However, recombinant GmPDIL-3a and GmPDIL-3b did not function as oxidoreductases or as molecular chaperones in vitro, although a proportion of each protein formed complexes in both thiol
Trang 1protein disulfide isomerase family proteins with nonclassic active center motifs
Kensuke Iwasaki1, Shinya Kamauchi1,*, Hiroyuki Wadahama1, Masao Ishimoto2, Teruo Kawada1 and Reiko Urade1
1 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan
2 National Agricultural Research Center for Hokkaido Region, Sapporo, Japan
Introduction
Secretory, organelle and membrane proteins are
synthe-sized and folded with the assistance of molecular
chap-erones and other folding factors in the endoplasmic
reticulum (ER) In many cases, the process of protein
folding is accompanied by N-glycosylation and the
for-mation of disulfide bonds [1] Disulfide bonds are
essential for structural stabilization and for regulation
of the functions of many secretory and plasma mem-brane proteins [2,3] The formation and isomerization
of disulfide bonds are catalyzed by protein disulfide isomerase (PDI) and other PDI family proteins located
in the ER [4,5] PDI has two thioredoxin domains containing the redox active site CGHC (a and a¢) and two inactive domains (b and b¢) [6] Other PDI family
Keywords
cotyledon; disulfide bond; endoplasmic
reticulum; protein disulfide isomerase;
soybean
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 GmPDIL-3a, GmPDIL-3b and
genomic GmPDIL-3b are available in the
DDBJ ⁄ EMBL ⁄ GenBank databases under
accession numbers AB189468, AB189469
and AB303863, respectively
(Received 2 April 2009, revised 14 May
2009, accepted 29 May 2009)
doi:10.1111/j.1742-4658.2009.07123.x
Protein disulfide isomerase (PDI) and other PDI family proteins are mem-bers of the thioredoxin superfamily and are thought to play important roles
in disulfide bond formation and isomerization in the endoplasmic reticulum (ER) The exact functions of PDI family proteins in plants remain unknown In this study, we cloned two novel PDI family genes from soy-bean leaf (Glycine max L Merrill cv Jack) The cDNAs encode proteins of
520 and 523 amino acids, and have been denoted GmPDIL-3a and GmP-DIL-3b, respectively GmPDIL-3a and GmPDIL-3b are the first plant ER PDI family proteins reported to contain the nonclassic redox center motif CXXS⁄ C, and both proteins are ubiquitously expressed in the plant body However, recombinant GmPDIL-3a and GmPDIL-3b did not function as oxidoreductases or as molecular chaperones in vitro, although a proportion
of each protein formed complexes in both thiol-dependent and thiol-inde-pendent ways in the ER Expression of GmPDIL-3a and GmPDIL-3b in the cotyledon increased during seed maturation when synthesis of storage proteins was initiated These results suggest that GmPDIL-3a and GmPDIL-3b may play important roles in the maturation of the cotyledon
by mechanisms distinct from those of other PDI family proteins
Structured digital abstract
l MINT-7137566 : Bip (uniprotkb: Q587K1 ), GmPDIL-3b (genbank_nucleotide_g: 51848586 ) and GmPDIL-3a (genbank_nucleotide_g: 51848584) colocalize ( MI:0403 ) by cosedimentation through density gradients ( MI:0029 )
Abbreviations
ER, endoplasmic reticulum; PDI, protein disulfide isomerase; PDILT, testis-specific protein disulfide isomerase-like protein; PVDF,
poly(vinylidene difluoride).
Trang 2members contain one or more thioredoxin domains [7].
PDI family proteins containing the redox active center
transfer the disulfide bond between the two cysteine
residues of their active site to the substrate protein [8]
Recently, it has been shown that PDI family proteins
containing nonclassic redox motifs, such as yeast
Eug1p and mammalian testis-specific PDI-like protein
(PDILT) and ERp44, may function in protein folding,
retention or transport of ER proteins, or regulation of
ER calcium channel activity [9–12]
In plants, a set of 22 orthologs of known PDI family
proteins was discovered by a genome-wide search of
Arabidopsis thaliana, and was separated into 10
phylo-genetic groups [13] However, little is known about the
physiological roles of plant PDI family members
Stud-ies investigating their contribution to protein folding,
transport and quality control are only now beginning
Soybean seeds contain large amounts of protein,
especially in their cotyledon cells, where large
quanti-ties of storage proteins such as glycinin and
b-conglyci-nin are synthesized and folded in the ER during seed
development [14,15] PDI family proteins are predicted
to function in collaboration with other molecular
chaperones during the folding of these proteins
Previ-ously, we identified and characterized five soybean
PDI family proteins belonging to group I (GmPDIL-1),
group II (GmPDIL-2), group IV (GmPDIS and
GmP-DIS-2), and group V (GmPDIM) [16–18], which all
contain two classic CGHC motifs All of these proteins
had thiol oxidoreductase activity in vitro and were
ubiquitously expressed in the body of the plant
GmP-DIL-1, GmPDIM and GmPDIS-1 are unfolded protein
response genes, and were upregulated by the
accumula-tion of unfolded proteins in the ER GmPDIS-1 and
GmPDIM associate with proglycinin (glycinin
precur-sor prior to proteolytic processing), and GmPDIL-1
and GmPDIL-2 associate with proglycinin and
b-con-glycinin in the ER, suggesting that they may play
important roles in folding and in formation and
rearrangement of disulfide bonds in the storage
proteins
Group III PDI family proteins have not been
stud-ied Putative amino acid sequences obtained from
Arabidopsis genome sequence predict the typical PDI
domain structure a–b–b¢–a¢, but that both the
a-domain and a¢-domain contain nonclassic CXXS ⁄ C
motifs as opposed to the more traditional CGHC
sequence In this study, we describe soybean group III
PDI family ER proteins, namely GmPDIL-3a and
GmPDIL-3b, and identify nonclassic redox center
CXXS⁄ C motifs in each Characterization of
GmP-DIL-3a and GmPDIL-3b and changes in their
expression during seed development are described In
addition, our data suggest that GmPDL-3a and GmPDIL-3b form protein complexes in both thiol-dependent and thiol-inthiol-dependent ways in the ER
Results
cDNA cloning of GmPDIL-3a and GmPDIL-3b
In order to clone the soybean orthologs of Arabidopsis PDI-like1-5 and PDI-like1-6 (group III PDIs) [13], we first obtained their nucleotide sequences from the Insti-tute for Genomic Research Soybean Index and used them in blast searches We identified the tentative consensus sequence TC183516, and primer sets were designed on the basis of this sequence Two cDNAs were cloned using RNA extracted from young soybean leaves by 5¢-RACE and 3¢-RACE using these primers Genomic GmPDIL-3b was cloned and sequenced, whereas the genomic sequence data of GmPDIL-3a were obtained from phytozome v3.1.1 (Department of Energy Joint Genome Institute and the Center for Integrative Genomics, http://www.phytozome.net/soy-bean#A) Comparison of the genomic sequences of GmPDIL-3a and GmPDIL-3b with those of the Ara-bidopsis and rice orthologs showed conservation of exon⁄ intron structure across these plant species (Fig S1)
Two AUG codons (AUG1 and AUG2) were found upstream of the putative functional translation initia-tion codon (AUG3) Initiainitia-tion of translainitia-tion from AUG3 produces 520 or 523 amino acid proteins named GmPDIL-3a and GmPDIL-3b, respectively, in both mRNAs (Figs 1A and S2) To determine whether AUG3 in both mRNAs was the authentic initiation codon, in vitro translation reactions were performed using in vitro transcribed wild-type mRNA,
or an mRNA containing an AUG codon mutant(s)
A 54 kDa polypeptide was generated when wild-type GmPDIL-3a mRNA was used (Fig 1B, lane 2), but was not detected when a mutant GmPDIL-3a mRNA that contained AGG in place of AUG3 was used (lane 6) However, this polypeptide was translated when both AUG1 and AUG2 were changed to AGG, confirming that neither AUG1 nor AUG2 is the authentic initiation codon (Fig 1B, lanes 3–5) A lar-ger amount of the 54 kDa polypeptide was generated from the GmPDIL-3a mRNA with the AUG2 muta-tion (Fig 1B, lanes 4 and 5) than from that contain-ing only the AUG1 mutation (lane 3) or wild-type mRNA (lane 2), suggesting that initiation events can also begin at AUG2, but are unproductive because of the stop codon located just upstream of AUG3 On the other hand, AUG1 in GmPDIL-3a mRNA may
Trang 3not be used as efficiently for translation initiation,
and therefore may not interfere with translation from
AUG3 For GmPDIL-3b, a 59 kDa polypeptide was
generated from wild-type mRNA, and from mRNA
that contained AGG in place of both AUG1 and
AUG2 (Fig 1C, lanes 2–5), whereas it was not
trans-lated from mutant mRNA in which both AUG3 and
AUG4 were changed to AGG (lane 6) The amino
acid sequence identity shared between GmPDIL-3a
and GmPDIL-3b, excluding the signal peptides, was
92% The structure of GmPDIL-3a and GmPDIL-3b
was predicted to contain the four domains a–b–b¢–a¢
(Fig 1D) GmPDIL-3a and GmPDIL-3b have two
predicted thioredoxin domains between amino acids
65–164 and 403–481, and 68–167 and 406–508,
respectively, corresponding to the a-domain and
a¢-domain of PDI [7] Notably, both GmPDIL-3a and
GmPDIL-3b lack the two classic PDI redox-active
CGHC motifs within the a-domain and a¢-domain
Instead, they both contain the sequence CPRS in the
a-domain and CMNC or CINC in the a¢-domain GmPDIL-3a and GmPDIL-3b contain a C-terminal KDEL sequence that probably functions in ER reten-tion⁄ retrieval [19], and one putative N-glycosylation site
Recombinant GmPDIL-3a and GmPDIL-3b have neither thiol oxidoreductase nor chaperone activities in vitro
Many types of PDI family proteins have oxidative refolding activity on unfolded polypeptides and⁄ or the ability to reduce disulfide bonds [7,8] To determine whether GmPDIL-3a or GmPDIL-3b possesses these activities, recombinant mature forms of each pro-tein were expressed in Escherichia coli and purified (Fig S3A,B) Both proteins were soluble and eluted in
a monomeric form from a gel filtration column (data not shown) It was confirmed by far-UV CD experi-ments that the two proteins were folded (Fig S3C)
A
D
Fig 1 Identification of the initiation codons
in GmPDIL-3a and Gm-PDIL-3b mRNAs (A) Schematic representation of the structure of GmPDIL-3a and GmPDIL-3b mRNAs The putative ORFs (gray boxes) of GmPDIL-3a and GmPDIL-3b are indicated AUG1, AUG2, AUG3 and AUG4 indicate the first, second, third and fourth AUG codons from the 5¢-termini, respectively Crosses indicate ter-mination codons (B) In vitro translation of GmPDIL-3a Translation reactions were per-formed without (lane 1) or with (lane 2) 1 lg
of wild-type GmPDIL-3a mRNA or mutant GmPDIL-3a mRNA, of which the first (lane 3), second (lane 4), first and second (lane 5)
or third AUG (lane 6) was replaced with AGG Products were separated by SDS ⁄ PAGE and detected by fluorography (C) In vitro translation of GmPDIL-3b Trans-lation reactions were performed without (lane 1) or with (lane 2) 1 lg of wild-type GmPDIL-3b mRNA, or with mutant GmP-DIL-3b mRNA, of which the first (lane 3), second (lane 4), first and second (lane 5)
or third and fourth AUGs (lane 6) were replaced with AGG (D) Putative domain structure of GmPDIL-3a and GmPDIL-3b The boxes indicate the domain boundaries predicted by an NCBI conserved domain search Black boxes in domain-a and domain-a¢ represent the CPRS and CXXC motifs A closed circle with a bar represents
an N-glycosylation consensus site SP, signal peptide.
Trang 4As shown in Fig 2A,B, neither protein was able to
catalyze the oxidation of thiol residues on the synthetic
peptide and the oxidative refolding of reduced and
denatured RNase A In addition, neither protein
reduced the disulfide bond in insulin (Fig 2C) As it
has been reported that mammalian PDI family
pro-teins function together with other PDI family propro-teins
or with molecular chaperones to effectively fold
nas-cent proteins [20–23], we next tested the ability of
GmPDIL-3a and GmPDIL-3b to work in concert with
the other soybean PD1 proteins Gm-PDIL-1 and
GmPDIL-2 [16] However, as shown in Fig 2B,
GmP-DIL-3a and GmPDIL-3b had no stimulatory effect on
the oxidative refolding of RNase A by GmPDIL-1 and
GmPDIL-2 when mixed together, further confirming
that the functional properties of GmPDIL-3a and GmPDIL-3b are probably unique
Among other soybean PDI proteins, GmPDIL-1 and GmPDIL-2 function as molecular chaperones, and prevent the aggregation of unfolded rhodanese [16]
We next tested whether GmPDIL-3a and GmPDIL-3b function in a similar manner Aggregation of unfolded rhodanese occurred over 14 min in the absence of PDI, and was partially inhibited by GmPDIL-2 (Fig 2D) On the other hand, GmPDIL-3a and GmP-DIL-3b did not inhibit the aggregation of rhodanese, even at concentrations up to 1.2 lm (3 : 1 molar ratio, PDI to rhodanese), suggesting that they do not func-tion as molecular chaperones like other PDI family proteins
Fig 2 3a and 3b have neither oxidoreductase nor chaperone activity (A) Thiol oxidase activities of recombinant GmPDIL-3a (open circles), GmPDIL-3b (solid circles) and bovine PDI (solid squares) were assayed using the synthetic peptide as a substrate as described in Experimental procedures (B) Oxidative refolding activity of recombinant GmPDIL-3a (3a), GmPDIL-3b (3b), GmPDIL-1 (L-1), L-1 plus 3a or 3b, GmPDIL-2 (L-2), or L-2 plus 3a or 3b Activity was assayed by measuring the RNase activity produced through the regeneration of the active form of reduced RNase A Data represent the mean ± standard deviation for three experiments (C) Thiol reductase activities of recombinant GmPDIL-3a (open circles), GmPDIL-3b (solid circles) and bovine PDI (solid squares) were assayed using insulin as a substrate (D) Chaperone activities of recombinant GmPDIL-3a, GmPDIL-3b and GmPDIL-2 were assayed by measuring the aggregation of rhodanese in the absence (open triangles) or presence of GmPDIL-3a (open circles), GmPDIL-3b (solid circles), or GmPDIL-2 (solid squares).
Trang 5Expression of GmPDIL-3a and GmPDIL-3b in
soybean tissue
We next prepared antiserum directed against
recombi-nant GmPDIL-3a and a synthetic peptide containing
sequences found in 3b, but not in
GmPDIL-3a Anti-GmPDIL-3a serum recognized both
recombi-nant GmPDIL-3a and GmPDIL-3b (Fig 3A, lanes 1
and 2), whereas anti-GmPDIL-3b serum reacted
exclu-sively with recombinant GmPDIL-3b (Fig 3A, lanes 4
and 5) Anti-GmPDIL-3a serum reacted with both a
55 kDa and a 59 kDa band in western blot analysis
of cotyledon proteins (Fig 3A, lane 3) Both the 55
and 59 kDa bands were N-glycosylated, as digestion
experiments using glycosidase F resulted in the
bands shifting to 53 and 57 kDa, respectively (Fig 3B)
The cotyledon proteins that were deglycosylated
with glycosidase F and detected with the serum were
characterized by two-dimensional gel electrophoresis and western blot analysis Two spots of 53 and
57 kDa, with isoelectric points of 5.3 and 5.1, respec-tively, were detected with anti-GmPDIL-3a serum (Fig 3C, upper panel) The isoelectric point of the
53 kDa spot (5.3) was identical to a pI value calcu-lated from the amino acid sequence of GmPDIL-3a, and the isoelectric point of the 57 kDa spot (5.1) was consistent with that from the amino acid sequence of GmPDIL-3b GmPDIL-3b antiserum detected the
57 kDa spot, but not the 53 kDa spot (Fig 3C, lower panel), suggesting that the 53 and 57 kDa spots are GmPDIL-3a and GmPDIL-3b, respectively Samples from different parts of the soybean plant were then prepared and analyzed by western immunoblot GmPDIL-3a and GmPDIL-3b were expressed in roots, stems, trifoliolate leaves, flowers, and cotyledons (Fig 3D), suggesting that it is a ubiquitously expressed protein that probably performs a function common to all of these tissues
Both GmPDIL-3a and GmPDIL-3b have N-termi-nal sigN-termi-nal sequences that target these proteins to the
A
B D
C
Fig 3 Expression of GmPDIL-3a and GmPDIL-3b in soybean
tissues (A) Purified recombinant GmPDIL-3a (lanes 1 and 4),
GmPDIL-3b (lanes 2 and 5) and proteins extracted from the
cotyle-don (lane 3) were analyzed by western blot using anti-GmPDIL-3a
serum (lanes 1–3) or anti-GmPDIL-3b serum (lanes 4 and 5).
(B) GmPDIL-3a and GmPDIL-3b are N-glycosylated in soybean The
proteins extracted from the cotyledon were treated without (lane 1)
or with (lane 2) glycosidase F Proteins were analyzed by western
blot using anti-GmPDIL-3a serum (C) Cotyledon proteins were
treated with glycosidase F, separated by two-dimensional
electro-phoresis, and analyzed by western blot using anti-GmPDIL-3a
serum (upper panel) or anti-GmPDIL-3b serum (lower panel) pI,
iso-electric point (D) Thirty micrograms of protein extracted from the
cotyledon (80 mg bean) (lane 1), root (lane 2), stem (lane 3), leaf
(lane 4) and flower (lane 5) were analyzed by western blot using
anti-GmPDIL-3a serum.
A
B
Fig 4 Localization of GmPDIL-3a and GmPDIL-3b in the ER lumen (A) Microsomes were isolated from cotyledons (100 mg bean), and were fractionated on isopyknic sucrose gradients in the presence of MgCl2or EDTA Proteins from each fraction were ana-lyzed by western blot using anti-GmPDIL-3a serum or anti-BiP serum The top of the gradient is on the left, and 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 blot using anti-GmPDIL-3a serum.
Trang 6ER, and a C-terminal ER retention sequence (KDEL).
To confirm localization of 3a and
GmPDIL-3b to the ER, microsomes were prepared from
cotyle-don cells and were separated by sucrose gradient
centrifugation in the presence of MgCl2or EDTA, and
fractions were collected and analyzed by western blot
(Fig 4A) Peaks corresponding to GmPDIL-3a and
GmPDIL-3b were detected at a density of 1.21 gÆmL)1
in the presence of MgCl2 In the presence of EDTA,
which releases ribosomes from the rough ER [24], the
peaks of GmPDIL-3a and GmPDIL-3b were shifted to
the lighter sucrose fractions and therefore had a
reduced density of 1.16 gÆmL)1, suggesting that
GmP-DIL-3a and GmPDIL-3b localize to the rough ER To
confirm the presence of GmPDIL-3a and GmPDIL-3b
in the ER lumen, microsomes were prepared from
cotyledon cells and were treated with proteinase K in
the absence or presence of Triton X-100 Both
GmP-DIL-3a and GmPDIL-3b were resistant to protease
treatment in the absence of detergent, and were
degraded when detergent was added (Fig 4B),
suggest-ing that they are both luminal proteins
Generally, PDI family proteins play important roles
in folding and quality control of nascent polypeptides
[25] In soybean cotyledon, large amounts of seed
stor-age proteins such as glycinin and b-conglycinin are
synthesized and translocated into the ER lumen during
the maturation stage of embryogenesis Therefore, we
next measured the mRNA and protein levels of
GmP-DIL-3a and GmPDIL-3b by real-time RT-PCR and
western blotting, respectively, during different stages of
development The amounts of pro-b-conglycinin and
proglycinin are considered to be nearly equivalent to
the synthesis levels of both b-conglycinin and glycinin,
as b-conglycinin and proglycinin are transient
pro-tein forms that are present in the ER prior to
process-ing in the protein storage vacuoles The synthesis of
proglycinin and pro-b-conglycinin was initiated when
the seeds achieved a mass of 50 mg (Fig 5A, lanes 2
and 4) The amount of GmPDIL-3a and GmPDIL-3b
proteins increased until the seeds grew from 40 to
80 mg (Fig 5A, lane 1) Thereafter, the level remained
constant This event correlated with the amount of
GmPDIL-3a mRNA, although the amount of
GmP-DIL-3b mRNA was not consistent with the amount of
GmPDIL-3b protein expression (Fig 5B)
Expression of many ER-resident proteins can be
upregurated by ER stress in plant cells [26–28]
There-fore, we next measured the amounts of GmPDIL-3a
and GmPDIL-3b mRNA in cotyledon cells under
stress by treatment with tunicamycin or dithiothreitol
The amount of neither mRNA was affected by either
treatment, whereas the mRNA of BiP, which is a
representative unfolded protein response gene, was dramatically upregulated (data not shown) These data suggest that expression of neither GmPDIL-3a nor GmPDIL-3b is influenced by cellular stress
GmPDIL-3a and GmPDIL-3b form protein complexes in the ER
Many ER proteins form complexes with other ER resi-dent proteins, and associate with nascent polypeptides during folding [22,29] We next determined whether GmPDIL-3a or GmPDIL-3b forms complexes in the
ER Cotyledon proteins were extracted with digitonin
A
B
Fig 5 Expression of GmPDIL-3a and GmPDIL-3b in soybean coty-ledons during maturation (A) Cotyledon proteins (25 lg) were sepa-rated by SDS ⁄ PAGE and immunostained with anti-GmPDIL-3a serum (lane 1), anti-pro-b-conglycinin a¢ serum (lane 2), anti-b-con-glycinin a¢ serum (lane 3), and anti-anti-b-con-glycinin acidic subunit serum (lanes 4 and 5) 3a, GmPDIL-3a; 3b, GmPDIL-3b; Pro 7S-a¢, pro-b-conglycinin a¢; 7S-a¢, mature-pro-b-conglycinin a¢; Pro 11S, proglyci-nin; and 11S-A, mature glycinin acidic subunit (B) GmPDIL-3a mRNA (upper panel) and GmPDIL-3b mRNA (lower panel) were quantified by real-time RT-PCR Each value was normalized by dividing it by that for actin mRNA Values were calculated as a percentage of the highest value obtained during maturation Data represent the mean ± standard deviation of four experiments.
Trang 7and were separated by blue native PAGE, which
pro-vides analysis of native proteins and protein complexes
[30] Blue native gels were subjected to SDS⁄ PAGE as
a second-dimension separation, followed by western
blot analysis (Fig 6A) Multiple complexes containing
GmPDIL-3a or GmPDIL-3b with molecular sizes
larger than those of monomeric GmPDIL-3a or
GmPDIL-3b were detected in the region of 130–
300 kDa (Fig 6A) A proportion of GmPDIL-3a or
GmPDIL-3b in these complexes was detected as mixed
disulfides When western blots were performed in the
presence of N-ethylmaleimide to trap any
disulfide-bound intermediates under nonreducing conditions,
trace amounts of GmPDIL-3a and GmPDIL-3b
mole-cules were found to be engaged in intermolecular, disulfide-linked complexes of approximately 130 kDa (Fig 6B, lane 3) As these mixed disulfide bonds disap-peared under reducing conditions (Fig 6B, lane 1), it
is likely that GmPDIL-3a or GmPDIL-3b interacts with proteins in the ER through a redox-dependent mechanism When nonreducing experiments were per-formed after crosslinking treatment of associated pro-teins with dithiobis(succinimidyl propionate), the
130 kDa complexes decreased in abundance, whereas complexes ranging in size from 200 kDa to greater than 250 kDa appeared (Fig 6B, lane 4) This could suggest that a proportion of the 130 kDa disulfide-linked complexes associated noncovalently with other proteins Partner proteins for GmPDIL-3a or GmP-DIL-3b in these complexes remain to be identified
Discussion
In this report, we characterized new members of the plant PDI family, which we now refer to as GmP-DIL-3a and GmPDIL-3b The conserved exon struc-ture of the GmPDIL-3a and GmPDIL-3b genomic sequences suggests that both genes may have arisen
by gene or chromosome duplication The conservation
of GmPDIL-3a and GmPDIL-3b orthologs in higher plants suggests that they play important physiological roles in these systems In the cotyledon, maximal expression of GmPDIL-3a and GmPDIL-3b in the late stage of seed development suggests that they per-form a unique role in folding or in accumulation of storage proteins, which are synthesized during this stage Both GmPDIL-3a and GmPDIL-3b have the same domain architecture, a–b–b¢–a¢, as the soybean group I and group II PDI family proteins GmPDIL-1 and GmPDIL-2 GmPDIL-3a and GmPDIL-3b share 30% identity with GmPDIL-2, but contain the non-classic redox motif CXXS⁄ C as opposed to the more common CGHC motif Atypical CXXS⁄ C motifs in thioredoxin domains have been noted in some PDI family proteins of yeast and animals [9–12], but this
is the first report to confirm expression of such pro-teins in the ER of plants A CXXS motif and a CXXC motif in the N-terminal and C-terminal thi-oredoxin domains and the surrounding sequences are extremely conserved between plant orthologs of GmP-DIL-3a and GmPDIL-3b, suggesting an important functional role for these regions PDI requires both cysteine residues present in the redox active site for oxidase activity, but the N-terminal cysteine is suffi-cient for isomerase function [31,32] Recombinant GmPDIL-3a and GmPDIL-3b showed no oxidase activity in vitro, although they have a CXXC motif in
A
B
Fig 6 GmPDIL-3a or GmPDIL-3b form protein complexes in a
thiol-dependent or thiol-independent manner in the ER (A)
Cotyle-don proteins (100 mg bean) were extracted with digitonin and
ana-lyzed by two-dimensional electrophoresis on blue native (BN) PAGE
and SDS ⁄ PAGE and western blot using anti-GmPDIL-3a serum (B)
Cotyledon proteins (100 mg bean) treated with (lanes 2 and 4) or
without (lanes 1 and 3) dithiobis(succinimidyl propionate) (DSP)
were lysed in the presence of N-ethylmaleimide and analyzed by
10% reducing (R) or nonreducing (NR) SDS ⁄ PAGE and western blot
using anti-GmPDIL-3a serum.
Trang 8their a¢-domain Additionally, GmPDIL-3a and
GmP-DIL-3b showed no reductase activity Replacement of
the second and third amino acids in classic
redox-active CGHC motifs with methionine or isoleucine
and asparagine in GmPDIL-3a and GmPDIL-3b may
be the cause of the lack of such enzymatic activities
Alternatively, the lack of other amino acids, such as
arginine, which is important for the regulation of the
active site redox potential in human PDI [8,33], may
cause the lack of enzymatic activity Mammalian
PDILT, which has the same domain structure as
PDI, but lacks oxidoreductase activity, has been
dem-onstrated to have chaperone activity in vitro [34] As
PDILT forms a complex with the calnexin homolog
calmegin in vitro, this protein is thought to function
as a redox-inactive chaperone for glycoprotein folding
in testis However, neither GmPDIL-3a nor
GmP-DIL-3b showed chaperone activity in vitro, although
it was demonstrated that they formed noncovalent
complexes with unidentified proteins in the ER In
addition, interaction between GmPDIL-3a or
GmP-DIL-3b and storage proteins such as proglycinin and
b-conglycinin, and other ER molecular chaperones
such as calnexin, calreticulin, BiP and PDI family
proteins, in vivo was not detected (data not shown),
suggesting that GmPDIL-3a and GmPDIL-3b may
not act as chaperones in the ER
A proportion of GmPDIL-3a and GmPDIL-3b
formed mixed disulfide complexes with an
unidenti-fied protein in the ER Mammalian PDI family
pro-tein ERp44 forms transient intermolecular bonds
with substrate proteins or with the disulfide donor
Ero1s ERp44 cannot be an oxidoreductase, because
it has CRFS instead of CGHC However, the
cyste-ine in this motif forms transient mixed disulfide
bonds with IgM subunits, adiponectin, and
formyl-glycine-generating enzyme, which are devoid of ER
retention signals, to regulate their transport [35–38]
ERp44 also functions to retain Ero1a and Ero1b into
the ER by forming a mixed disulfide bond and by
controlling the ratio of redox isoforms of Ero1a
[12,38,39] Instead, GmPDIL-3a and GmPDIL-3b
may function as retention or redox devices, like
ERp44, rather than as chaperones In any case,
iden-tification of partner proteins in the mixed disulfide
complex and noncovalent complexes of GmPDIL-3a
and GmPDIL-3b will be required to establish their
physiological function
Little is known about the coordinated function of
ER chaperones in the plant Previously, we observed
that at least four types of PDI family proteins
(GmP-DIL-1, GmPDIL-2, GmPDIM, and GmPDIS) were
expressed ubiquitously in the plant body [16–18] Thus,
it may be difficult to substitute other PDI family pro-teins for GmPDIL-3a or Gm-PDIL-3b, as they proba-bly have unique functions in the plant The details of how PDI family proteins contribute to ER function and protein folding are beginning to emerge, and, importantly, knowledge concerning GmPDIL-3a and GmPDIL-3b can now be applied to the understanding
of how divergent PDI family proteins contribute to quality control in the ER, and how this process influ-ences vital plant function
Experimental procedures
Plants Soybean seeds (Glycine max L Merrill cv Jack) were planted in 5 L pots and grown in a controlled environment chamber at 25C 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
Cloning of GmPDIL-3a and GmPDIL-3b Cloning of the cDNAs for GmPDIL-3a and GmPDIL-3b was performed by 3¢-RACE and 5¢-RACE Soybean trifolio-late center leaves were frozen under liquid nitrogen and then ground into a fine powder with an SK-100 micropestle (Tokken, Inc., Chiba, Japan) Total RNA was isolated using the RNeasy Plant Mini kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer’s protocol mRNA was isolated from total RNA with the PolyATtract mRNA Isolation System (Promega Corporation, Madison, WI, USA) The 3¢-RACE method was performed using the SMART RACE cDNA Amplification kit (Clontech Labora-tories, Inc., Mountain View, CA, USA), according to the manufacturer’s protocol, using the primer 5¢-ACTCTCC TGAATCTTGTTAAC-3¢ The amplified DNA fragments were subcloned into the pT7Blue vector (TaKaRa Bio Inc., Shiga, Japan), and the inserts in the plasmid vectors were sequenced using the fluorescence dideoxy chain termination method and an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) The 5¢-RACE method was performed using the primer 5¢-GAAGCGTGG GGTAGTCATTCACTTGCAG-3¢, which was designed on the basis of the sequence obtained by 3¢-RACE The ampli-fied DNA fragments were subcloned into the pT7Blue vector and sequenced as described above
In vitro translation Plasmids containing the cDNA fragments encoding GmP-DIL-3a or GmPDIL-3b with mutations in the ATG codons
Trang 9were constructed as follows DNA fragments with mutations
were amplified from cDNA of wild-type GmPDIL-3a or
GmPDIL-3b as template by PCR, using a mutagenic primer
and a forward primer (Table S1) Then, the second PCR
was performed using the reaction mixture of the first PCR
and a reverse primer (Table S1) Wild-type and mutagenic
DNA fragments were subcloned at the SpeI restriction site
into pT7Blue (TaKaRa Bio Inc.) and sequenced Plasmids
were linearized by digestion with KpnI, and were
tran-scribed in vitro using a RiboMax Large Scale RNA
produc-tion systems kit (Promega Corporaproduc-tion) In vitro translaproduc-tion
reactions were performed in in a total volume of 25 lL
containing 1 lg of mRNA, 555 kBq of L-[35S] in vitro cell
labeling mix (37 TBqÆmmol)1; GE Healthcare BioSciences
Corporation, Piscataway, NJ, USA), 80 lm cysteine⁄
methi-onine-free amino acid mixture, 0.8 units of RNasin
ribo-nuclease inhibitor, 120 mm potassium acetate, and 12.5 lL
of wheat germ extract (Promega Corporation) at 25C
for 90 min Proteins were separated by 10% SDS⁄ PAGE,
and were detected by fluorography with ENLIGHTNING
(Perkin Elmer Life Sciences, Boston, MA, USA)
Construction of expression plasmids
Expression plasmids encoding mature GmPDIL-3a (Thr24–
Leu520) and GmPDIL-3b (Ser27–Leu523), excluding the
putative signal peptides, were constructed as follows DNA
fragments were amplified from 3a and
GmPDIL-3b cDNAs by PCR using the primers 5¢-GACGACGACA
AGATGGAGGTTAAGGATGAGTTG-3¢ and 5¢-GAG
GAGAAGCCCGGTCTATAACTCATCTTTGAGTAC-3¢
for GmPDIL-3a, and 5¢-GACGACGACAAGATGGAGG
TTGAGGATGAGTTGG-3¢ and 5¢-GAGGAGAAGCCCG
GTTCATAACTCATCTTTGACGAC-3¢ for GmPDIL-3b
Amplified DNA fragments were subcloned into pET46Ek⁄
LIC (EMD Biosciences, Inc., San Diego, CA, USA) and
sequenced The recombinant proteins have the His-tag
linked to the N-terminus
Expression and purification of recombinant
GmPDIL-3a and GmPDIL-3b
E coli BL21(DE3) cells were transformed with the
expres-sion vectors described above The expresexpres-sion of
recombi-nant GmPDIL-3a was induced in the presence of 0.4 mm
isopropyl thio-b-d-galactoside at 4C for 5 days, whereas
the expression of recombinant GmPDIL-3b was induced in
the presence of 0.4 mm isopropyl thio-b-d-galactoside at
30C for 4 h Extraction and purification of recombinant
proteins was performed as described previously [18] The
concentration of each protein was determined by measuring
the absorbance at 280 nm using the molar extinction
coeffi-cient of 31 830 m)1Æcm)1 for both proteins, as calculated
according to the modified method of Gill and von Hippel
[40] The concentration of the proteins extracted from
soybean tissues was measured by RC-DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA)
Enzymatic activity assays Thiol oxidative refolding activity was assayed as previously described by measuring RNase activity produced through the regeneration of the active form from reduced and denatured RNase A in the presence of 0.5 lm recombinant GmPDIL-3a, GmPDIL-3b, GmPDIL-1, or GmPDIL-2 [41,42] Thiol reductase activity was measured as previously described, where the glutathione-dependent reduction of insulin was measured by Morjana and Gilbert [43] Briefly, 50 lg of bovine PDI (TaKaRa Bio Inc.), recombinant GmPDIL-3a or recombinant GmPDIL-3b was incubated in 1 mL of 0.2 m sodium phosphate buffer (pH 7.5) containing 5 mm EDTA, 3.7 mm reduced glutathione, 0.12 mm NADPH, 16 U of glu-tathione reductase (Sigma-Aldrich Inc., St Louis, MO, USA) and 30 lm insulin (Sigma-Aldrich Inc.) at 25C, and absor-bance was monitored at 340 nm Oxidase activity was assayed using a synthetic peptide, NH2 -NRCSQGSCWN-COOH, as described by Alanen et al [44] Briefly, 0.5 lm bovine PDI, recombinant GmPDIL-3a or recombinant GmPDIL-3b was incubated in 0.2 m sodium phosphate⁄ citrate buffer (pH 6.5), 2 mm reduced glutathione, 0.5 mm oxidized glutathione and 5 lm synthetic peptide at 25C, and fluorescence was monitored at 350 nm with excitation at
280 nm on a Hitachi F-3000 fluorescence spectrophotometer (Hitachi Ltd, Tokyo, Japan)
Chaperone activity assays Chaperone activity was assayed as described previously [45] Briefly, aggregation of 0.4 lm rhodanese (Sigma-Aldrich Inc.) during refolding was measured spectrophoto-metrically at 320 nm (25C) in the absence or presence
of 1.2 lm recombinant GmPDIL-3a, GmPDIL-3b, and GmPDIL-2
Antibody production Guinea pig antiserum specific for GmPDIL-3a and rabbit antiserum specific for a GmPDIL-3b peptide were prepared using recombinant GmPDIL-3a and the synthetic peptide GSVTEAEKFLRKY, which was conjugated to keyhole limpet hemocyanin by Operon Biotechnologies, K.K (Tokyo, Japan) Antisera specific for pro-b-conglycinin, b-conglycinin, glycinin acidic subunit and BiP were prepared as described previously [18]
Western blot analysis Proteins were extracted from plant tissues by boiling in SDS⁄ PAGE buffer [46] To cleave N-linked glycans,
Trang 10proteins were extracted from the cotyledons in buffer
containing 0.02% SDS, 0.1 m Tris⁄ HCl (pH 8.6), and 1%
Nonidet P-40 Approximately 0.4 mg of protein was treated
with 10 mU of glycosidase F (Sigma-Aldrich Inc.) at 37C
for 16 h For crosslinking of proteins, slices of cotyledons
were homogenized with 20 strokes of a Dounce
homo-genizer at 4C in 3 mL of buffer containing 20 mm Hepes
(pH 7.2), 150 mm NaCl, 1% protease inhibitor cocktail for
plant cells (Sigma-Aldrich Inc.), and 20 mm
N-ethylmale-imide, in the presence or absence of 20 mm
dithiobis(succin-imidyl propionate) The homogenate was placed on ice for
2 h, and crosslinking was terminated by the addition of
20 mm glycine for 20 min on ice Proteins were then
sus-pended in SDS⁄ PAGE buffer and subjected to SDS ⁄ PAGE
[46], and were transferred to poly(vinylidene difluoride)
(PVDF) membranes For two-dimensional separation by
isoelectric focusing and SDS⁄ PAGE, SDS was removed
from the samples with the 2D clean-up kit (GE Healthcare
UK Ltd) Proteins (100 lg) were applied to 7 cm
Ready-Strip IPG Ready-Strips (Bio-Rad Laboratories), and isoelectric
focusing was performed using a Protean IEF Cell (Bio-Rad
Laboratories) The strips were then subjected to SDS⁄
PAGE, and proteins on the gel were transferred to PVDF
membranes For two-dimensional electrophoresis of blue
native PAGE [30] and SDS⁄ PAGE, slices of cotyledons
were homogenized with 20 strokes of a Dounce
homo-genizer in ice-cold buffer containing 50 mm Bis-Tris (pH
7.2), 50 mm NaCl containing 10% (w⁄ v) glycerol, 0.001%
ponceau S, and 1% digitonin After standing at 4C for
1 h, the homogenate was centrifuged for 30 min at
14 000 g Five per cent Coomassie Brilliant Blue G-250
solution was added to the supernatant to a final
concentra-tion of 0.25%, and the supernatant was subjected to
3–12% polyacrylamide gradient gel electrophoresis
accord-ing to the manufacturer’s protocol for the Native PAGE
Novex Bis-Tris Gel System (Invitrogen Corporation,
Carls-bad, CA, USA) Blue native PAGE gels were then
sub-jected to SDS⁄ PAGE, and proteins on the gel were
transferred to PVDF membranes Membranes were
incu-bated with primary antibody, followed by a horseradish
peroxidase-conjugated IgG secondary antibody (Promega
Corporation), and were developed using Western Lightning
Chemiluminescence Reagent (Perkin Elmer Life Science) as
previously described [18]
Real-time RT-PCR
Measurement of mRNA was performed as described
previ-ously [18] Briefly, total RNA was isolated using the
RNeasy Plant Mini kit (Qiagen Inc.) mRNA was
quanti-fied by real-time RT-PCR with a Thermal Cycler Dice Real
Time System (TaKaRa Bio Inc.) Forward primers 5¢-CG
TTTGAAGGGTGAGGAGGAAAA[FAM]G-3¢ and 5¢-CA
CAAGAGAGTTCTGCGATAACCTTG[FAM]G-3¢
(Invi-trogen Corporation) and reverse primers 5¢-AAGTAGGCA
ACAAAACAACG-3¢ and 5¢-GTTTTCCCGACAATAA-CATGG-3¢ were used for detecting GmPDIL-3a and GmPDIL-3b, respectively Primers for quantification of actin mRNA were as described previously [18] Each value was normalized by dividing it by that for actin mRNA
Proteinase K treatment of microsomes Microsomes were prepared from cotyledons as described previously [17], and treated with 0.5 mgÆmL)1proteinase K (Sigma-Aldrich Inc.) in the presence or absence of 1% Tri-ton X-100 for 5 min at 4C Proteins were precipitated with 10% trichloroacetic acid for 30 min at 4C and analyzed by western blot
ER fractionation Slices of cotyledons were homogenized with 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) sucrose, and either 5 mm MgCl2 or 5 mm EDTA Homo-genates were centrifuged for 10 min at 1000 g at 4C Next,
600 lL of the supernatant was loaded onto a 12 mL linear 21–56% (w⁄ w) sucrose gradient prepared in the same buffer Samples were centrifuged at 154 400 g for 2 h at 4C, and
1 mL fractions were collected and assayed by western blot
Acknowledgements
We thank Dr M Kito for critical reading of the man-uscript and warm encouragement This work was supported by a grant from the Program for Promotion
of Basic Research Activities for Innovative Biosciences, and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18658055)
References
1 Helenius A & Aeb M (2004) Roles of N-linked glycans
in the endoplasmic reticulum Annu Rev Biochem 73, 1019–1049
2 Wittrup KD (1995) Disulfide bond formation and eukaryotic secretory productivity Curr Opin Biotechnol
6, 203–208
3 Hogg PJ (2002) Biological regulation through protein disulfide bond cleavage Redox Rep 7, 71–77
4 Freedman RB, Hirst TR & Tuite MF (1994) Protein disulphide isomerase: building bridges in protein folding Trends Biochem Sci 19, 331–336
5 Creighton TE, Zapun A & Darby NJ (1995) Mecha-nisms and catalysts of disulfide bond formation in proteins Trends Biotechnol 13, 18–23