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 Bios
Trang 1homologous to animal P5 – molecular cloning and
characterization as a functional protein for folding of
soybean seed-storage proteins
Hiroyuki Wadahama1,*, Shinya Kamauchi1,*,†, Yumi Nakamoto2, Keito Nishizawa2,
Masao Ishimoto2, Teruo Kawada1and Reiko Urade1
1 Graduate School of Agriculture, Kyoto University, Uji, Japan
2 National Agricultural Research Center for Hokkaido Region, Sapporo, Japan
Secretory, organellar and membrane proteins are
syn-thesized and folded with the assistance of molecular
chaperones and other folding factors in the
endoplas-mic reticulum (ER) In many cases, protein folding in
the ER is accompanied by N-glycosylation and the for-mation of disulfide bonds [1] Forfor-mation of disulfide bonds between correct pairs of cysteine residues in a nascent polypeptide chain is thought to be catalyzed
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: urade@kais.kyoto-u.ac.jp
†Present address
Osaka Bioscience Institute, Suita, Japan
Database
The nucleotide sequence data for the cDNA
of GmPDIM and genomic GmPDIM have
been submitted to the DDBJ ⁄ EMBL ⁄
Gen-Bank databases under accession numbers
AB189994 and AB295118, respectively
*These authors contributed equally to this
article
(Received 11 October 2007, revised 18
November 2007, accepted 20 November
2007)
doi:10.1111/j.1742-4658.2007.06199.x
The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER) In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L Mer-rill cv Jack) mRNA The cDNA encodes a protein called GmPDIM It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5 Recombinant GmPDIM expressed in Escheri-chia coli displayed an oxidative refolding activity on denatured RNase A The genomic sequence of GmPDIM was also cloned and sequenced Com-parison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon⁄ intron struc-ture Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon It localized to the lumen of the ER Data from co-immunopre-cipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin In addi-tion, GmPDIM associated with the a¢ subunit of b-conglycinin, a seed-storage protein in the presence of tunicamycin These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone
Abbreviations
DSP, dithiobis[succinimidylpropionate]; ER, endoplasmic reticulum; ERSE, ER stress responsive element; PDI, protein disulfide isomerase.
Trang 2by the protein disulfide isomerase (PDI) family of
pro-teins [2–4] In humans, 17 genes of the PDI family
have been identified [5] The physiological role of each
PDI protein and their interactions with each other and
with other ER-resident molecular chaperones have
been partially elucidated P5, an animal PDI family,
was first discovered in Chinese hamster [6] P5 has
both thiol-oxidoreductase activity and chaperone
activ-ity [7,8] In addition, roles for P5 other than the
fold-ing of nascent proteins have been reported in animal
cells Zebrafish P5 is involved in the production of
midline-derived signals required to establish left⁄ right
asymmetry [9] In human tumor cells, cell-surface
P5 was required for shedding of the soluble major
his-tocompatibility complex class I-related ligand, resulting
in the promotion of tumor immune evasion [10] In
plants, a set of 22 orthologs of known PDI-like
pro-teins was discovered using a genome-wide search of
Arabidopsis thaliana and these were separated into
10 phylogenetic groups [11] Among these groups,
group V genes show structural similarities to
ani-mal P5 However, group V gene products in plant cells
have not been identified
Large quantities of storage protein are synthesized
in the ER during seed development in soybean
cotyle-don cells [12] Approximately 70% of seed-storage
proteins are composed of the two major globulins
glycinin and b-conglycinin They are folded and
assembled into trimers in the ER, and then
trans-ported and deposited in the protein storage vacuoles
[13] Glycinin is synthesized as a 60 kDa precursor
polypeptide and is proteolytically processed into
40 kDa acidic and 20 kDa basic subunits in the
pro-tein storage vacuoles [14–16] A1aB1b, a major
glyci-nin, possesses two intradisulfide bonds between
Cys12–Cys45 and Cys88–Cys298 These disulfide
bonds are required for assembly into hexamers and
for the structural stability of the protein [17–19]
Thus, proper folding and disulfide bond formation is
important for the effective deposition of glycinin in
the vacuoles ER-resident PDI proteins may play a
central role in this folding process Previously, we
identified two novel PDI proteins belonging to
group IV, GmPDIS-1 and GmPDIS-2, and
demon-strated that GmPDIS-1 is associated with proglycinin
in the ER [20] However, involvement of the other
PDI proteins in the folding of storage proteins
remains unclear
In this study, we isolated cDNA clones and genomic
sequences encoding a soybean group V gene of the
PDI family The tissue distribution and cellular
locali-zation of GmPDIM and changes in its expression
dur-ing seed development are described In addition, our
data suggest that GmPDIM and proglycinin or b-con-glycinin associate during the course of the folding process
Results
cDNA cloning of GmPDIM
To clone the soybean ortholog of group V Arabidopsis PDI-like2-2 or PDI-like2-3 [11], a blast search was performed using the nucleotide sequences of these cDNAs from the Institute for Genomic Research Soybean Index The tentative consensus sequence BU926832 was obtained Using primer sets designed from this sequence, we cloned a cDNA from the RNA extracted from young soybean leaves by 3¢-RACE and 5¢-RACE This cDNA encoded GmPDIM, a protein
of 438 amino acids (supplementary Fig S1) containing
a putative N-terminal secretary signal sequence and a C-terminal tetrapeptide, KDEL, which acts as a signal for retention in the ER [21,22] GmPDIM possesses two tandem thioredoxin-like motifs, each containing a CGHC active site Arginine residues R126 and R255, which are involved in the regulation of the active site redox potential in human PDI [5,23], were conserved
In addition, glutamic acid residues E58 and E186, which have been suggested to facilitate ‘the escape’ of the cystein residue of the active site from a mixed disulfide bond with substrate [5,23], were also con-served The amino acid sequence of GmPDIM and orthologs from other plant species were 80% simi-lar, excluding the putative N-terminal signal peptide The amino acid sequence identity between GmPDIM and human P5 was 46%
Recombinant GmPDIM was expressed in Escherichia coli as a soluble protein and was purified by affinity-column and gel-filtration chromatography (supplemen-tary Fig S2A) Recombinant GmPDIM had a CD spectrum typical of a folded protein (supplementary Fig S3) The domain structure of GmPDIM was pre-dicted to be a linear sequence of three domains in an a–a¢–b from the sequence homology to the conserved domains Therefore, we subjected the recombinant GmPDIM protein to limited proteolysis with either trypsin or V8 protease to determine their domain boundaries The native recombinant protein was digested to give smaller peptide fragments after treat-ment with either protease The sites of proteolytic cleavage were determined to be Lys150 (K150) and R255 by N-terminal sequencing of the trypsin peptide fragments The N-terminal sequences of other frag-ments generated by protease digestion were AHHHHH and corresponded to the N-terminal histidine tag of
Trang 3the recombinant protein We next determined the
C-terminal amino acid residues of the peptide
frag-ments by measuring their masses by MALDI-TOF
MS Most cleavage sites resided in two narrow regions,
overlapping the putative boundary regions in
GmPDIM between a and a¢, and a¢ and b, respectively
(Fig 1) These results show that GmPDIM has a
lin-ear sequence of three domains in an a–a¢–b pattern
similar to animal P5 [5]
We next determined the activity of recombinant
GmPDIM, which catalyzed oxidative refolding of the
reduced and denatured RNase A The specific activity
of GmPDIM was 45 mmol RNaseAÆmin)1Æmol)1
(sup-plementary Fig S2B) Despite the fact that human P5
has molecular chaperone activity [7], no such activity
was detected with recombinant GmPDIM, (data not
shown)
Cloning of genomic sequences of GmPDIM
The genomic sequence encoding GmPDIM was cloned
and sequenced Alignment and comparison with the
cDNA sequence showed that GmPDIM contains nine
exons (supplementary Fig S4) Nucleotide sequence of
the ORF of the GmPDIM gene was identical to that
of the cDNA Comparison of the soybean genomic
sequence of GmPDIM with those of A thaliana (AGI
number At1g04980 and At2g32920) and Oryza sativa
(MOsDb number Os09g27830) identified significant
conservation in the exon⁄ intron structure across these
species Moreover, all introns matched degenerate
con-sensus sequence of branch points of plants (YTNAN)
upstream of the 3¢ splice site [24]
We next analyzed the promoter region of GmPDIM,
2340 bp upstream of the translational initiation codon
ATG A search of the database of plant promoters
(PLACE: http://www.dna.affrc.go.jp/PLACE/) using
the sequences upstream of the coding region of
GmPDIMas the query detected an ER stress responsive
elements (ERSE; CCAAT-N9-CCACG) [25] and a
number of cis-acting regulatory elements involved in
the regulation of endosperm specific genes (Table 1)
Expression of GmPDIM in soybean tissues
We next prepared antiserum against recombinant GmPDIM Anti-GmPDIM serum immunoreacted to recombinant GmPDIM by western immunoblot (Fig 2A, lane 1), and also to two bands from cotyle-don cell extract of 50 and 52 kDa (Fig 2A, lane 2) The intensity of these bands decreased when anti-GmPDIM serum was pre-incubated with purified recombinant GmPDIM (Fig 2A, lanes 3–5), suggest-ing that the antibodies specifically immunoreacted with GmPDIM or a protein homologous to GmPDIM Further western immunoblot analyses indicated that GmPDIM is expressed ubiquitously in roots, stems, trifoliolate leaves, flowers and cotyledons (Fig 2B) The approximate quantity of this protein in leaves decreased during leaf expansion
Large amounts of seed-storage proteins are syn-thesized and are translocated to the ER during the maturation stage of embryogenesis Previously, we demonstrated that the synthesis of glycinin was initi-ated when the seeds achieved a mass of 50 mg and increased gradually until they grew to 300 mg We also demonstrated that the synthesis of b-conglycinin was initiated when the seeds achieved a mass of 40 mg, increased until the seeds grew to 70 mg, and then decreased [20] Under such conditions, the folding machinery comprised of molecular chaperones and other functional proteins must be strengthened in response to the increased de novo synthesis of seed-storage proteins Therefore, we next measured the mRNA and protein levels of GmPDIM using real-time RT-PCR or western immunoblot, respectively The rel-ative level of GmPDIM mRNA was higher in the early stages of seed development and subsequently decreased (Fig 3A) The amount of GmPDIM protein was also higher in the early stages, but decreased until the seed grew to 100 mg Expression of GmPDIM increased in the late stage of seed development (Fig 3B) These results suggest that upregulation of the expression of GmPDIM occurs at a time when the requirement for molecular chaperones is high
Fig 1 Putative domain structure of GmPDIM Schematic representation of cleavage sites in GmPDIM by limited proteolysis The upper line represents recombinant protein The lower boxes indicate the domain boundaries predicted by an NCBI conserved domain search The arrows indicate cleavage sites Black boxes in domain a and a’ represent the CGHC motif SP, signal peptide.
Trang 4Upregulation of GmPDIM by ER stress
Many ER-resident chaperones are upregulated by the
accumulation of unfolded protein in the ER (i.e
ER stress) [26–29] Because the consensus sequences
to ERSE were found within the promoter region of
GmPDIM, we next tested whether expression of
GmPDIM responded to ER stress When ER stress
was induced by treatment with tunicamycin or
l-azetidine-2-carboxylic acid in soybean cotyledons, GmPDIM mRNA increased (Fig 4A,B) Upregula-tion of mRNA of GmPDIM was detected by DNA array analysis with a genechip (Affymetrix, Santa Clara, CA, USA) designed from soybean expression sequence tags (data not shown) In addition, protein levels of GmPDIM, BiP and calreticulin were also increased in the cotyledons treated with tunicamycin (Fig 4C)
Table 1 Putative regulatory motifs found within the promoter sequences of GmPDIM.
Motif
Consensus
Distance from ATG Sequencea
ERSE CCAAT-N9-CCACG Putative cis-acting element involved
in unfolded protein response
from the B-hordein gene of barley and the alpha-gliadin, gamma-gliadin, and low molecular weight glutenin genes of wheat
DPBFcore Dc3 ACACNNG bZIP transcription factors, DPBF-1
and 2 (Dc3 promoter-binding factor-1 and 2) binding core sequence;
Found in the carrot (D.c.) Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by ABA
Brassica napus Sequence is also known as RRE (R response element) Conserved in many storage-protein gene promoters
GCN4 motif TGAGTCA cis-acting element required for
endosperm-specific expression
Prolamine box TGCAAAG cis-acting element involved in
quantitative regulation of the GluB-1 gene
complex containing the two RY repeats and the G-box) of napA gene in Brassica napus; Required for seed specific expression
in the 5’-upstream region of b-conglycinin gene
in the 5’-upstream region of b-conglycinin gene
a Conserved bases of the motifs are in large letters.
Trang 5GmPDIM is an ER luminal protein
GmPDIM has an N-terminal signal sequence for
tar-geting it to the ER, and a C-terminal ER-retention
sig-nal sequence KDEL We performed a magnesium-shift
assay to confirm the localization of GmPDIM in the
rough ER Microsomes were prepared from the
cotyle-dons and centrifuged through a sucrose gradient in the
presence of magnesium or EDTA The buoyant density
of rough ER is decreased by dissociation of ribosomes
in the presence of EDTA Fractions were collected
from the sucrose gradient and were analyzed by
wes-tern immunoblot The peak of GmPDIM at a density
of 1.21 gÆmL)1 in the presence of magnesium was
shifted to fractions of lighter sucrose (1.16 gÆmL)1) in
the presence of EDTA (Fig 5A), indicating that
Gm-PDIM localized in the rough ER Next, microsomes
were purified from cells and treated with proteinase K
in the absence or presence of Triton X-100 GmPDIM
was resistant to protease treatment in the absence of detergent (Fig 5B, lane 3), but when the microsomal membranes first were disrupted by Triton X-100, Gm-PDIM was degraded (Fig 5B, lane 4) These results indicate that GmPDIM is an ER luminal protein
Association of GmPDIM with proglycinin and b-conglycinin a’ in the cotyledon
GmPDIM has oxidative folding activity in vitro and localizes to the ER lumen of the cotyledon, suggesting that it may function on folding of glycinin [17] Because nascent polypeptides and molecular chaper-ones transiently associate with each other in the ER,
we next attempted to detect an interaction between GmPDIM and proglycinin, which is translocated into the lumen of the ER for folding Because a transient association between a chaperone and nascent polypep-tide is generally unstable, immunoprecipitation experi-ments were carried out after treatment with the protein cross-linker dithiobis[succinimidylpropionate] (DSP) GmPDIM was detected in the immunoprecipitate with
B
A
Fig 2 Expression of GmPDIM in soybean tissues (A) Purified
recombinant GmPDIM (20 ng) (lane 1) and proteins extracted from
the cotyledon (30 lg) (lanes 2–5) were analyzed by western
immu-noblot with anti-GmPDIM serum (1 lL) treated without (lanes 1, 2)
or with 16 lg (lane 3), 80 lg (lane 4) or 400 lg (lane 5) purified
recombinant GmPDIM (B) Thirty micrograms of protein extracted
from the cotyledon (80 mg bean) (lane 1), root (lane 2), stem
(lane 3), 3 cm leaf (lane 4), 6 cm leaf (lane 5), 9 cm leaf (lane 6)
and flower (lane 7) were analyzed by western immunoblot with
anti-GmPDIM serum.
A
B
Fig 3 Expression of GmPDIM in soybean cotyledons during matu-ration (A) GmPDIM mRNA was quantified by real time RT-PCR Each value was standardized by dividing the value by that for actin mRNA Values are calculated as a percentage of the highest value obtained during maturation Data represent the mean ± SD for four experiments (B) Proteins (25 lg) extracted from cotyledons were analyzed by western immunoblot with anti-GmPDIM serum.
Trang 6anti-GmPDIM serum by western immunoblot
(Fig 6A) The efficiency of immunoprecipitation of
GmPDIM was not influenced by cross-linking with
DSP We next tried to detect an association between
GmPDIM with proglycinin In order to detect trace
amounts of nascent proglycinin, cotyledons were
meta-bolically labeled with [35S]-methionine and [35
S]-cyste-ine After labeling, microsomes were prepared from
the cotyledons in the presence of DSP, were
solubi-lized, and were subjected to immunoprecipitation
with anti-GmPDIM serum or non-immunized serum
Immunoprecipitates were treated with dithiothreitol to
reduce the disulfide bonds formed by cross-linking
with DSP, and were then subjected to a second
immu-noprecipitation using anti-glycinin acidic subunit
serum No precipitation of proglycinin with
non-immunized serum, was confirmed (Fig 6B, lanes 1
and 2) Proglycinin was detected in the
immunoprecipi-tate using anti-GmPDIM serum (Fig 6B, lane 4) These results suggest that GmPDIM may associate with proglycinin in the ER
A
C
B
Fig 4 Responses of the GmPDIM gene to ER stress induced by
reagent treatment Cotyledons from 137–142 or 210–263 mg beans
were divided into two halves and incubated in the absence or
pres-ence of tunicamycin (TM) for 24 h (A) or L -azetidine-2-carboxylic
acid for 18 h (B) GmPDIM mRNA was quantified by real time
RT-PCR Each value was standardized by dividing the value by that for
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 Data represent the mean ± SD for three
experi-ments BiP* and CRT* (calreticulin) are from Wadahama et al [20].
(C) Proteins (15 lg) extracted from cotyledons treated without
(lanes 1, 3 and 5) or with (lanes 2, 4 and 6) tunicamycin for 24 h
were analyzed by western immunoblot with anti-GmPDIM serum
(lanes 1 and 2), anti-BiP serum (lanes 3 and 4) and anti-calreticulin
serum (lanes 5 and 6).
A
B
Fig 5 Localization of GmPDIM in the ER lumen (A) Microsomes were isolated from cotyledons (100 mg beans), and microsomes were fractionated on isopyknic linear sucrose gradients in the pres-ence of MgCl 2 or EDTA Proteins from each gradient fraction were analyzed by western immunoblot with anti-GmPDIM serum The top of the gradient is on the left Density (gÆmL)1) is indicated on the top (B) Microsomes were treated without (lanes 1 and 2) or with (lanes 3 and 4) proteinase K, in the absence (lanes 1 and 3)
or presence (lanes 2 and 4) of Triton X-100 Microsomal proteins (10 lg) were analyzed by western immunoblot with anti-GmPDIM serum.
Fig 6 Co-immunoprecipitation of GmPDIM and proglycinin (A) Confirmation of immunoprecipitation of GmPDIM with anti-GmP-DIM serum Microsomes were isolated from cotyledons (150 mg beans) and treated with (+) or without ( )) DSP Proteins were extracted and immunoprecipitated with anti-GmPDIM serum Mi-crosomes (lane 1) and the immunoprecipitants (lanes 2 and 3) were analyzed by western immunoblot with anti-GmPDIM serum (B) Co-immunoprecipitation experiments Cotyledons were pretreated without (lanes 1–4) or with (lanes 5 and 6) dithiothreitol and labeled with Pro-mix L-[ 35 S] in vitro labeling mix for 1 h After labeling, mi-crosomes were isolated and treated with (+) or without ( )) DSP The extracts from the microsomes were subjected to immunopre-cipitation with non-immuninized serum (lanes 1 and 2) or anti-GmPDIM serum (lanes 3–6) The precipitants were subjected to a second immunoprecipitation with anti-glycinin acidic subunit serum The final precipitants were subjected to SDS ⁄ PAGE and analyzed
by fluorography The positions of proglycinins (pro 11S) are indi-cated on the right.
Trang 7Because co-immunoprecipitation between
proglyci-nin and GmPDIM was dependent on cross-linking by
DSP, it is possible that GmPDIM associates
non-cova-lently with proglycinin in the ER To test this, similar
experiments were performed using cotyledon cells
trea-ted with dithiothreitol Dithiothreitol is a
membrane-permeable reducing agent that inhibits disulfide bond
formation in the ER Therefore, it was expected that
unfolded proglycinin would increase in the ER in the
presence of dithiothreitol A small amount of
proglyci-nin was detected in immunoprecipitates using
cotyle-don cells that were not treated with DSP (Fig 6B,
lane 5), whereas a large amount of proglycinin was
detected in immunoprecipitates from cotyledon cells
treated with DSP (Fig 6B, lane 6)
The PDI family of proteins play roles not only as
thiol-oxidereductases, but also as molecular chaperones
in the ER [7,8,30] To test whether GmPDIM also
functions as a molecular chaperone in the folding of
b-conglycinin, we examined if GmPDIM associates
with the b-conglycinin a¢ subunit No precipitation of
the b-conglycinin a¢ subunit with non-immunized
serum was confirmed (Fig 7, lanes 1 and 2) The
b-conglycinin a¢ subunit was barely detected in the
immunoprecipitate with anti-GmPDIM serum (Fig 7,
lanes 3 and 4) We next performed the
immunoprecipi-tation experiment using cotyledon cells treated with
tunicamycin Tunicamycin may increase the amount of
unfolded b-conglycinin a¢ subunit in the ER, because the folding efficiency of glycoproteins like b-conglyci-nin is reduced by inhibition of N-glycosylation [1] A small amount of b-conglycinin a¢ subunit was detected
in the immunoprecipitate with anti-GmPDIM serum from cotyledon cells untreated with DSP (Fig 7, lane 5), but larger amounts were detected when the cells were treated with DSP (Fig 7, lane 6) These results suggest that GmPDIM may associate with an unglycosylated form of the b-conglycinin a¢ subunit in the ER in the presence of tunicamycin
Discussion
In this study, we cloned the cDNA of GmPDIM and characterized it as a member of the PDI family of pro-teins GmPDIM has two tandem redox-active thio-redoxin-like domains, a and a’, and a redox-inactive thioredoxin-like domain, b The amino acid sequence
of GmPDIM is highly similar to that of the animal thiol-oxidoreductase P5 [7] The recombinant form of GmPDIM was active as it could refold RNase A that had been reduced and denatured The specific activity
of GmPDIM was very similar to those of GmPDIS-1 and GmPDIS-2 (other soybean PDI family proteins) [20] However, the specific activities of GmPDIM, GmPDIS-1 and GmPDIS-2 were relatively low and corresponded to 10% of bovine PDI activity [20] Because the amino acid sequences of the two thio-redoxin domains and a region essential for oxidative refolding activity were conserved in GmPDIM, Gm-PDIS-1 and GmPDIS-2, their low activities may be due to their low affinities for unfolded RNase A Specific antiserum against recombinant GmPDIM reacted with two bands in the soybean cell lysate, and both migrated to similar positions on the SDS⁄ PAGE gel It is unclear whether both proteins were products from a single GmPDIM gene or from two separate genes, as gene duplications are common in higher plants In A thaliana, two group V genes (AtPDIL2-2 and AtPDIL2-3) were identified (supplementary Fig S1) Therefore, it is possible that one of the bands
is GmPDIM and the other is a homolog However, it cannot be excluded that the second band seen on immunoblotting is due to modification of GmPDIM Expression of GmPDIM mRNA was upregulated by
ER stress Likewise, the DNA microarray analysis dem-onstrated that expression of 2 and
AtPDIL2-3were also upregulated by ER stress [26,27] The ERSE consensus sequence was identified in the promoter regions of both GmPDIM and AtPDIL2-2; this cis-acting regulatory element is frequently found in genes responsive to ER stress [26–28] In addition, the
Fig 7 Co-immunoprecipitation of GmPDIM and the b-conglycinin
a’ subunit Cotyledons were pretreated without (lanes 1–4) or with
(lanes 5 and 6) 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 microsomes were
subjected to immunoprecipitation with non-immuninized serum
(lanes 1 and 2) or anti-GmPDIM serum (lanes 3–6) The precipitants
were subjected to a second immunoprecipitation with
anti-b-con-glycinin a’ subunit serum The final precipitants were subjected to
SDS ⁄ PAGE and analyzed by fluorography The position of the
b-conglycinin a’ subunit (7S-a’) is indicated on the right.
Trang 8A thaliana transcription factor, AtbZIP60, activates
transcription from ERSE [31] Therefore, GmPDIM and
AtPDIL2-2 may be unfolded protein responsive genes
regulated by AtbZIP60 and may play an important role
in maintaining homeostasis of the ER under ER stress
Consensus sequences regulating seed-specific
expres-sion were found in the promoter region of GmPDIM
For example, RY repeat, which was reported to
func-tion for seed-specific transcripfunc-tion of b-conglycinin,
was found [32] However, mRNA expression patterns
of GmPDIM and b-conglycinin [12] were different,
suggesting that expression of these genes in the
cotyle-don is regulated in a different manner It is not known
how the level of GmPDIM mRNA is controlled in the
cotyledon cells The protein levels of GmPDIM
increased dramatically during seed maturation,
sug-gesting that it may also play an important role in this
process The regulation of GmPDIM protein levels
during this stage was a post-transcriptional event
rather than a transcriptional one It is obscure how the
protein levels of GmPDIM are controlled
PDI family proteins are thought to catalyze the
formation of disulfide bonds on nascent polypeptide
chains in the ER Therefore, GmPDIM was assumed
to relate to proglycinin folding that accompanies the
formation of disulfide bonds in the ER of cotyledon
cells Because the interaction between these two
pro-teins was detected only in microsomes treated with
DSP, it is likely that the majority of GmPDIM
associ-ates non-covalently with proglycinin Interaction
between GmPDIM and proglycinin was also detected
in the presence of dithiothreitol, which inhibits
disul-fide bond formation in the ER and may cause
accumu-lation of unfolded proglycinin Because the active
center (CGHC) of GmPDIM is reduced in the
pres-ence of dithiothreitol, it cannot form disulfide bonds
with the cysteine residues in proglycinin Therefore, it
is possible that GmPDIM associates non-covalently
with proglycinin in the presence of dithiothreitol This
also suggests that GmPDIM may function as a
mole-cular chaperone for proglycinin However, because the
chaperone activity of GmPDIM for rhodanase was not
detected (data not shown), it is likely that GmPDIM
recognizes specific protein elements other than
hydro-phobic structures exposed in unfolded proteins No
association of GmPDIM with the b-conglycinin a¢
subunit was detected under normal conditions, but a
positive interaction was detected in the presence of
tunicamycin Tunicamycin inhibits N-glycosylation and
may cause accumulation of the unfolded b-conglycinin
a¢ subunit in the ER GmPDIM may play a role as a
molecular chaperone for the b-conglycinin a¢ subunit,
because the mature form possesses no disulfide bonds
[33,34] However, it is not clear if GmPDIM can interact with both the glycosylated and non-glycosylated forms
of the b-conglycinin a¢ subunit, or only with the non-glycosylated form Alternatively, it is possible that GmPDIM catalyzes disulfide bond formation during folding, because five cysteine residues are present in the pro-sequence of the b-conglycinin a¢ subunit, which are subsequently removed in the post-ER compartment The results obtained using the anti-GmPDIM serum must be interpreted cautiously, because association between both GmPDIM and a homolog of GmPDIM with proglycinin or the b-conglycinin a¢ subunit may
be detected, because anti-GmPDIM serum immuno-reacted to two similar cotyledon proteins (Fig 2A) Future experiments with antibodies specific to individ-ual proteins will clarify this result
Molecular chaperones in the ER are believed to col-laborate with each other in different ways so that they adapt to each substrate protein Exhaustive study of the interaction between the PDI family and other molecular chaperones, including their recognition sites
on substrate polypeptides, is necessary to clarify the folding mechanism of each protein
Experimental procedures
Plants
Soybean (Glycine max L Merrill cv Jack) seeds were planted in 5-L pots and grown in a controlled environ-mental chamber at 25C under 16 : 8 h day ⁄ 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
DNA cloning of GmPDIM
The cloning of GmPDIM cDNA was performed by 3¢- and 5¢-RACE 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, Valencia, CA, USA) according to the manufac-turer’s protocol Messenger RNA was isolated from total RNA with the PolyATtract mRNA Isolation System (Promega, Madison, WI) The 3¢-RACE method was
kit (Clontech, Mountain View, CA) according to the manu-facturer’s protocol using the primer 5¢-TCCTCACCCGTG CTTCAACTCACTCC-3¢ The 5¢-RACE method was per-formed using the primer 5¢-CTGTTGGCTGAATGCT CATTGATAGGG-3¢, which was designed based on the
Trang 9sequence obtained by 3¢-RACE The amplified DNA
frag-ment was subcloned into the pT7Blue vector (TaKaRa Bio
Inc., Shiga, Japan) The inserts in the plasmid vectors were
sequenced using the fluorescence dideoxy chain termination
method and an ABI PRISM 3100-Avant Genetic
Ana-lyzer (Applied Biosystems, Foster City, CA, USA)
Cloning of genomic sequences encoding
GmPDIM
Genomic sequence encoding GmPDIM was isolated from
the transformation-competent artificial chromosome (TAC;
pYLTAC7) library of soybean variety ‘Misuzudaizu’ by the
three-dimensional screening system [35,36] Screening was
performed by PCR using the primer set 5¢-CAATTGA
TGCTGATGCTCATCCGTC-3¢ and 5¢-CATGGCCCAG
TTTAACCTTCCCTT-3¢
Construction of His-tagged expression plasmid
Expression plasmid encoding His-tagged GmPDIM without
the putative signal peptide was constructed as follows The
DNA fragment was amplified from GmPDIM cDNA by
amplified DNA fragment was subcloned into the
ligation-independent cloning site of the pET46Ek⁄ LIC vector
(EMD Biosciences, San Diego, CA, USA) The
recombi-nant protein has the His-tag linked to the N-terminus
Expression and purification of recombinant
GmPDIM
BL21(DE3) cells were transformed with the His-tagged
expression vector described above Expression of
recombi-nant protein was induced by the addition of 0.4 mm
isopro-pyl thio b-d-galactoside at 30C for 4 h Cells from 1 L of
culture were collected by centrifugation, disrupted by
soni-cation in 8 mL of 20 mm Tris⁄ HCl (pH 8.0) containing
5 mm imidazole and 0.5 m NaCl Affinity-column
chroma-tography using His-Bind resin and gel-filtration
chromato-graphy were carried out as described previously [20] The
concentration of purified recombinant GmPDIM was
deter-mined by absorbance values at 280 nm using the molar
extinction coefficient calculated by the modified method of
Gill and von Hippel [37] An extinction coefficient of
53 400 m)1Æcm)1was used for recombinant GmPDIM
Limited proteolysis of GmPDIM
in100 mm Tris⁄ HCl (pH 8 0) with either trypsin (1 lg)
(Sigma-Aldrich Co., St Louis, MO, USA) or V8 protease
(2 lg) (Sigma-Aldrich) at 25C for 30 or 120 min, respec-tively The cleavage sites of the resulting peptides were mapped as described previously [20]
Oxidative refolding assay with reduced RNase A
PDI activity was assayed by measuring RNase activity pro-duced through the regeneration of the active form from reduced and denatured RNase A Reduced and denatured RNase A was prepared as described previously by Creigh-ton [38] Each reaction mixture contained 200 mm [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (pH 7.5),
150 mm NaCl, 2 mm CaCl2, 0.5 mm glutathione disulfide,
0.25 mgÆmL)1 recombinant GmPDIM and was incubated
at 25C An aliquot (16 lL) of the reaction mixture was removed and RNase A activity was measured spectrophoto-metrically at 284 nm with cytidine 2¢,3¢-cyclic monophos-phate as the substrate [39] Reactivation of reduced RNase A in the absence of recombinant protein was sub-tracted from reactivation in the presence of GmPDIM
Antibodies
Anti-GmPDIM serum was prepared using recombinant
Japan) Preparation of antibody specific to BiP, calreticulin, the glycinin acidic subunit and the b-conglycinin a¢ subunit has been described previously [20]
Western immunoblot analysis
Soybean tissues that had been frozen under liquid nitrogen were ground into fine powders with a micropestle SK-100 Proteins were extracted by boiling for 5 min in SDS⁄ PAGE buffer [40] containing a 1% cocktail of protease inhibitors (Sigma-Aldrich) Protein concentration in the sample was measured with a protein assay kit (RC DC protein assay, Bio-Rad, Hercules, CA, USA) Proteins were subjected to SDS⁄ PAGE [40] and blotted onto a poly(vinylidene difluo-ride) membrane Blots were immunostained with specific antibodies as described in the text, and with horseradish peroxidase-conjugated IgG antiserum (Promega) as second-ary Blots were developed with the Western Lightning Chemiluminescence Reagent (Perkin Elmer Life Sciences, Boston, MA, USA)
Real time RT-PCR analysis
Measurement of mRNA was performed as described previ-ously [20] Briefly, 250 lgÆmL)1tunicamycin or 50 lm l-az-etidine-2-carboxylic acid (Sigma-Aldrich) was administered
to the inner surface of the divided half of the cotyledon and incubated at 25C Total RNA was isolated from
Trang 10plant tissues using an RNeasy Plant Mini kit
Quantifica-tion of mRNA was performed by real-time RT-PCR with a
Thermal Cycler Dice Real Time System (TaKaRa Bio
Inc.) The forward primer 5¢-CGGAACCAAAACATGC
5¢-CGTTACAGGCA-ACTTGTTTCTCA-3¢ were used for detection of GmPDIM
mRNA
ER fractionation
Slices of cotyledons were homogenized by 20 strokes of a
Dounce homogenizer in ice-cold buffer containing 100 mm
Tris⁄ HCl (pH 7.8), 10 mm KCl containing 12% (w ⁄ v)
homogenate was centrifuged for 10 min at 1000 g at 4C
Approximately 600 lL of supernatant was loaded onto a
12 mL linear 21–56% (w⁄ w) sucrose gradient made in the
same buffer After centrifugation for 2 h at 154 400 g and
4C, 1 mL fractions were collected from the bottom of the
gradient and assayed by western immunoblot using the
anti-GmPDIM serum The sucrose concentration of each
fraction was measured with a refractometer NAR-1T
(ATAGO CO., LTD, Tokyo, Japan)
Proteinase K treatment of microsomes
Microsomes were prepared from cotyledons as described
previously [20], and were treated with 0.5 lgÆmL)1
protein-ase K in the presence or absence of 1% Triton X-100 for
5 min at 4C Proteins were precipitated with 10%
trichlo-roacetic acid for 30 min at 4C, and were analyzed by
wes-tern immunoblot with anti-GmPDIM serum
Immunoprecipitation experiments
Six pairs of cotyledons were isolated, divided into two
halves and labeled flat-side up in a Petri dish at 25C for
1 h with a mixture of 1.48 MBq per 4 mL Pro-mix L-[35S]
in vitro labeling mix (37 TBqÆmmol)1) (GE Healthcare,
Little Chalfont, UK) and 6 mL FN Lite [41] For treatment
of cotyledons under ER-stress conditions, a cotyledon was
pretreated with or without 250 lgÆmL)1 tunicamycin or
1 mm dithiothreitol at 25C for 5 h and labeled in the
presence of the stress reagent The cotyledons were rinsed
with FN Lite containing 10 mm cold methionine and
cyste-ine three times and with 20 mm sodium pyrophosphate
buf-fer (pH 7.5) containing 0.3 m mannitol (bufbuf-fer A), after
which slices from the flat side of each cotyledon were cut
The slices were homogenized by 20 strokes of a Dounce
homogenizer at 4C in 3 mL buffer A with or without
1 mgÆmL)1 DSP The homogenate was placed on ice for
2 h The cross-linking reaction was terminated by the
addition of 2 mm glycine for 30 min on ice The
micro-somes were prepared as described previously [20] Proteins were extracted from the microsomes with 50 mm Tris⁄ HCl buffer (pH 7.5) containing 150 mm NaCl, and pre-cleared with protein A-conjugated Sepharose beads (50% slurry) (Sigma-Aldrich) Initial immunoprecipitation was per-formed at 4C for 16 h with non-immmunized serum or affinity-purified anti-GmPDIM sera The immunoprecipi-tate was dissolved in 2% SDS and 0.4 m dithiothreitol The second immunoprecipitation was performed with anti-glycinin acidic subunit serum or anti-b-conglycinin a¢ subunit serum at 4 C for 16 h The antigen–antibody complexes were subjected to SDS⁄ PAGE, and radiolabeled proteins were detected by fluorography with ENLIGHTING (NEN Life Science Products, Inc., Boston, MA, USA) Part
of the immunoprecipitant obtained in the first immunopre-cipitation using anti- GmPDIM serum was subjected to SDS⁄ PAGE and analyzed by western immunoblot using anti-GmPDIM and the One-StepTM Complete IP-Western kit (GenScript Co., Piscataway, NJ, USA)
Acknowledgements
We thank Ms Masatoshi Izumo for identifying the C-terminal amino acid of the peptide produced by pro-tease digestion We thank Ms Kensuke Iwasaki and
Ms Akie Ko for assaying the oxidative refolding activ-ity This study was supported by a grant from the Prog-ram for Promotion of Basic Research Activities for Innovative Biosciences and a Grant-in-Aid for Explora-tory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18658055)
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