Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with pro-glycinin, a precursor of the seed storage protein pro-glycinin, and the a¢-subunit of
Trang 1protein disulfide isomerases as molecular chaperones for seed storage proteins
Shinya Kamauchi1,*,†, Hiroyuki Wadahama1,*, Kensuke Iwasaki1, 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, organelle and membrane proteins are folded
with the assistance of molecular chaperones and other
folding factors in the endoplasmic reticulum (ER) In
many cases, protein folding in the ER is accompanied
by N-glycosylation and the formation of disulfide bonds
[1] The directed formation of disulfide bonds in a
nas-cent polypeptide chain is thought to be catalyzed by
protein disulfide isomerase (PDI; EC 5.3.4.1) and PDI-related family proteins that belong to the thioredoxin superfamily [2–4] Animal PDI has been shown to act not only as a thiol-oxidoreductase enzyme, but also as a molecular chaperone [5] PDI is thought to bind poly-peptides through its hydrophobic region and to form, break and isomerize disulfide bonds in these
polypep-Keywords
endoplasmic reticulum; molecular
chaperone; protein disulfide isomerase;
soybean; storage protein
Correspondence
R Urade, Graduate School of Agriculture,
Kyoto University, Uji, Kyoto 611-0011, Japan
Fax: +81 774 38 3758
Tel: +81 774 38 3757
E-mail: urade@kais.kyoto-u.ac.jp
†Present address
Osaka Bioscience Institute, Suita, Japan
*These authors contributed equally to this
work
Database
The nucleotide sequence data for the cDNA
of GmPDIL-1 and GmPDIL-2 and genomic
GmPDIL-1 and GmPDIL-2 are available in
the DDBJ ⁄ EMBL ⁄ GenBank databases under
accession numbers AB182628, AB185851,
AB300660 and AB300661, respectively
(Received 26 December 2007, revised 22
February 2008, accepted 18 March 2008)
doi:10.1111/j.1742-4658.2008.06412.x
Protein disulfide isomerase family proteins play important roles in the fold-ing of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum In this study, we cloned two similar protein disul-fide isomerase family genes from soybean leaf (Glycine max L Merrill cv Jack) The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively Recombinant versions of GmP-DIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA Genomic sequences of both GmPDIL-1and GmPDIL-2 were cloned and sequenced The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon–intron structure across plant spe-cies The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response GmP-DIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with pro-glycinin, a precursor of the seed storage protein pro-glycinin, and the a¢-subunit
of b-conglycinin, a seed storage protein found in cotyledon cells under con-ditions that disrupt the folding of glycinin or b-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones
Abbreviations
AZC, L -azetidine-2-carboxylic acid; DSP, dithiobis(succinimidylpropionate); ER, endoplasmic reticulum; ERSE, endoplasmic reticulum stress-responsive element; PDI, protein disulfide isomerase; PVDF, poly(vinylidene difluoride).
Trang 2tides [6] In plants, a genome-wide search of
Arabidop-sis thaliana identified a set of 22 PDI orthologs
sepa-rated into 10 phylogenetic groups [7] Groups I and II
have two thioredoxin domains and show structural
simi-larities to PDI of other eukaryotes Genes encoding
group I or II PDIs are present in several plants [8–16]
Among them, group I PDIs of rice and Rubiaceae were
shown to affect folding of proglutelin in vivo and cystine
knot defense proteins in vitro [15,17]
In soybean cotyledon cells, large quantities of
stor-age protein are synthesized in the ER during seed
development to reserve carbon and nitrogen for
germi-nation and early growth [18] Primary soybean seed
storage proteins are globulins called glycinin and
b-conglycinin They are folded and assemble into
tri-mers in the ER, and are then transported to, and
deposited in, protein storage vacuoles [19] Glycinin is
synthesized as a precursor subunit that undergoes two
proteolytic processing steps; the first is a removal of an
N-terminal signal peptide in the ER, and the second is
fragmentation of the precursor into 40 kDa acidic and
20 kDa basic subunits in protein storage vacuoles
[20,21] The second processing step is required for
assembly into hexamers [22] A major glycinin,
A1aB1b, possess two intramolecular disulfide bonds,
Cys12–Cys45 and Cys88–Cys298, which are required
for hexamer assembly and structural stability [23–25]
Folding and the formation of the disulfide bonds of
glycinin are predicted to be facilitated by some PDI
family members Previously, we identified novel PDI
family proteins belonging to group IV (GmPDIS-1
and GmPDIS-2) and group V (GmPDIM), and
showed that GmPDIS-1 and GmPDIM associated with
proglycinin in the ER [26,27] However, involvement
of other PDI family proteins in the folding of the
stor-age proteins remains a topic of investigation
In this study, we isolated cDNA clones and genomic
sequences encoding soybean group I and II PDI family
members We present the tissue distribution, cellular
localization and modulation of expression of the
proteins encoded by genes from each of these two
groups during soybean seed development We provide
evidence of an association between GmPDIL-1 or
GmPDIL-2 and proglycinin or b-conglycinin during
the course of the folding process of these proteins
Results
Cloning and expression of GmPDIL-1 and
GmPDIL-2
To clone the soybean ortholog of Arabidopsis
PDI-like 1-1 and PDI-like 1-3 categorized in groups I
and II [7], a blast search was performed using the nucleotide sequence of PDI-like 1-1 or PDI-like 1-3 cDNA from the Institute for Genomic Research Soy-bean Index As a result, tentative consensus sequences, TC188262 from PDI-like 1-1 and TC176115 from PDI-like 1-3, were found Using primer sets designed from their nucleotide sequences, we cloned cDNAs derived from young soybean leaves by RT-PCR These cDNAs encoded proteins, named GmPDIL-1 and GmPDIL-2, which were 525 and 551 amino acids long, respectively (supplementary Figs S1 and S2) Both pro-teins possess a putative N-terminal secretory signal sequence and two thioredoxin-like motifs with a CGHC active site Arginines (Arg128 and Arg482 of GmPDIL-1 and Arg163 and Arg505 of GmPDIL-2) known to be involved in the regulation of the active site redox potential in human PDI [28,29] were con-served In addition, glutamic acid residues (Glu67 and Glu412 of GmPDIL-1, and Glu95 and Glu434 of GmPDIL-2), suggested to facilitate the release of the active site from a mixed disulfide with substrate [30], were also conserved GmPDIL-1 and GmPDIL-2 pos-sessed C-terminal, KDEL-related sequences that func-tion in ER retenfunc-tion [31,32] The amino acid sequence identity of GmPDIL-1 and GmPDIL-2 to each other, minus the signal peptides, was 30%
Recombinant GmPDIL-1 and GmPDIL-2 proteins were expressed in Escherichia coli and purified (Fig 1A,B) Both recombinant proteins were soluble and eluted in a monomeric form from a gel filtration column (data not shown) To examine the helical con-tent, far-UV CD was performed Both GmPDIL-1 and GmPDIL-2 yielded CD spectra that reflected folded globular protein, and the calculated a-helical content was 34% and 28% for PDIL-1 and PDIL-2, respec-tively (data not shown) The domain structures of GmPDIL-1 and GmPDIL-2 were predicted to form a linear sequence of four domains in an a–b–b¢–a¢ orien-tation beginning at the region of conserved domain sequence homology We subjected the recombinant GmPDIL-1 and GmPDIL-2 proteins to limited prote-olysis with either trypsin or V8 protease to determine their domain boundaries After proteolysis for various time periods, the native recombinant proteins were gradually degraded, resulting in the generation of smaller-sized peptide fragments (data not shown) The sites of proteolytic cleavage were determined to be Lys77, Lys152, Lys162 and Glu39 of GmPDIL-1 by N-terminal sequencing of the trypsin peptide fragments and the V8 protease peptide fragments, respectively In addition, four cleavage sites were identified by measur-ing the masses of the peptide fragments by MALDI-TOF MS Among the cleavage sites, four resided in
Trang 3two narrow regions, overlapping the putative
bound-ary regions in GmPDIL-1 between a and b and b¢ and
a¢ (Fig 1C) In the case of GmPDIL-2, Lys175, Glu43,
Glu62, Glu68 and Glu135 were identified by
N-termi-nal sequencing of the enzymatic digest fragments,
respectively, and Glu541 was identified by
MALDI-TOF MS These cleavage sites were located in regions
of N-terminal extension, in the C-terminal tail, and in
the putative boundary regions between a and b
(Fig 1D) As lysine, arginine or glutamic acid residues
are present in the putative boundary region between b
and b¢ in GmPDIL-1, and between b and b¢ and b¢
and a¢ in GmPDIL-2, the structure of these regions
may be protease resistant
The activity of recombinant GmPDIL-1 and
GmP-DIL-2, which catalyze oxidative refolding of reduced
and denatured RNaseA, was examined The specific
activities of GmPDIL-1 and GmPDIL-2 were 472 and
300 mmol RNaseAÆmin)1Æmol)1, respectively (Fig 2A)
Several mammalian and yeast PDI family proteins are
known to function as molecular chaperones [5] We measured the molecular chaperone activity, which pre-vents the aggregation of unfolded rhodanese Aggrega-tion occurred over 14 min without PDI, but was inhibited by GmPDIL-2 in a concentration-dependent manner (Fig 2C) In the presence of 2.4 lm
GmPDIL-2 (molar ratio of 6 : 1 to rhodanese), 30% of the rhodanese aggregation was inhibited for at least
14 min GmPDIL-1 exhibited slight, but significant, chaperone activity at a molar ratio of 6 : 1 to rhoda-nese (Fig 2B)
Cloning of GmPDIL-1 and GmPDIL-2 genomic sequences
Genomic sequences encoding 1 or
GmPDIL-2 were cloned and sequenced The alignment and comparison with the corresponding cDNA showed that GmPDIL-1 and GmPDIL-2 were composed of
10 and 12 exons, respectively (supplementary Fig S3)
A
C
D
B
Fig 1 Prediction of the GmPDIL-1 and GmPDIL-2 domain structures Recombinant GmPDIL-1 (A) and GmPDIL-2 (B) in E coli (lane 1) were purified by His-tag column chromatography (lane 2), followed by gel filtration chromatography (lane 3) Proteins in each effluent were sepa-rated by 10% SDS ⁄ PAGE and stained with Coomassie Blue (C,D) Schematic representation of cleavage sites in recombinant GmPDIL-1 (C) and GmPDIL-2 (D) by limited proteolysis with trypsin and V8 protease The upper line represents recombinant protein The boxes below indi-cate the domain boundaries predicted by an NCBI conserved domain search The arrows indiindi-cate the determined cleavage sites Black boxes in domains a and a¢ represent the CGHC motif A closed circle with a bar represents an N-glycosylation consensus site SP, signal peptide.
Trang 4Nucleotide sequences of the ORF of both PDI genes
were exactly the same as those of cDNAs cloned
in this study Comparisons of soybean genomic
sequences of GmPDIL-1 or GmPDIL-2 with those of
Arabidopsis (AGI number At1g77510), rice (MOsDb
number Os04g35600) and wheat (accession number
AJ277377) [33] or Arabidopsis (AGI number
At3g54960) and rice (MOsDb number Os02g01010)
showed a significant conservation of the exon–intron
structure across these species The exon–intron
structure across GmPDIL-1 and GmPDIL-2 was
considerably different All introns of GmPDIL-1 and
GmPDIL-2 matched the branchpoint consensus
sequence in plants (YTNAN) upstream of the 3¢-site
The second, third, fifth, sixth and seventh introns
of GmPDIL-1 had the plant branchpoint consensus
sequence TTNAN, whereas the first, fourth, eighth
and ninth introns of GmPDIL-1 had the consensus
sequence CTNAN [34] In the case of GmPDIL-2,
10 of the 11 introns had TTNAN, and the eighth
intron possessed CTNAN
Promoter regions of around 1 kb and 1.5 kb
upstream of each start codon of GmPDIL-1 and
GmP-DIL-2were analyzed A search for elements upstream
of the coding region of GmPDIL-1 in the database of
plant promoters (PLACE: http://www.dna.affrc.go.jp/
PLACE/) detected ER stress-responsive element
(ER-SEs), CCAAT-N9-CCACG [35], and a number of
cis-acting regulatory elements involved in
endosperm-specific gene regulation, including G-box, DPBF core
Dc3, E-box, SEF 1 motif and SEF 4 motifs (Table 1)
In the promoter region of GmPDIL-2, cis-acting
regu-latory elements involved in the regulation of
endo-sperm-specific genes, AACA motif, DPBF core Dc3,
E-box, RY-repeat and SEF 1 motif, were found
(Table 2) However, no ER stress regulatory element
was found
GmPDIL-1 mRNA, but not GmPDIL-2 mRNA, is upregulated by ER stress
Expression of genes encoding ER-resident proteins is known to be upregulated by the accumulation of unfolded protein in the ER (i.e ER stress) in plant cells [36,37] As the consensus sequence of the ERSE was found in the promoter region of GmPDIL-1, the potential for GmPDIL-1 response to ER stress was
Fig 2 Activity of recombinant GmPDIL-1 and GmPDIL-2 (A)
Oxi-dative refolding activity of the recombinant GmPDIL-1, GmPDIL-2,
GmPDIS-1, GmPDIS-2 and GmPDIM Activity was assayed by the
measurement of RNase activity produced through the regeneration
of the active form of reduced RNaseA Data represent the
mean ± SD for four experiments The data for GmPDIS-1,
GmP-DIS-2 and GmPDIM are from Wadahama et al [26,27] (B,C)
Chap-erone activity of GmPDIL-1 (B) and GmPDIL-2 (C) The aggregation
of rhodanese (0.4 l M ) was measured without (solid circles) or with
0.8 l M GmPDIL-2 (open triangles), 2.4 l M GmPDIL-1 (open
squares) or 2.4 l M GmPDIL-2 (open squares) Each value
repre-sents the mean of three experiments Bars represent SD The
statistical significance of difference was determined between
aggregations in the reaction with and without GmPDIL1 or
GmPDIL-2 by the unpaired Student t-test *P < 0.01.
Trang 5examined First, we performed microarray analysis of
transcripts from soybean cotyledons treated with or
without an ER-stress inducing reagent, tunicamycin,
on a GeneChip (Affymetrix, Santa Clara, CA, USA)
designed from soybean-expressed sequence tags Many
(488) probe sets showed a mean variation of ‡ 4-fold
under tunicamycin treatment (supplementary
Table S1) In total, 178 genes, including some encoding
PDI family members, GmPDIL-1, GmPDIS-1 [26] and
GmPDIM [27], were identified by homology search of
these probe sets Probes sets for GmPDIL-2 exhibited
little variation (data not shown) To confirm the
induc-tion of GmPDIL-1 mRNA by ER stress, the cotyledon
was treated with or without tunicamycin or
l-azeti-dine-2-carboxylic acid (AZC), and mRNA levels of
GmPDIL-1 and GmPDIL-2 were measured by
real-time RT-PCR Expression of GmPDIL-1 was
upregu-lated by treatments with tunicamycin and AZC in a
similar manner to expression of the well-known
unfolded protein response genes BiP and calreticulin
(Fig 3) On the other hand, the expression of
GmP-DIL-2 was not affected by treatment with either
tunicamycin or AZC
Tissue distribution and cellular localization of GmPDIL-1 and GmPDIL-2
Anti-GmPDIL-1 and anti-GmPDIL-2 sera specifically recognized recombinant GmPDIL-1 and GmPDIL-2, respectively (Fig 4A, lanes 1 and 4) Anti-GmPDIL-1 and anti-GmPDIL-2 sera did not immunoreact with recombinant GmPDIL-2 and GmPDIL-1, respectively (data not shown) Anti-GmPDIL-1 serum immunore-acted with bands of 60 and 63 kDa (Fig 4A, lane 2), whereas anti-GmPDIL-2 serum immunoreacted with a single 72 kDa band in western analysis of cotyledon proteins (Fig 4A, lane 5) These bands were not detected with anti-GmPDIL-1 and anti-GmPDIL-2 sera pretreated with purified recombinant GmPDIL-1 and GmPDIL-2, respectively (Fig 4A, lanes 3 and 6), suggesting that such antibodies specifically immunore-acted with GmPDIL-1 and GmPDIL-2 in the cotyle-don GmPDIL-1 and GmPDIL-2 have two and five consensus sequences for N-glycosylation, respectively (Fig 1C,D) When cotyledon proteins were digested with glycosidase F, the mass of bands that immuno-reacted with GmPDIL-1 or GmPDIL-2 sera became
Table 1 Putative regulatory motifs found within the promoter sequences of GmPDIL-1 Base substitutions are in lower-case letters.
Distance from ATG Sequence ERSE CCAAT-N9-CCACG Putative cis-acting element involved in
unfolded protein response
CACGTG
motif (G-box)
CACGTG Essential for expression of b-phaseolin
gene during embryogenesis in bean, tobacco and Arabidopsis
DPBFcore Dc3 ACACNNG bZIP transcription factors, Dc3
promoter-binding factor-1 and fator-2 binding core sequence; found in the carrot Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can
be induced by abscisic acid
Brassica napus Sequence is also known as RRE (R response element).
Conserved in many storage protein gene promoters
SEF 1 motif ATATTTAWW Sequence found in 5¢-upstream region
of soybean b-conglicinin gene
SEF 4 motif RTTTTTR Sequence found in the 5¢-upstream
region of soybean b-conglycinin gene
Trang 6smaller, shifting from 60 and 63 kDa to 59 and
62 kDa, or from 72 kDa to 63 kDa (Fig 4B),
suggest-ing N-glycosylation of these proteins in soybean
Isoelectric points (pI) of GmPDIL-1 and GmPDIL-2
deglycosylated with glycosidase F were determined by
two-dimensional electrophoresis (Fig 4C) Two spots
of 60 and 63 kDa with similar pI values (5.0 and 5.1),
corresponding to bands detected on SDS⁄ PAGE, were detected with anti-GmPDIL-1 serum in cotyledon pro-teins These pI values are almost consistent with that
of the recombinant GmPDIL-1 A single spot was detected with anti-GmPDIL-2 serum The pI of the spot (4.9) was consistent with that of the recombinant GmPDIL-2 GmPDIL-1 and GmPDIL-2 were found
Table 2 Putative regulatory motifs found within the promoter sequences of GmPDIL-2 Base substitutions are in lower-case letters.
Motif
Consensus
Distance from ATG Sequence AACA motif AACAAAC Core of AACA motifs found in rice glutelin genes, involved in
controlling endosperm-specific expression
DPBFcore Dc3 ACACNNG bZIP transcription factors, Dc3 promoter-binding factor-1 and
factor-2 binding core sequence; found in the carrot Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by abscisic acid
E-box CANNTG E-box of napA storage protein gene of Brassica napus.
Sequence is also known as RRE (R response element).
Conserved in many storage protein gene promoters
RY repeat CATGCAY RY repeat in seed storage protein genes in legumes such
as soybean
SEF 1 motif RTTTTTR Sequence found in the 5¢-upstream region of soybean
b-conglycinin gene
Fig 3 Response of GmPDIL-1 and
GmP-DIL-2 gene expression to ER stress
Cotyle-dons from 137–142 mg or 210–263 mg
beans were divided into two halves and
incubated in the absence or presence of
tunicamycin (TM) for 24 h (A) or AZC for
18 h (B), respectively The mRNAs of
GmP-DIL-1, GmPDIL-2, BiP or calreticulin (CRT)
were quantified by real-time RT-PCR Each
value was standardized by correcting for
actin mRNA Fold expression change was
calculated as the ratio of mRNA in the
sam-ples treated with the stress reagent to that
in the untreated sample Data represent the
mean ± SD for three experiments Data for
*BiP and *CRT are from Wadahama et al.
[26].
Trang 7to be expressed in roots, stems, trifoliolate leaves,
flow-ers and cotyledons by immunodetection (Fig 4D)
Expression in leaves decreased during leaf expansion
Levels of GmPDIL-1 increased until the seeds grew
to 70 mg (Fig 5B) Thereafter, the level remained
almost constant Thus, GmPDIL-1 may be expressed
to enhance the machinery for the folding of seed
stor-age proteins However, this event appeared to be
inde-pendent of transcriptional regulation, as the amounts
of GmPDIL-1 mRNA did not correlate with the levels
of GmPDIL-1 protein expression (Fig 5A) Levels of
GmPDIL-2 and GmPDIL-2 mRNA did not correlate
with the synthesis of storage proteins (Fig 5C,D)
GmPDIL-1 and GmPDIL-2 have both an
N-termi-nal sigN-termi-nal sequence for targeting to the ER and a
C-terminal ER retention sequence (KDEL) To confirm
localization of GmPDIL-1 and GmPDIL-2 to the ER, microsomes prepared from cotyledon cells were sepa-rated by sucrose gradient in the presence of MgCl2 or EDTA and analyzed by western blotting (Fig 6A) Peaks for GmPDIL-1, GmPDIL-2 and BiP, well known as an ER lumen protein, were detected at a density of 1.21 gÆmL)1 in the presence of MgCl2 In the presence of EDTA, which causes release of ribo-somes from the rough ER, the peaks of GmPDIL-1, GmPDIL-2 and BiP displayed a similar shift to the lighter sucrose fractions (density of 1.16 gÆmL)1) This suggests localization of GmPDIL-1 and GmPDIL-2 to the rough ER Then, to confirm residence of GmP-DIL-1 and GmPDIL-2 in the ER lumen, microsomes prepared from cotyledon cells were treated with pro-teinase K in the absence or presence of Triton X-100
A
B
D
C
Fig 4 Expression of 1 and 2 in soybean tissues (A) Cross-reactivity of antiserum prepared against recombinant
GmPDIL-1 or GmPDIL-2 with recombinant GmPDIL-GmPDIL-1 (20 ng) (lane GmPDIL-1) and GmPDIL-2 (20 ng) (lane 4) and the cotyledon proteins (GmPDIL-10 lg) (lanes 2, 3, 5 and 6) Anti-GmPDIL-1* and anti-GmPDIL-2* represent anti-GmPDIL-1 serum (1 lL) and anti-GmPDIL-2 serum (1 lL) treated with the recom-binant GmPDIL-1 (4 lg) and the recomrecom-binant GmPDIL-2 (10 lg), respectively (B) GmPDIL-1 and GmPDIL-2 were N-glycosylated proteins in soybean The proteins extracted from the cotyledon were treated without (lane 1) or with (lane 2) glycosidase F The cotyledon proteins (10 lg) and recombinant GmPDIL-1 (20 ng) or GmPDIL-2 (20 ng) (lane 3) were separated by SDS ⁄ PAGE and immunostained with anti-GmP-DIL-1 or anti-GmPDIL-2 serum (C) Separation of recombinant GmPanti-GmP-DIL-1 and GmPDIL-2 and GmPanti-GmP-DIL-1 and GmPDIL-2 expressed in the soy-bean cotyledon by two-dimensional electrophoresis (D) Detection of GmPDIL-1 and GmPDIL-2 in soysoy-bean tissues Thirty micrograms of protein extracted from the cotyledon (80 mg bean), root, stem, 3 cm leaf, 6 cm leaf, 9 cm leaf and flower were separated by 10% SDS ⁄ PAGE and immunostained with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum.
Trang 8Both GmPDIL-1 and GmPDIL-2 were resistant to
protease treatment in the absence of detergent, and
were degraded in the presence of Triton X-100
(Fig 6B), suggesting that GmPDIL-1 and GmPDIL-2
are luminal proteins
Association of GmPDIL-1 and GmPDIL-2 with proglycinin and b-conglycinin a¢-subunit in cotyledon cells
GmPDIL-1 and GmPDIL-2 were shown to have oxi-dative folding activity in vitro and to localize to the cotyledon ER, suggesting that they may function in proglycinin folding that is accompanied by the forma-tion of intramolecular disulfide bonds We sought to detect an association between GmPDIL-1 or GmP-DIL-2 and glycinin in cotyledon cells by immunopre-cipitation from a cotyledon microsomal extract pretreated with the protein crosslinker dithiobis(succin-imidylpropionate) (DSP) First, we confirmed the immunoprecipitation of GmPDIL-1 and GmPDIL-2 from the microsomal extract with anti-GmPDIL-1 serum and anti-GmPDIL-2 serum, respectively (Fig 7A,B) Each immunoprecipitant was analyzed by western blotting with anti-GmPDIL-1 or anti-GmP-DIL-2 serum GmPDIL-1 and GmPanti-GmP-DIL-2 were immu-noprecipitated irrespective of crosslinking treatment Following metabolic labeling of nascent proteins with [35S]methionine and [35S]cysteine, glycinin was immu-noprecipitated with anti-(glycinin acidic subunit) serum and detected by fluorography (Fig 7C, lanes 3 and 4) Most of the label was found in proglycinin After labeling, microsomes from the cotyledons were crosslinked, solubilized, and immunoprecipitated with preimmune serum, GmPDIL-1 serum or anti-GmPDIL-2 serum The immunoprecipitants were trea-ted with dithiothreitol to reduce the disulfide bonds formed by crosslinking, and subjected to a second immunoprecipitation with anti-(glycinin acidic subunit) serum No band was observed in the preimmune serum sample (Fig 7C, lanes 1 and 2) Little proglycinin was detected in the immunoprecipitant with anti-GmPDIL-1
A C
D
B
Fig 5 Expression of GmPDIL-1 and
GmP-DIL-2 in soybean cotyledons during
matura-tion mRNA of GmPDIL-1 (A) and GmPDIL-2
(C) was quantified by real-time RT-PCR.
Each value was standardized with actin
mRNA Values are calculated as a
percent-age of the highest value obtained during
maturation Bars represent SD of four
experiments Thirty micrograms of proteins
extracted from the cotyledons was
sepa-rated by 10% SDS ⁄ PAGE and
immuno-stained with anti-GmPDIL-1 serum (B) and
anti-GmPDIL-2 serum (D).
A
B
Fig 6 Localization of GmPDIL-1 and GmPDIL-2 in the ER lumen.
(A) Microsomes were isolated from cotyledons (100 mg beans) and
fractionated on isopicnic linear sucrose gradients in the presence of
MgCl2or EDTA Proteins from each gradient fraction were analyzed
by western blotting with anti-GmPDIL-1 serum, anti-GmPDIL-2
serum, and anti-BiP serum, respectively The top of the gradient is
on the left Density (gÆmL)1) is indicated at the top (B) Microsomes
were treated without (lanes 1 and 2) or with (lanes 3 and 4)
pro-teinase K in the absence (lanes 1 and 3) or presence (lanes 2 and
4) of Triton X-100 Micosomal proteins (10 lg) were analyzed by
western blotting with anti-GmPDIL-1 serum and anti-GmPDIL-2
serum, respectively.
Trang 9serum or anti-GmPDIL-2 serum (data not shown).
Sufficient detection of proglycinin associated with
GmPDIL-1 and GmPDIL-2 was difficult, as only a
few proglycinin molecules may associate transiently
with PDI proteins in the ER Then, similar
experi-ments were performed with dithiothreitol-treated
coty-ledons Unfolded proglycinin may increase in the ER
in the presence of dithiothreitol Some proglycinin was
detected in the immunoprecipitant from the cotyledon
untreated with DSP, but much more proglycinin was
detected in the immunoprecipitant with anti-GmPDIL-1
and anti-GmPDIL-2 sera from the cotyledon treated
with DSP (Fig 7C, lanes 5–8) These results suggest
that GmPDIL-1 and GmPDIL-2 molecules associate
with unfolded proglycinin in the lumen of the ER in
the presence of dithiothreitol
Recombinant proteins, especially GmPDIL-2, exhib-ited chaperone activity in vitro, raising the question of whether they act as chaperones for proteins such as b-conglycinin, which have no intramolecular disulfide bonds We then looked for an association between GmPDIL-1 or GmPDIL-2 and b-conglycinin a¢-sub-unit [35S]b-conglycinin a¢-subunit was confirmed to be immunoprecipitated with anti-(b-conglycinin a¢-sub-unit) serum (Fig 7D, lanes 3 and 4) b-Conglycinin a¢-subunit was hardly detected in the immunoprecipi-tant with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum (data not shown) Then, the immunoprecipita-tion was performed with the cotyledons treated with tunicamycin Tunicamycin may increase unfolded b-conglycinin a¢-subunit in the ER, as the folding effi-ciency of glycoproteins such as b-conglycinin a¢-sub-unit may be lowered by inhibition of N-glycosylation b-Conglycinin a¢-subunit was detected in the immuno-precipitants with anti-GmPDIL-1 serum only from the cotyledons treated with DSP (Fig 7D, lane 6) Some b-conglycinin a¢-subunit was detected in the immuno-precipitants with anti-GmPDIL-2 serum from cotyle-dons untreated with DSP (Fig 7D, lane 7) Much more b-conglycinin a¢-subunit was detected in the immunoprecipitants with anti-GmPDIL-2 serum from the cotyledons treated with DSP (Fig 7D, lane 8) These results suggest that GmPDIL-1 and GmPDIL-2 associate with b-conglycinin a¢-subunit in the lumen of the ER in the presence of tunicamycin
A
C
B
D
Fig 7 Coimmunoprecipitation of GmPDIL-1 or GmPDIL-2 and pro-glycinin or b-conpro-glycinin a¢-subunit Confirmation of immunoprecipi-tation of GmPDIL-1 and GmPDIL-2 with each specific antibody Microsomes were isolated from cotyledons (150 mg beans) and treated with (+) or without ( )) DSP Proteins were extracted and immunoprecipitated with anti-GmPDIL-1 serum or anti-GmPDIL-2 serum The proteins extracted from the ER (lane 1) and the immu-noprecipitants (lanes 2 and 3) were analyzed by western blotting with anti-GmPDIL-1 serum (A) or anti-GmPDIL-2 serum (B) Aster-isks indicate rabbit serum immunoglobulins recovered by the first immunoprecipitation in the immunoprecipitant (C,D) Coimmunopre-cipitation experiments Cotyledons were pretreated with dithiothrei-tol (C) or tunicamycin (D) and labeled with Pro-mix L -[ 35 S] in vitro labeling mix for 1 h After labeling, microsomes were isolated and treated with (+) or without ( )) DSP The extracts from the micro-somes were subjected to immunoprecipitation with preimmune serum (lanes 1 and 2), anti-(glycinin acidic subunit) serum (C, lanes 3 and 4) anti-(b-conglycinin a¢-subunit) serum (D, lanes 3 and 4), anti-GmPDIL-1 serum (lanes 5 and 6), or anti-GmPDIL-2 serum (lanes 7 and 8) The precipitants were subjected to a second immu-noprecipitation with (glycinin acidic subunit) serum (C) or anti-(b-conglycinin a¢-subunit) serum (D) The final precipitants were subjected to SDS ⁄ PAGE and analyzed by fluorography The position
of proglycinins (pro11S) or b-conglycinin a¢-subunit (7S-a¢) is indi-cated on the right.
Trang 10In this study, we cloned and characterized the cDNAs
of GmPDIL-1 and GmPDIL-2 as members of the PDI
family The amino acid sequences and putative domain
structures of GmPDIL-1 and GmPDIL-2 were similar
to each other, and both recombinant proteins exhibited
thiol-oxidoreductase and chaperone activities
Antise-rum against recombinant GmPDIL-1 immunoreacted
with two soybean proteins of comparable masses that
had similar pIs It is unclear whether either protein
was the product of the GmPDIL-1 gene or of different
genes As the masses of both bands appeared to be
smaller to similar extents after treatment with
glycosi-dase F, it is unlikely that the differences in size were
due to differences in glycosylation In other plant
spe-cies, gene duplications are found at high frequencies
Previously, we found other soybean PDI family genes,
those encoding GmPDIS-1 and GmPDIS-2, which
might have arisen by gene duplication [26] Kainuma
et al purified a 63 kDa soybean protein from
cotyle-dons and characterized it as a PDI family protein [38]
N-terminal amino acid sequences of the peptide
frag-ments from the purified 63 kDa protein were analyzed,
and the sequence of a 63 amino acid fragment was
determined Within the sequence, 58 amino acids were
identical to the amino acid sequence of GmPDIL-1,
suggesting that PDIL-1 and the 63 kDa PDI are
homologous proteins encoded by different genes
Therefore, it seems likely that the doublet band may
be the 63 kDa PDI and GmPDIL-1
GmPDIL-1, GmPDIS-1 and GmPDIM mRNAs,
but not GmPDIL-2 and GmPDIS-2 mRNAs, were
ele-vated after ER stress [26,27] Expression of the
Arabid-opsis orthologs of GmPDIL-1, GmPDIS-1 and
GmPDIM has been revealed to be induced by ER
stress by DNA microarray analysis [36,37,39] In the
promoter regions of GmPDIL-1, GmPDIM [27], and
their Arabidopsis orthologs, consensus sequences of
ERSE were found Consensus sequences of ERSE were
frequently found in the promoter region of other
Ara-bidopsisgenes responsive to ER stress [36,37] In
addi-tion, a novel Arabidopsis transcription factor,
AtbZIP60, has been shown to activate transcription
from ERSE [40] Therefore, genes of these PDI family
members may be unfolded protein-responsive genes
that play important roles in maintaining homeostasis
of the ER under conditions of stress
The consensus sequences for seed-specific expression
were found in the promoter regions of both GmPDIL-1
and GmPDIL-2 However, the mRNA expression
patterns of GmPDIL-1 and GmPDIL-2 were different,
suggesting that the expression of these genes in
cotyle-dons is regulated differently and varies from that observed for storage proteins [18] A large amount of soybean storage proteins is synthesized in cotyledon cells during seed maturation [18], suggesting that abun-dant, nascent, unfolded proteins are translocated to the ER lumen A rapid increase in the workload of the
ER, as a result of the synthesis of storage proteins, may elicit an ER stress response However, it seems unlikely that ER stress arises during the normal matu-ration process of soybean seeds as decreases in the mRNA levels of GmPDIL-1, GmPDIS-1 [26] and GmPDIM [27] were observed during the accumulation
of the storage proteins Expression of certain seed stor-age proteins changed as a function of sulfur supply Under conditions of no sulfur, expression of glycinin,
a sulfur-rich storage protein, was decreased In contrast, expression of the b-conglycinin b-subunit, a sulfur-poor storage protein, was elevated [41] Sulfur regulation by such proteins is mediated by
O-acetyl-l-serine levels [42] On the other hand, the levels of GmPDIL-1 and GmPDIL-2 mRNAs were not affected
by the level of sulfur (supplementary Fig S4), suggest-ing that the levels of these mRNAs are not regulated
in a manner responsive to the expression levels of stor-age proteins The protein levels of both GmPDIL-1 and GmPDIL-2 were also differentially regulated in cotyledons during seed development Protein levels of GmPDIL-1, GmPDIS-1 and GmPDIM dramatically increased during seed maturation, but GmPDIL-2 and GmPDIS-2 were expressed at low levels during the same stage These results may reflect the importance of GmPDIL-1 in seed maturation
In general, the PDI family proteins catalyze the for-mation of disulfide bonds on nascent polypeptide chains in the ER Hence, GmPDIL-1 and GmPDIL-2 may support proglycinin folding that accompanies the formation of disulfide bonds in the ER of cotyledon cells However, the association of GmPDIL-1 or GmP-DIL-2 and proglycinin was barely detectable under normal conditions, whereas this association was detected in the presence of dithiothreitol, which inhib-its disulfide bond formation in the ER and may cause the accumulation of unfolded proglycinin As the active sites of the PDI family proteins are reduced in the ER in the presence of dithiothreitol, neither GmP-DIL-1 nor GmPDIL-2 forms a mixed disulfide bond with the cysteine residues of proglycinin Therefore, GmPDIL-1 and GmPDIL-2 could noncovalently asso-ciate with proglycinin in the presence of dithiothreitol, suggesting that GmPDIL-1 and GmPDIL-2 may func-tion as molecular chaperones for proglycinin rather than thiol-oxidoreductases The chaperone activity of GmP-DIL-1 for rhodanese was low (Fig 2B) GmPGmP-DIL-1