-
one proteins in the ER.
It was suggested that the expression of both Gm-
PDIS-1 and GmPDIS-2 was differentially regulated in
the cotyledons during seed development. The level of
GmPDIS-1 was dramatically increased in the late sta-
ges of seed maturation, in contrast to GmPDIS-2,
which was present at a low level during the same
stage. These results may suggest the importance of
GmPDIS-1 in the folding of proglycinin. The regula-
tion of GmPDIS-1 levels during this stage was a post-
transcriptional event rather than a transcriptional one.
It is unknown how the levels of GmPDIS-1 are con-
trolled.
The transcriptional responses of the GmPDIS-1 and
GmPDIS-2 genes to ER stress were also different.
Thus, GmPDIS-1 mRNA, but not GmPDIS-2 mRNA,
was induced by treatment with tunicamycin, dithio-
threitol, and AZC. Only one ortholog to GmPDISs
[PDI-like2-1 (At2g47470)], a UPR gene [11,12], is
present on nonduplicated region of chromosome 1 in
Arabidopsis [10], and the amino acid sequence identi-
ties between PDI-like2-1 and GmPDIS-1 or GmPDIS-2
were 75% and 70%, respectively. Hence, it is pre-
sumed that GmPDIS-1 is the soybean ortholog of
PDI-like2-1 and that GmPDIS-2 may be a paralogous
gene generated from GmPDIS-1 by a gene duplication

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

E
F
G
H
Fig. 10. Expression of GmPDIS-1 and GmP-
DIS-2 in soybean cotyledons during matur-
ation. Thirty micrograms (A–E) or 5 lg (F–H)
of proteins extracted from cotyledons were
separated by 10% SDS ⁄ PAGE and immuno-
stained with specific antibodies against
GmPDIS-1 (A), GmPDIS-2 (B), BiP (C), cal-
reticulin (D), the prosequence of b-conglyci-
nin a¢ (E), b-conglycinin a¢ (F), and glycinin
acidic subunits, respectively. Proglycinin (G)
and mature glycinin acidic subunits (H) were
the 53 and 35 kDa bands on the same blot.
Soybean protein disulfide isomerase family H. Wadahama et al.
696 FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS
than differences in the ability to sense ER stress or
transcription capacity in the cotyledons.
A large quantity of soybean storage proteins is syn-
thesized in the cotyledon cells during seed maturation
[54]. This means that abundant nascent unfolded pro-
teins are translocated to the lumen of the ER. A rapid
increase in the workload of the ER, as a result of the
synthesis of these storage proteins, may elicit an ER
stress. However, it seems unlikely that ER stress arises
in the normal maturation process of soybean seeds, as
no notable increases in the mRNAs of UPR genes,
such as those encoding BiP, calreticulin, and GmP-

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

)1
tunicamycin (TM) for
24 h, 1 m
M dithiothreitol (DTT) for 5 h, or 50 mM AZC for 18 h. The
mRNAs of GmPDIS-1 (A), GmPDIS-2 (B), BiP (C) or calreticulin (D)
were quantified by real time RT-PCR. Each value was standardized
by dividing by the value of actin mRNA. Fold-expression change
was calculated as the ratio of mRNA in the samples treated with
the stress reagent to that in the untreated sample.
H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 697
Experimental procedures
Plants
Soybean (Glycine max L. Merrill cv. Jack) seeds were
planted in 5 L pots and grown in a controlled environmen-
tal chamber at 25 °C under 16 h day ⁄ 8 h night cycles.
Roots were collected from plants 10 days after seeding.
Flowers, leaves and stems were collected from plants
45 days after seeding. All samples were immediately frozen
and stored in liquid nitrogen until use.
For treatments of cotyledons under ER-stress conditions,
a cotyledon was divided into two halves. Two hundred
microliters of 1% dimethylsulfoxide, with or without
250 lgÆmL
)1
tunicamycin, or distilled water, with or without
1mm dithiothreitol, or 50 mm AZC (Sigma-Aldrich Inc.,
St Louis, MO), were administered to the inner surface of
the divided half of the cotyledon and incubated at 25 °C.
cDNA cloninig of GmPDIS-1, GmPDIS-2, BiP,

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

Corporation, Madison, WI). 5¢-RACE was performed with
the SMART RACE cDNA Amplification kit (Clontech
Laboratories, Inc., Mountain View, CA) according to the
manufacturer’s protocol, using the oligonucleotide primer
5¢-GCAGTTTTCCACGGTCTGCTTATCTAGGG-3¢,which
was designed from the nucleotide expressed sequence tag
contig sequence TC204381 from The Institute for Genomic
Research So ybean I ndex ( />T_index.cgi?species¼soybean). The amplified DNA fragment
was subcloned into the pT7Blue vector. The inserts in the plas-
mid vectors were seque nced using the fluores cence dideoxy
chain termination method and an ABI PRISM 3100-Avant
Genetic Analyzer (Applied Biosyst ems, Foster City, CA).
Construction of His-tagged expression plasmids
Expression plasmids encoding His-tagged mature GmPDIS-
1, GmPDIS-2, BiPA [21,22] or calreticulin without the
putative signal peptide were constructed as follows. The
DNA fragment was amplified from cDNAs of GmPDIS-1,
GmPDIS-2, BiP and calreticulin by PCR using the oligo-
nucleotide primers 5¢-GACGACGACAAGATGGACGA
CGTCGTTGTGCTCTCTG-3¢ and 5¢-GAGGAGAAGC
CCGGTTCAAGCCGCATATGTCGACAAGATAT-3 ¢ for
GmPDIS-1, 5¢-GACGACGACAAGATGGACGACGTCG
TTGCACTCACAGAG-3¢ and 5¢-GAGGAGAAGCCCGGT
TCAAGCAAAGATAGATAAGATGTTC-3¢ for GmPDIS-2,
5¢-GACGACGACAAGATGAAGGAGG AAGCCACCAA
GTTGGGG-3¢ and 5¢-GAGGAGAAGCCCGGTCTAGA
GCTCGTCATGAGAATC-3¢ for BiP, and 5¢-GACGACG
ACAAGATGGAAGAGCGTTTCGATGACGGGTGGG-3¢
and 5¢-GAGGAGAAGCCCGGTCTAGAGTTCATCATG
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
were collected by centrifugation, disrupted by sonication in

8mL of 20mm Tris ⁄ HCl buffer (pH 8), containing 5 mm
imidazole and 0.5 m NaCl (binding buffer), and then centri-
fuged at 10 000 g for 30 min at 4 °C on an RA-200J rotor by
a Kubota 1710 (Kubota, Tokyo, Japan). The supernatant
was applied to a column packed with His-Bind resin (EMD
Biosciences, Inc.). After washing of the column with binding
buffer containing 60 mm imidazole, recombinant proteins
were eluted with binding buffer containing 1 m imidazole,
concentrated with a Centriplus YM-10 (Millipore, Billerica,
MA), and then subjected to gel filtration chromatography on
a TSK gel G3000SW column (Tosoh, Tokyo, Japan) equili-
brated with 20 mm Tris ⁄ HCl buffer (pH 7.4) containing
0.15 m NaCl and 10% glycerol. Recombinant proteins
that eluted in the inside volume fractions were collected.
The amino acid sequence was confirmed by N-terminal
sequencing using a Procise Protein Sequencer 492 (Applied
Biosystems). The concentrations of purified recombinant
GmPDIS-1 and GmPDIS-2 were determined from their
absorbances at 280 nm using the molar extinction coefficients
that were calculated by the modified method of Gill & von
Hippel [55]. An extinction coefficient of 37 360 m
)1
Æcm
)1
was
used for recombinant GmPDIS-1, and an extinction coeffi-
cient of 38 850 m
)1
Æcm
)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
(GmPDIS-1) or 120 min (GmPDIS-2). The peptides pro-

duced were separated by SDS ⁄ PAGE (15% gel) [58],
transferred to a polyvinylidene difluoride membrane (Bio-Rad
Laboratories, Hercules, CA), and stained with Ponceau S.
N-terminal amino acid sequencing of each peptide was car-
ried out. Mass values of the peptides produced by limited
proteolysis were determined by MALDI-TOF MS. The
digestion solution was diluted with nine volumes of 20 mm
Tris ⁄ HCl buffer (pH 7.4) containing 0.15 m NaCl and 10%
glycerol, concentrated to the original volume in a Microcon
YM-10 (Millipore), and subjected to AXIMA-CFR
MALDI-TOF MS plus (Shimadzu Biotech, Kyoto, Japan).
Analysis of aggregation of Ab
The aggregation of Ab(1–40) in vitro was determined [59–
62]. Ab(1–40) (Peptide Institute, Osaka, Japan) was dis-
solved in 0.02% w ⁄ w ammonia solution at a concentration
of 500 lm.AnAb fibril seed was prepared by the incuba-
tion of 50 lm Ab peptide in NaCl ⁄ P
i
for 7 days at 37 °C.
Ab peptide (50 lm) and 10 lgÆmL
)1
seed were incubated at
37 °C in the absence or presence of GmPDIS-1 or Gm-
PDIS-2 in NaCl ⁄ P
i
.Ab aggregations were monitored in an
F-3000 fluorescence spectrophotometer (Hitachi Ltd,
Tokyo, Japan) at excitation and emission wavelengths of
446 nm and 490 nm, respectively, after the components had
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
then immunostained with specific antibodies and horserad-

ish peroxidase-conjugated IgG antibody (Promega Corpora-
tion), using the Western Lightning Chemiluminescence
Reagent (Perkin Elmer Life Sciences, Boston, MA).
Real time RT-PCR analysis
Total RNA was isolated from plant tissues using an
RNeasy Plant Mini kit. Relative quantification of mRNA
was carried out by real-time RT-PCR and an ABI PRISM
7000 Sequence Detection System (Applied Biosystems).
Forward primers 5¢-CACGCAAACAAGTGATTCAGC
AGTGCG[FAM]G-3¢,5¢-CAGCCGAAACATCATCAGA
GCAATGGC[FAM]G-3¢,5¢-CGGATTTCCGCTCACTCT
TTATC[FAM]G-3¢ and 5¢-GACCTCCCATAACACCAG
TATGTCGAGG[JOE]C-3¢ (Invitrogen Corporation, Carls-
bad, CA) were used for detection of the mRNAs of GmP-
DIS-1, GmPDIS-2, calreticulin, and actin 3 (GenBank
accession number V00450), respectively.
Reverse primers 5¢-TGCAGCAAGTATGGAGTCTCT
GG-3¢,5¢-AGGCGAGGATCGTTCACATAA-3¢,5¢-GAC
GGAAGCTGGAGAAGACACC-3¢ and 5¢-AGGCAGGA
TTTGCTGGTGAC-3¢ (Invitrogen Corporation) were used
for detection of the mRNAs of GmPDIS-1, GmPDIS-2,
calreticulin, and actin 3, respectively.
A forward primer, 5¢-GCACAAGGATTCCAAAGGTA
CAA-3¢, a reverse primer, 5¢-CACCCTCTCCACTCAA
AATGCT-3¢, and a TaqMan probe, 5¢-FAM-TGCACT
GCAGCACCATAGGCAACTG-TAMURA-3¢ (Applied
Biosystems), were used for detection of BiP mRNA.
Laser scanning immunofluorescence microscopy
Cotyledon tissues from developing soybean seeds were cut
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
100 000 g for 1 h at 4 °C on an RP10AT4 rotor by a CS120

(Hitachi Koki, Tokyo, Japan) to separate cytosol and micro-
somes. The obtained microsomal pellet was resuspended in
buffer A. The suspension was treated with 0.5 mgÆmL
)1
pro-
teinase K in the presence or absence of 1% Triton X-100 for
5 min at 4 °C. Proteins in the samples treated without Tri-
ton X-100 were precipitated with 10% trichloroacetic acid
for 30 min at 4 °C. Proteins in the cytosol and microsomes
were analyzed by western blotting as described above.
Labeling of cotyledons
Six pairs of cotyledons (150 mg weight) were isolated, divi-
ded into two halves and labeled flat side up in a Petri
dish at 25 ° C for 6 h with 1.48 MBq ⁄ 4 lL Pro-mix L-[
35
S]
in vitro labeling mix (37 TBqÆmmol
)1
) (GE Healthcare Bio-
Sciences Corp.) and 6 lL of FN Lite [63]. In the case of
pulse-chase experiments, cotyledons were labeled at 25 °C
for 15 min. The cotyledons were rinsed with FN Lite con-
taining 10 m m cold methionine and cysteine three times and
incubated at 25 °C for 1–6 h. The radiolabeled cotyledons
were rinsed at 4 °C in buffer A, and slices from the flat side
of each cotyledon were cut. The slices were homogenized by
20 strokes of a Dounce homogenizer at 4 °C in 3 mL of buf-
fer A with or without 1 mgÆmL
)1
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-
ered as described above and dissolved in SDS ⁄ PAGE buffer

[58] at 100 °C for 5 min. The proteins were separated on a
10% SDS-polyacrylamide gel. Radiolabeled proteins were
detected by fluorography with ENLIGHTNING (Perkin
Elmer Life Sciences).
Acknowledgements
We thank M. Kito, Emeritus Professor of Kyoto
University, for the critical reading of the manuscript,
valuable advice, and warm encouragement. We thank
T. Kanekiyo, Department of Pediatrics, Osaka
University Graduate School of Medicine, for excellent
support with the analysis of molecular chaperone
activity. This study was supported by a Grant from
the Program for Promotion of Basic Research
Activities for Innovative Biosciences.
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H. Wadahama et al. Soybean protein disulfide isomerase family
FEBS Journal 274 (2007) 687–703 ª 2006 The Authors Journal compilation ª 2006 FEBS 703