Cooperation of Soybean PDI Family Members and ERO1 Reiko ...10 Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 11 12 One sentence summary: Two

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Cooperation of Soybean PDI Family Members and ERO1 1

Reiko Urade, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-2

0011, Japan, +81 774 38 3760, [email protected] 3

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Plant Physiology Preview. Published on December 8, 2015, as DOI:10.1104/pp.15.01781

Copyright 2015 by the American Society of Plant Biologists

www.plantphysiol.orgon February 16, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and 5

ERO1 in Soybean 6

Motonori Matsusaki, Aya Okuda, Taro Masuda, Katsunori Koishihara, Ryuta Mita, 7

Kensuke Iwasaki, Kumiko Hara, Yurika Naruo, Akiho Hirose, Yuichiro Tsuchi, Reiko 8

Urade* 9

Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 10

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One sentence summary: Two soybean protein thiol disulfide oxidoreductases achieve 12

cooperatively and efficient oxidative protein folding by relaying an oxidizing equivalent 13

supplied from endoplasmic reticulum oxidoreductin-1a. 14

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1 This work was supported by a Grant in Aid for Scientific Research (No. 18658055, No. 18

26660111) from the Japan Society for the Promotion of Science, a grant from the Program 19

for Promotion of Basic Research Activities for Innovative Biosciences, and a grant from the 20

Takano Life Science Research Foundation. 21

* Address correspondence to [email protected]. 22

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Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via 31

disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide 32

isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER 33

oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidation is 34

equivalent to the active centers of PDI. In this study, we expressed recombinant soybean 35

Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER 36

oxidoreductases, in contrast to mammalian Ero1s having a high specificity for protein 37

disulfide isomerase. One of these ER oxidoreductases, GmPDIM, associated in vivo and in 38

vitro with GmPDIL-2, which was unable to be oxidized by GmERO1a. We therefore 39

pursued the possible cooperative oxidative folding by GmPDIM, GmERO-1a, and 40

GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative 41

refolding. In this process, GmERO1a preferentially oxidized the active center in the a’ 42

domain among the a, a' and b domain of GmPDIM. A disulfide bond introduced into the 43

active center of the a’ domain of GmPDIM was shown to be transferred to the active center 44

of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active 45

centers of both the a or a’ domain of GmPDIL-2. Therefore, we propose that the relay of an 46

oxidizing equivalent from an ER oxidoreductase to another may play an essential role in 47

cooperative oxidative folding by multiple ER oxidoreductases in plants. 48

49



5

In eukaryotes, many secretory and membrane proteins fold via disulfide bond 50

formation in the endoplasmic reticulum (ER). Seed storage proteins of major crops, such as 51

wheat, corn, rice, and beans, that are important protein sources for humans and domestic 52

animals, are synthesized in the ER of the endosperm or cotyledon. A number of seed 53

storage proteins fold by the formation of intramolecular disulfide bonds (oxidative folding) 54

and are transported to and accumulate in protein bodies (Kermode and Bewley, 1999; 55

Jolliffe et al., 2005). In contrast to normally folded proteins, misfolded and unfolded 56

proteins are retained in the ER and degraded by an ER-associated degradation or vacuolar 57

system (Smith et al., 2011; Pu and Bassham, 2013). Therefore, quick and efficient oxidative 58

folding of nascent seed storage proteins is needed for their accumulation in protein bodies. 59

During this process, protein disulfide isomerase (PDI) (EC 5.3.4.1) and other ER 60

protein thiol disulfide oxidoreductases (ER oxidoreductases) are thought to catalyze the 61

formation and isomerization of disulfide bonds in nascent proteins (Hatahet and Ruddock, 62

2009; Feige and Hendershot, 2011; Lu and Holmgren, 2014). After phylogenetic analysis of 63

the Arabidopsis thaliana genome, 10 classes of ER oxidoreductases (classes I–X) were 64

identified (Houston et al., 2005). Among them, class I ER oxidoreductases, a plant PDI 65

ortholog, has been studied in a wide variety of plants. Class I ER oxidoreductases have two 66

catalytically active domains a and a’, containing active centers composed of Cys-Gly-His-67

Cys and two catalytically inactive domains b and b’. An Arabidopsis ortholog of class I ER 68

oxidoreductases is required for proper seed development and regulates the timing of 69

programmed cell death by chaperoning and inhibiting cysteine proteases (Andème 70

Ondzighi et al., 2008). OaPDI, a PDI from Oldenlandia affinis, a coffee family (Rubiaceae) 71

plant, is involved in the folding of knotted circular proteins (Gruber et al., 2007). The rice 72

ortholog (PDIL1-1) was suggested to be involved in the maturation of the major seed 73

storage protein glutelin (Takemoto et al., 2002). Furthermore, rice PDIL1-1 plays a role in 74

regulatory activities for various proteins that are essential for the synthesis of grain 75

components as determined by analysis of a T-DNA insertion mutant (Satoh-Cruz et al., 76

2010). 77



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The oxidative refolding ability of class I ER oxidoreductases was confirmed in 78

recombinant soybean (GmPDIL-1) and wheat proteins produced by an E. coli expression 79

system established from cDNAs (Kamauchi et al., 2008; Kimura et al., 2015). Class II and 80

III ER oxidoreductases have an a–b–b’–a’ domain structure. Class II ER oxidoreductases 81

have an acidic amino acid-rich sequence in the N-terminal region ahead of the a domain. 82

Recombinant soybean (GmPDIL-2) and wheat class II ER oxidoreductases have oxidative 83

refolding activities similar to that of class I (Kamauchi et al., 2008; Kimura et al., 2015). 84

Class III ER oxidoreductases contain the nonclassical redox-center Cys-X-X-Ser/Cys 85

motifs, as opposed to the more traditional CGHC sequence, in the a and a’ domains. 86

Recombinant soybean (GmPDIL-3) and wheat proteins lack oxidative refolding activity in 87

vitro (Iwasaki et al., 2009; Kimura et al., 2015). Class IV ER oxidoreductases are unique to 88

plants and have an a–a’–ERp29 domain structure, which is homologous to the C-terminal 89

domain of mammalian ERp29 (Demmer et al., 1997). Recombinant soybean class IV ER 90

oxidoreductases (GmPDIS-1 and GmPDIS-2) and wheat class IV ER oxidoreductase 91

possess an oxidative refolding activity that is weaker than that of classes I and II 92

(Wadahama et al., 2007; Kimura et al., 2015). Class V ER oxidoreductases are plant 93

orthologs of mammalian P5 and have an a–a’–b domain structure. A rice class V ER 94

oxidoreductase, PDIL2;3, plays an important role in the accumulation of the seed storage 95

protein Cys-rich 10-kDa prolamin (crP10) (Onda et al., 2011). Recombinant soybean class 96

V ER oxidoreductase, GmPDIM and wheat class V ER oxidoreductase possess an oxidative 97

refolding activity similar to that of class IV (Wadahama et al., 2008; Kimura et al., 2015). 98

In the soybean, GmPDIL-1, GmPDIL-2, GmPDIM, GmPDIS-1, and GmPDIS-2 were 99

found to associate transiently with a seed storage precursor protein, proglycinin, in the ER 100

of the cotyledon by coimmunoprecipitation experiments, suggesting that multiple ER 101

oxidoreductases are involved in the folding of the nascent proglycinin. 102

The disulfide bond in the active center of ER oxidoreductases is reduced as a result of 103

catalyzing disulfide bond formation in an unfolded protein. The reduced active center of 104

PDI was discovered to be oxidized again by ER oxidoreductin-1 (Ero1p) in yeast (Frand 105

and Kaiser, 1998; Pollard et al., 1998). Ero1p orthologs are present universally in 106



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eukaryotes. Yeast and flies have a single copy of the ERO1 gene, which is essential for 107

survival (Frand and Kaiser, 1998; Pollard et al., 1998; Tien et al., 2008). Mammals have 108

two genes encoding Ero1-α (Cabibbo et al., 2000) and Ero1-β (Pagani et al., 2000) that 109

function as major disulfide donors to nascent proteins in the ER, but are not critical for 110

survival (Zito et al., 2010). Domain a of yeast PDI is the most favored substrate of yeast 111

Ero1p (Vitu et al., 2010), whereas a’ of human PDI is specifically oxidized by human Ero1-112

α (Chambers et al., 2010) and Ero1-β (Wang et al., 2011). Electrons from cysteine residues 113

of the active centers of PDI are transferred to oxygen by Ero1 (Tu and Weissman, 2004; 114

Sevier and Kaiser, 2008). The reaction mechanisms of yeast Ero1p and human Ero1s have 115

been intensively investigated; their regulation by PDI has been extensively studied as well 116

(Tavender and Bulleid, 2010; Araki and Inaba, 2012; Benham et al., 2013; Ramming et al., 117

2015). Only rice Ero1 (OsERO1) has been identified as a plant ortholog of Ero1p (Onda et 118

al., 2009). OsERO1 is necessary for disulfide bond formation in rice endosperm. The 119

formation of native disulfide bonds in the major seed storage protein proglutelin was 120

demonstrated to depend upon OsERO1 by RNAi knockdown experiments. However, no 121

plant protein thiol disulfide oxidoreductases that are oxidized by a plant Ero1 ortholog have 122

been identified to date. 123

In this study, we show that multiple soybean ER oxidoreductases can be activated by a 124

soybean Ero1 ortholog (GmERO1a). In addition, we propose a synergistic mechanism by 125

which GmPDIM and GmPDIL-2 cooperatively fold unfolded proteins using oxidizing 126

equivalents provided by GmERO1 in vitro. 127

. 128

129

RESULTS 130

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Identification and Characterization of Soybean Ero1 132



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The GmERO1a cDNA and GmERO1b cDNA encoding the soybean Ero1 orthologs 133

were cloned. GmERO1a and GmERO1b encoded proteins composed of 465 amino acids 134

containing 19 cysteine residues (Fig. 1A and Supplemental Fig. S1). GmERO1 proteins 135

were expressed as a single 53-kDa band in soybean root, stem, leaf, and cotyledon (Fig. 136

1B). The band shifted to 46-kDa under nonreducing SDS-PAGE, suggesting that GmERO1 137

proteins have intramolecular disulfide bonds. We confirmed that GmERO1 proteins, which 138

have a putative transmembrane region (Trp16 - Ser34) near the N-termini and two putative 139

N-glycosylated asparagine residues, are type I membrane-bound glycoproteins targeted to 140

the ER. When the extract from the cotyledon was treated with endoglycosidase H or F, the 141

size of GmERO1 proteins decreased from 53 kDa to 50 kDa (Fig. 1C), suggesting that one 142

or more high-mannose type N-glycan(s) were attached to GmERO1 proteins. GmERO1 143

proteins were recovered in the precipitate of the cell homogenate, but not the supernatant, 144

after ultra-centrifugation at 100,000 × g, and pretreatment with Triton X-100 caused 145

solubilization of GmERO1 proteins in the supernatant along with soybean calnexin (Fig, 146

1D). In confocal microscopic images, GmERO1 was observed upon immunostaining to 147

colocalize with GmPDIS-1, which is localized in the ER (Wadahama et al., 2007) (Fig. 1E). 148

The expression levels of GmERO1 proteins and their mRNAs were high when seed 149

storage proteins β-conglycinin and glycinin were synthesized (70–100 mg and 150–200 mg 150

cotyledon weight, respectively) (Fig. 1F and G). GmERO1a and GmERO1b mRNAs were 151

upregulated following ER stress induced by tunicamycin (Fig. 1H). The ER stress response 152

elements (Oh et al., 2003; Iwata and Koizumi, 2005; Iwata et al., 2008; Hayashi et al., 153

2013; Sun et al., 2013) were not found within the 2500-bp upstream and downstream 154

sequences of the transcriptional region of either GmERO1a or GmERO1b. 155

156

Recombinant GmERO1a Oxidizes Various ER Oxidoreductases 157

The soluble and folded 48-kDa recombinant GmERO1a, which is missing 60 N-158

terminal residues including the putative transmembrane region and the C-terminal 23 159



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residues (Fig. 2A), was expressed in Escherichia coli. Its mobility as visualized by SDS-160

PAGE shifted to 38 kDa under nonreducing conditions, suggesting that the recombinant 161

protein has intramolecular disulfide bonds (Fig. 2B). The recombinant protein had circular 162

dichroism (CD) spectra typical of a well-folded protein (Fig 2C). The UV-visible 163

absorption spectrum of the recombinant GmERO1a showed a typical unresolved flavin 164

envelope with a maximum absorbance at 454 nm and a shoulder at 485 nm (Fig. 2D). The 165

spectrum, identical to that of authentic flavin adenine dinucleotide (FAD), was obtained 166

after denaturation with guanidine hydrochloride, indicating that FAD is noncovalently 167

bound to recombinant GmERO1a. The molar ratio of GmERO1a to bound FAD was 168

calculated to be 1:1 with a molecular extinction coefficient of 12.9 mM-1cm-1. The FAD 169

moiety bound to GmERO1a was reduced by DTT (Fig. 2E), suggesting that FAD bound to 170

GmERO1a acts as a cofactor to transfer electrons from a substrate. 171

We next determined the ability of recombinant GmERO1a to oxidize five soybean ER 172

oxidoreductases (Fig. 3A). Oxidation of ER oxidoreductases by GmERO1a was monitored 173

by oxygen consumption (Fig. 3B). The reaction was performed in the presence of GSH, as 174

a substrate for oxidation by ER oxidoreductases oxidized by GmERO1a. In contrast to 175

human Ero1α and Ero1β, which predominantly oxidize PDI, GmEro1a oxidized GmPDIL-1, 176

GmPDIS-1, GmPDIS-2, and GmPDIM to comparable levels (Fig. 3C and Supplemental 177

Fig. S2). GmPDIL-2 was negligibly oxidized by GmERO1a. 178

Oxidative refolding by these ER oxidoreductases was determined in the presence of 179

GmERO1a. ER oxidoreductases other than GmPDIL-2 were able to refold reduced and 180

denatured RNase A (Fig. 4A). Lag times prior to the initiation of refolding (Fig. 4B, white 181

bars) and refolding rates (black bars) differed between the ER oxidoreductases. The shortest 182

lag times and the highest refolding rates were observed in the presence of GmPDIL-1. The 183

rate of refolding by GmPDIL-1 increased with increasing concentrations of GmERO1a. 184

The lag time of refolding by GmPDIL-1 did not change at different concentrations of 185

GmERO1a. The rates of refolding by GmPDIM, GmPDIS-1, and GmPDIS-2 were slower 186

than that by GmPDIL-1, and underwent very little change at different concentrations of 187



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GmERO1a. In contrast to the refolding rates, the lag times decreased with increasing 188

concentrations of GmERO1a. These results suggest that dithiol oxidation is rate limiting in 189

the reaction by GmPDIL-1, whereas disulfide isomerization was rate limiting in the 190

reactions by GmPDIM, GmPDIS-1, and GmPDIS-2. 191

This observation was confirmed by analysis of intermediates during refolding. RNase A 192

molecules during refolding were separated by nonreducing SDS-PAGE after modification 193

of the free thiol groups with 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS) 194

(Fig. 4C). GmPDIL-1 converted most of the reduced and denatured RNase A molecules 195

(Dred) into an intermediate with non-native disulfide bonds (Dox) within the first 5 min. 196

During the next 15 min, "Dox" was converted into a native form with four disulfide bonds 197

(N) (Fig. 4D, left). GmPDIM, GmPDIS-1, or GmPDIS-2 converted most of the "Dred" into 198

"Dox" within the first 5 min, and conversion of "Dox" into "N" was slower than by 199

GmPDIL-1 (Fig. 4C and D, right). Neither "Dox" nor "N" was generated by GmPDIL-2 (Fig. 200

4C). 201

202

GmPDIL-2 and GmPDIM Cooperatively Refold Denatured RNase A 203

It was previously found that GmPDIS-1, GmPDIM, GmPDIL-1, and GmPDIL-2 204

associate with nascent proglycinin in the ER, suggesting that these ER oxidoreductases 205

cooperatively fold glycinin in vivo (Wadahama et al., 2007; Kamauchi et al., 2008; 206

Wadahama et al., 2008). Associations between these ER oxidoreductases in the ER were 207

detected by coimmunoprecipitation. GmPDIM and GmPDIS-1, GmPDIM and GmPDIS-2, 208

GmPDIS-1 and GmPDIS-2, and GmPDIM and GmPDIL-2 were coimmunoprecipitated 209

(Fig. 5A). Therefore, we examined the refolding of RNase A when these ER 210

oxidoreductases were present simultaneously. No combination of GmPDIM, GmPDIS-1, 211

and GmPDIS-2 showed an additive effect on the refolding rate (Supplemental Fig. S3). In 212

contrast, the refolding rate of denatured RNase A when GmPDIM and GmPDIL-2 were 213

present simultaneously was almost five times greater than that for GmPDIM alone, 214



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although GmPDIL-2 alone had no refolding activity (Fig. 5B). The refolding rate 215

drastically increased until a 1:1 molar ratio of GmPDIL-2 to GmPDIM was obtained (Fig. 216

5C). The association of GmPDIL-2 and GmPDIM was confirmed by far-western blot 217

analysis (Fig. 5D and E). 218

To identify the relevant domains for the binding of GmPDIM and GmPDIL-2, various 219

domain fragments of GmPDIL-2 (Supplemental Fig. S4) and GmPDIM (Supplemental Fig. 220

S5) were subjected to far-western blot analysis. Only the b’–a’ fragment of GmPDIL-2 221

bound to GmPDIM with affinity comparable to full GmPDIL-2 (Fig. 5D). The a–a’ 222

fragment of GmPDIM bound with high affinity to GmPDIL-2 (Fig. 5E). Taken together, the 223

binding sites of GmPDIL-2 and GmPDIM are composed of the b’ and a’ domains of 224

GmPDIL-2 and the a and a’ domains of GmPDIM. 225

226

The Active Centers of GmPDIL-2 are Important to their Cooperative Activity with 227

GmPDIM 228

The acceleration of oxidative refolding of RNase A by the cooperation of GmPDIL-2 229

and GmPDIM depends on the catalytic cysteine residues in the a and a’ domains of 230

GmPDIL-2. When four catalytic cysteine residues were replaced with alanine [GmPDIL-231

2(C101/104/440/443A)], the acceleration of oxidative refolding was eliminated (Fig. 6A 232

and B). Replacing two catalytic cysteine residues in either the a or a’ domain with alanine 233

[GmPDIL-2(C440/443A) or GmPDIL-2(C101/104A)] caused a decrease in oxidative 234

refolding activity to 55% or 69% of wild-type levels, respectively. In mutants in which the 235

C-terminal catalytic cysteine residue in the a’ and/or a domain was replaced by alanine 236

[GmPDIL-2(C104/443A), GmPDIL-2(C443A), and GmPDIL-2(C104A)], oxidative 237

refolding was slower than that of GmPDIL-2(C440/443A) or GmPDIL-2(C101/104A). The 238

effects of a, a’, a–b, a–b–b’, b–b’–a’, and b’–a’ fragments on the acceleration of oxidative 239

refolding were all very low (Fig. 6B). One reason for the small effects of the domain 240

fragments on the acceleration of oxidative refolding, other than the a–b–b’ fragment, may 241



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be their low oxidative refolding activities and disulfide reductase activities seen in the 242

presence of glutathione redox buffer without GmERO1a (Supplemental Fig. S4E). 243

Oxidative refolding by ER oxidoreductases is achieved through the formation of non-244

native disulfide bonds in substrate proteins and their subsequent rearrangement to native 245

bonds as seen in Fig. 4C. Therefore, it was predicted that GmPDIL-2 would stimulate the 246

formation and/or rearrangement of substrate disulfide bonds. The addition of GmPDIL-2 to 247

GmPDIM induced more rapid conversion of "Dred" into "Dox" and "N" than did GmPDIM 248

alone (Fig. 6C). Mutations in the active centers of GmPDIL-2 caused retardation in the 249

conversion into both "Dox" and "N." The rate of disulfide bond formation during the first 10 250

min of the addition of GmPDIL-2 to GmPDIM was twice that of GmPDIM alone (Fig. 6D), 251

suggesting that GmPDIL-2 stimulates the oxidation of GmPDIM by GmERO1. Mutant 252

GmPDIL-2(C101/104/440/443A) was devoid of the acceleration effect on refolding. Since 253

GmPDIL-2(C440/443A) and GmPDIL-2(C101/104A) showed about half the acceleration 254

effect of the wild type, the active centers of the a and a’ domain appeared to act 255

independently. The amount of native disulfide bonds formed during the reaction were 256

calculated from the amount of refolded RNase A. The formation rate of native disulfide 257

bonds during the cooperation of GmPDIL-2 and GmPDIM (Fig. 6D, red curve) was four-258

fold higher than GmPDIM alone (Fig. 6D, blue curve), suggesting that GmPDIL-2 259

accelerates both disulfide bond formation and the rearrangement accompanied with folding. 260

261

GmPDIL-2 Accelerates the Oxidation of GmPDIM by GmERO1a 262

From the results shown in Fig. 6, it was suggested that GmPDIL-2 accelerates the 263

oxidation of GmPDIM by GmERO1a. To confirm this possibility, oxidation of GmPDIM 264

by GmERO1a was measured in the presence of GmPDIL-2. In this experiment, GSH was 265

used as a substrate. Addition of GmPDIL-2 caused an increase in O2 consumption rate to 266

130% of that without GmPDIL-2 (Fig. 7A). The acceleration effect was lost upon mutation 267

of the active center of the a’; GmPDIL-2(C440/443A) and GmPDIL-268



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2(C101/104/440/443A) showed no acceleration effects. The acceleration effect of 269

GmPDIL-2(C101/104A) was lower than that of wild type GmPDIL-2. Similar effects by 270

GmPDIL-2 and its active-center mutations were observed in experiments coupled with 271

glutathione disulfide reductase and NADPH (Fig. 7B–D). Fragments a and a’ showed an 272

acceleration effect of one-half that of the wild type. Fragment b–b’ showed no effect. The 273

attachment of the b, b’, or b–b’ domain to the a or a’ domain had no positive effect. 274

275

GmERO1a Preferentially Oxidizes the Active Center in the a’ Domain of GmPDIM; 276

GmPDIM Oxidizes Both Active Centers in GmPDIL-2 277

From the results shown in Fig. 7A–D, we envisioned a pathway by which GmPDIM, 278

oxidized by GmERO1, transfers disulfide bonds to the active centers of GmPDIL-2. To test 279

this hypothesis, we determined which active center of GmPDIM is oxidized by GmERO1a 280

using active-center mutants of GmPDIM (Fig. 7E and F, black bars). Both 281

GmPDIM(C192/195A) and GmPDIM(C195A) were negligibly oxidized by GmERO1a. 282

Therefore, no disulfide bond formation in reduced and denatured RNase A by 283

GmPDIM(C192/195A) and GmPDIL-2 was detected (Fig. 6C and D). In contrast to 284

GmPDIM(C192/195A) and GmPDIM(C195A), GmPDIM(C64/67A) and GmPDIM(C67A) 285

were oxidized by GmERO1a, although at a slower rate than wild-type GmPDIM. These 286

findings suggest that GmERO1a specifically oxidizes the active center in the a’ domain of 287

GmPDIM. This finding was corroborated by the fact that the a’ fragment, but not the a 288

fragment, was oxidized at 74% of the oxidation rate of wild-type GmPDIM by GmERO1a. 289

The oxidation rate of the a’–b fragment was slightly slower than that of the a’ fragment, 290

and the a–a’ fragment was oxidized at almost same rate as wild-type GmPDIM, suggesting 291

that the b domain does not contribute to oxidation by GmERO1a. Oxidation of every 292

GmPDIM mutant by GmERO1 was accelerated by GmPDIL-2 (Fig. 7F, white bar). 293

Association of GmPDIM and GmERO1a was detected by far-western blot analysis (Fig. 294

7G). The a, a’, or b fragment of GmPDIM hardly associated with GmERO1a. However, 295



14

either the a–a’ or a’–b fragment associated with GmERO1a at an affinity comparable to 296

wild-type GmPDIM. 297

To confirm that the disulfide bond in the active center of the a’ domain of GmPDIM 298

formed by GmERO1a is transferred to the active center of GmPDIL-2 via the active center 299

of the a domain of GmPDIM, we analyzed mixed disulfide complexes of the active-center 300

mutants of GmPDIM and GmPDIL-2. When GmPDIM(C67A) and GmERO1a were 301

incubated with GmPDIL-2(C104/443A), two types of mixed disulfide complexes (L2-M1 302

and L2-M2) of GmPDIM(C67A) and GmPDIL-2(C104/443A), and the GmPDIM(C67A) 303

dimer were detected (Fig. 8A). When GmPDIM(C67A) and GmERO1a were incubated 304

with GmPDIL-2(C104A) or GmPDIL-2(C443A), only one mixed disulfide complex, L2-305

M2 (Fig. 8B) or L2-M1, (Fig. 8C) was formed. Neither the mixed disulfide complex nor the 306

GmPDIM(C67A) dimer was generated in the absence of GmERO1a (Fig. 8D). When wild-307

type GmPDIM and GmERO1a were incubated with GmPDIL-2(C104/443A), only a few 308

mixed disulfide complexes were detected (Fig. 8E). In addition, no mixed disulfide 309

complexes were formed from GmPDIM(C195A) (Fig. 8F). From these results, L2-M1 and 310

L2-M2 were deduced to be mixed disulfide complexes, which were linked by disulfide 311

bonds between Cys67 of GmPDIM and either Cys443 or Cys104 of GmPDIL-2. 312

The results shown in Fig. 7F suggest that a disulfide bond introduced into the active 313

center of the a’ domain of GmPDIM is transferred to the active center of the a domain of 314

GmPDIM. Then the oxidized GmPDIM can oxidize the active center of either the a or a’ 315

domain of GmPDIL-2 (Fig. 8A-F). The direction of a redox reaction between functional 316

groups depends on their redox potentials, i. e., a functional group with higher redox 317

potential oxidizes a functional group with lower redox potential. To obtain the redox 318

potentials of GmPDIM and GmPDIL-2, their redox equilibrium constants were determined. 319

Redox equilibrium constants of GmPDIM and GmPDIL-2 were 2.0 mM and 3.4 mM, 320

respectively (Fig. 8G and J). Since these are the mean values of two active sites, redox 321

equilibrium constants of the redox-inactive mutants of GmPDIM and GmPDIL-2 were 322

determined (Fig. 8H, I, K, and L). GmPDIM(C192/195A) showed the lowest redox 323



15

equilibrium constant (Keq = 0.7) among the mutant proteins. The reduction potential E0’ 324

values calculated from each redox equilibrium constant of GmPDIM(C192/195A), 325

GmPDIM(C64/67A), GmPDIL-2(C440/443A), and GmPDIL-2(C101/104A) were -147 mV, 326

-166 mV, -169 mV, and -163 mV, respectively. These values suggest that the a domain of 327

GmPDIM has a tendency to oxidize both the active centers of the a and a’ domains of 328

GmPDIL-2. 329

330

DISCUSSION 331

In this study, we found that the plant Ero1 ortholog has a broad substrate specificity. 332

GmERO1a oxidizes, not only the conserved typical ER oxidoreductase GmPDIL-1, but 333

also GmPDIM, GmPDIS-1, and GmPDIS-2. Human Ero1α and Ero1β preferentially 334

oxidize PDI. Therefore, another pathway that oxidizes other ER oxidoreductases likely 335

exists in human. Peroxiredoxin-4 primarily oxidizes two PDI family members, ERp46 and 336

P5, using H2O2 generated during the oxidation of PDI by human Ero1α (Sato et al., 2013). 337

Conversely, in the plant ER, a peroxiredoxin-4 ortholog has not been found. In rice, a lack 338

of OsEro1 causes the aggregation of glutelin because of deficient disulfide bond formation 339

(Onda et al., 2009), suggesting that disulfide bond formation depends on the Ero1 system 340

for protein folding in the plant ER. Therefore, plant Ero1 may induce efficient oxidative 341

folding in the ER by supplying disulfide bonds to multiple ER oxidoreductases. 342

Among the soybean ER oxidoreductases examined in this study, only GmPDIL-2 was 343

hardly oxidized by GmERO1a. The Keq values of the active centers in the a and a’ 344

domains of GmPDIL-2 were 4.33 and 2.66, respectively. Since these values are similar to 345

that of the a’ domain of GmPDIM (Keq = 3.53), which was preferentially oxidized by 346

GmERO1a, the lack of oxidation by GmERO1 is not due to the reduction potential of the 347

active centers of GmPDIL-2. One reason may be the low affinity between GmERO1a and 348

GmPDIL-2 (Fig. 3D). 349



16

We found GmPDIL-2 associate with GmPDIM in vivo. GmPDIL-2 can associate with 350

GmPDIM to cooperatively fold denatured RNase A in vitro. Oxidative folding proceeds via 351

two steps: (Step 1) introduction of transient, non-native disulfide bonds and (Step 2) their 352

isomerization into native disulfide bonds. The oxidative folding activity of GmPDIM was 353

lower than that of GmPDIL-2 in the presence of glutathione buffer without GmERO1a 354

(Supplemental Fig. S4E and Supplemental Fig. S5D). Therefore, there may be a pathway in 355

which GmPDIM forms non-native disulfide bonds in the substrate and then GmPDIL-2 356

folds and rearranges these disulfide bonds into native bonds (Fig. 9A). 357

In addition to synergistic effects on folding, the addition of GmPDIL-2 to GmPDIM 358

accelerated the formation of disulfide bonds in RNase A. GmPDIL-2 may increase the net 359

substrates for GmPDIM by reducing non-native disulfide bonds and stimulating folding. 360

However, even when GSH was used as a substrate, GmPDIL-2 increased the rate of 361

oxidation of GmPDIM by GmERO1, suggesting that increasing the rate of oxidation does 362

not depend on substrate folding only. We found that the active center of the a domain of 363

GmPDIM oxidized the active center of either the a or a’ domain of GmPDIL-2 (Fig. 8A–F). 364

The increase in the oxidation rate of GmPDIM by GmERO1a may be explained by the 365

transfer of disulfide bonds from GmPDIM to the associated GmPDIL-2, in addition to 366

substrate proteins. The absolute concentration of glutathione in the ER is unknown; 367

however, a consensus exists that the total glutathione concentration in the ER is around 9–368

10 mM and that the ratio of [GSH]2/[GSSG] in the ER is around 3:1 (Hatahet and Ruddock, 369

2009). From these values, the reduction potential for ER glutathione is estimated as -191 370

mV. Under these redox conditions in the ER, the active centers of GmPDIM and GmPDIL-371

2 would be largely reduced. This means that GmPDIM, once oxidized by GmERO1a, 372

would oxidize GmPDIL-2. 373

Using the active-center mutants, a model of the pathway of disulfide bond transfer from 374

GmPDIM to GmPDIL-2 can be constructed (Fig. 9B). First, GmERO1a entirely oxidizes 375

the a’ domain of GmPDIM (i). The preference of GmERO1a for the a’ domain of 376

GmPDIM is probably due to the reduction potential and the affinity between GmERO1a 377



17

and the a’ domain of GmPDIM. Since the reduction potential of the a’ domain was lower 378

than that of the a domain, the a’ domain will be more easily oxidized by GmERO1a. Then, 379

the oxidized GmPDIM transfers a disulfide bond to GSH or GmPDIL-2 mainly from the a 380

domain (ii), because the oxidation of GmPDIM(C64/67A) and GmPDIM(C67A)) was 381

significantly lower than that of wild-type GmPDIM (Fig. 7F). This finding suggests that the 382

disulfide bond transferred to the a’ domain of GmPDIM from GmERO1a is 383

intramolecularly transferred to the a domain. The oxidation of the a domain by the a’ 384

domain is thermodynamically contradictory, as the apparent redox equilibrium constant of 385

the a domain of GmPDIM(C192/195A) (0.73) was lower than that of the a’ domain of 386

GmPDIM(C64/67A) (3.53). If the a’ domain of GmPDIM is oxidized, the redox 387

equilibrium constant of the a and/or a’ domain may be altered by conformational changes 388

such that the a domain becomes susceptible to oxidation by the a’ domain. In human PDI, 389

the a’ domain was first oxidized by Ero1-α, which then oxidizes the a domain, and the 390

reduced a’ domain is oxidized by Ero1-α. During the sequential transfer of disulfide bonds, 391

PDI dynamically changes its structure from a closed form to an open form (Nakasako et al., 392

2010; Serve et al., 2010; Wang et al., 2012). Subsequently, disulfide bonds are transferred 393

from the GmPDIM to either the a or a’ domain of GmPDIL-2 (iii) and from the oxidized 394

GmPDIL-2 to a protein substrate (iv). 395

Human PDI has been shown to oxidize other ER oxidoreductases, such as ERp47, 396

ERp57, and P5 (Araki et al., 2013, Oka et al., 2015). Therefore, Ero1-α and PDI are 397

thought to constitute a regulatory hub for the regulation of the redox states of ER 398

oxidoreductases. In contrast, multiple soybean ER oxidoreductases are able to be oxidized 399

by GmERO1a. Therefore, in plants, it is thought that Ero1 and ER oxidoreductases 400

constitute a robust network for disulfide bond transfer and the regulation of redox 401

homeostasis in the ER. The relay of oxidizing equivalents from an ER oxidoreductase to 402

another may play an essential role for cooperative oxidative folding by multiple ER 403

oxidoreductases. 404

405



18

MATERIALS AND METHODS 406

407

Plants 408

Soybean (Glycine max L. Merrill cv. Jack) seeds were planted in 5-L pots and grown in a 409

controlled environmental chamber at 25°C under 16-h day/8-h night cycles. All samples taken were 410

immediately frozen and stored in liquid nitrogen until use. 411

412

cDNA Cloning of GmERO1a and GmERO1b 413

The cDNAs transcribed from GmERO1a and GmERO1b were amplified from total RNA 414

extracted from 100 mg immature soybean cotyledon by RT-PCR using forward primer 5’-415

CCTCAGTGTTCTTCAGCGATCTTCTTCTGGGTCTG-3’ and reverse primer 5’-416

CCAAACCACGACAGTGGTTTTCCTATGTCTAGTC-3’, and forward primer 5’-417

ATGGTGAAAGCGGAGATTGAGAAAAAGGGTTGCAGC-3’ and reverse primer 5’-418

CCACGACGGTGGTTTTCCTATGTCTAGTCTCTCTAG-3’, respectively. The amplified DNA 419

fragments were subcloned into the pUC19 vector. The inserts in the plasmid vectors were sequenced. 420

421

Preparation of Recombinant GmERO1a 422

An expression plasmid encoding GST-fused GmERO1a E70-G422 was constructed as follows. 423

The DNA fragment was amplified from GmERO1a cDNA by PCR using primers 5’-424

GCGATGGATCCTATGAAACTGTGGATCGTCTTAATG-3’ and 5’-425

GCGATGTCGACTATCCTCCTTCCATGATTCTTTCAGC-3’. The amplified DNA fragment was 426

subcloned into the pGEX6p-2 vector (GE Healthcare). The recombinant protein has glutathione-S-427

transferase (GST) linked to the amino terminus. Rosseta-gami cells (Takara Bio, Inc.) were 428

transformed with the GST-fusion vector described above. Expression of recombinant GST-429

GmERO1a was induced in LB containing 0.5 mM isopropyl thiogalactoside, 20 μM FAD, 100 430

μg/mL ampicillin, 15 μg/mL kanamycin, and 12.5 μg/mL tetracycline at 15°C for 120 h. The cells 431

were collected by centrifugation and disrupted by sonication in phosphate-buffered saline. 432



19

Recombinant GST-GmERO1a was adsorbed to a glutathione Sepharose 4B column and digested by 433

PreScission protease (GE Healthcare). The eluted recombinant GmERO1a was purified by gel 434

filtration chromatography on a TSK gel G3000SW column (Tosoh Co., Ltd.) equilibrated with 20 435

mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 10% glycerol as described previously 436

(Wadahama et al., 2007). The concentration of purified recombinant GmERO1a was determined by 437

amino acid analysis with norleucine as an internal standard. 438

439

Western Blot Analysis 440

Soybean (G. max L. Merrill. cv. Jack) seeds were planted in 5-L pots and grown in a controlled 441

environmental chamber at 25°C under 16-h day/8-h night cycles. Roots, leaves, and stems were 442

collected from 10-day-old seedlings. Seeds were collected from plants, and cotyledons were 443

isolated. Tissues were frozen in liquid nitrogen and then ground into a fine powder with a 444

micropestle SK-100 (Tokken, Inc.). Proteins were extracted by boiling for 5 min in SDS-PAGE 445

buffer (Laemmli, 1970) containing a 1% cocktail of protease inhibitors (Sigma-Aldrich, Inc.). To 446

cleave N-glycans, proteins were extracted from the cotyledons in 0.1% SDS/50 mM phosphate 447

buffer (pH 5.5). Protein concentration in the sample was measured with a RC DC protein assay kit 448

(Bio-Rad Laboratories). Proteins (0.4 mg) were treated with 10 mU endoglycosidase F or 449

endoglycosidase H (Sigma-Aldrich Inc.) at 37°C for 16 h. To separate the supernatant and 450

membrane fractions, the frozen cotyledons from three 100-mg seeds in liquid nitrogen were crushed 451

with a micropestle SK-100 and sonicated with a Sonifier II (Branson Ultrasonic Corporation) in 200 452

mM Tris-HCl, pH 7.8, five times for 30 sec on ice. The homogenate was placed into a cell strainer 453

(BD Biosciences) and centrifuged at 824 × g for 40 min at 4°C. The filtrated suspension was 454

divided two fractions, diluted with 200 mM Tris-HCl, pH 7.8, with or without 1% Triton X-100, 455

and centrifuged at 100,000 × g for 1 h at 4°C. Proteins were subjected to SDS-PAGE (Laemmli, 456

1970) and blotted onto a polyvinylidene difluoride membrane. Blots were immunostained first with 457

antiserum and then with horseradish peroxidase-conjugated IgG antiserum (Promega Corporation) 458

as the secondary antibody. Anti-GmERO1a serum and anti-GmCNX serum were prepared using 459

recombinant GmERO1a and recombinant C-terminal-truncated GmCNX (Supplemental Fig. S6), 460

respectively, by Operon Biotechnologies, K.K. Anti-GmPDIL-1 serum was prepared previously 461



20

(Kamauchi et al., 2008). Blots were developed with the Western Lightning Chemiluminescence 462

Reagent (Perkin Elmer Life Sciences). 463

464

Confocal Microscopy 465

Cotyledons from developing soybean seeds (195 mg) were cut into 3×3×1-mm cubes. The 466

pieces of tissue were fixed with 4% formaldehyde for 2 h at room temperature. The fixed cotyledon 467

pieces were dehydrated with a series of ethanol dilutions, embedded in Historesin (Leica 468

Microsystems), and sliced into sections. The sections were stained with anti-GmPDIS-1 rabbit 469

serum (Wadahama et al., 2007) and then biotin-anti-rabbit IgG goat serum (Cortex Biochem), 470

followed by incubation with Cy5-streptavidin (GE Healthcare). For detection of GmERO1, 471

specimens were stained with guinea pig antiserum against recombinant GmERO1a, followed by 472

staining with Cy3-conjugated anti-guinea pig IgG goat serum (Chemicon International). The 473

specimens were examined on a FV1000-D laser scanning confocal imaging system (Olympus). 474

475

Measurement of mRNA 476

Total RNA was isolated from young leaves treated with or without 5 μg/mL tunicamycin at 477

25°C for 5 h using an RNeasy Plant Mini kit (Qiagen Inc.). Quantification of mRNA was performed 478

by real-time PCR with a Thermal Cycler Dice™ Real Time System with SYBRⓇPremix Ex TaqTM 479

(TaKaRa Bio Inc.). Forward primers 5’- CATGCTCTCTTCTGAGTATTTGTCA-3’ and 5’-480

TCATCCAAGAAATGGGACCT-3’ and reverse primers 5’-481

CAGTGGTTTTGGTATGTCTAGTCTCTC-3’ and 5’-CGACGGTGGTTTTCCTATGT-3’ were 482

used for detection of GmERO1a mRNA and GmERO1b, respectively. 483

484

Far-UV CD Analysis 485

CD spectra of recombinant proteins in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM 486

NaCl and 10% glycerol were obtained using a J-720 spectropolarimeter (JASCO Corp.) in a 1-mm 487

path-length cell with a scan speed of 20 nm/min at 14°C. 488



21

489

ER Oxidoreductase Oxidation Assays 490

Oxygen consumption was measured using a Clark-type oxygen electrode system 491

(OXYT-1) (Hansatech Instruments Ltd.). All experiments were performed at 25°C in 100 492

mM [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (pH 7.5), 2 mM CaCl2, 493

and 150 mM NaCl. Catalytic oxygen consumption was initiated by the addition of 494

GmERO1a in a reaction mixture containing recombinant ER oxidoreductases or its variants, 495

and 10 mM GSH. 496

The activity was measured using a coupled assay following the decrease in absorbance 497

at 340 nm due to the consumption of NADPH by glutathione reductase (Sigma-Aldrich, 498

Inc.) with the reaction being started by the addition of GmERO1a to initiate the reaction 499

(Nguyen et al., 2011; Sato et al., 2013). A molar extinction coefficient of 6200 M-1cm- 1 for 500

NADPH was used for calculations. All experiments were performed in 100 mM HEPES 501

(pH 7.5) containing 150 mM NaCl and 2 mM CaCl2. 502

503

RNase A Refolding Assays 504

Thiol oxidative refolding activity was assayed as previously described by measuring 505

RNase activity following regeneration of the active form of the enzyme from its reduced 506

and denatured form in the presence of the recombinant proteins (Creighton, 1977; Lyles and 507

Gilbert, 1991). Reduced and denatured RNase A was prepared as described previously. 508

Each reaction mixture contained 100 mM HEPES buffer (pH 7.5), 150 mM NaCl, 2 mM 509

CaCl2, 0.5 mM glutathione disulfide, 2 mM glutathione, reduced RNase A, and 510

recombinant oxidoreductases and their variants, and were incubated at 25°C. The formation 511

of active RNase A was measured spectrophotometrically by monitoring the hydrolysis of 512

the RNase A substrate cCMP at 284 nm. The isomerase activities of oxidoreductases were 513

calculated as described previously (Kulp et al., 2006). Briefly, isomerase activity was 514

determined from the linear increase in the amount of enzymatically active RNase A with 515

time after a lag. Oxidation of free thiol residues to disulfides on reduced RNase A were also 516



22

determined by measuring the amounts of free thiol groups in the reaction mixtures (Ellman, 517

1959). 518

519

Gel-based RNase A Refolding Experiments 520

RNase A oxidation analyses were performed by the addition of 1 μM GmERO1 in 100 521

mM HEPES buffer (pH7.5), 150 mM NaCl, 2 mM CaCl2, and 3 μM recombinant ER 522

oxidoreductases and their variants containing 8 μM denatured and reduced RNase A. At the 523

indicated time points, free thiols were blocked by the addition of Laemmli's 4×SDS-loading 524

buffer (Laemmli, 1970) containing 8 mM 4-acetamido-4-maleimidylstilbene-2,2 disulfonic 525

acid and separated on a 15% polyacrylamide gel by SDS-PAGE (Laemmli, 1970) without 526

reducing reagent. Proteins were detected by Coomassie Brilliant Blue staining after 527

electrophoresis. 528

529

Immunoprecipitation Experiments 530

Cotyledons (each 100 mg) were frozen under liquid nitrogen and homogenized with a 531

Dounce homogenizer at 4°C in 20 mM HEPES buffer (pH 7.2) containing 150 mM NaCl, 532

1% digitonin, and 1% cocktail of protease inhibitors (Sigma-Aldrich). The homogenate was 533

placed on ice for 1 h and centrifuged for 30 min at 10,000 × g at 4°C. Immunoprecipitation 534

was performed at 4°C for 1 h with preimmune serum or anti-GmPDIL-1, anti-GmPDIL-2, 535

anti-GmPDIM, anti-GmPDIS-1, or anti-GmPDIS-2 serum. The immunoprecipitate was 536

collected with protein A-conjugated Sepharose beads (Sigma-Aldrich), washed with 20 mM 537

HEPES buffer (pH 7.2) containing 150 mM NaCl, and subjected to western blot analysis 538

using a specific antiserum against PDI family proteins as primary antibodies and 539

peroxidase-conjugated ImmunoPure Recombinant Protein A (Pierce Biotechnology). 540

541

Dot Far-western Blot Analysis 542

Purified recombinant ER oxidoreductases and their variants as prey proteins were 543

spotted onto a nitrocellulose membrane (GenScript) in a volume of 4 μL. The membrane 544



23

was dried, rinsed twice, and blocked with 20 mM Tris-HCl (pH 8) containing 150 mM 545

NaCl, 0.05% Tween 20, and 5% nonfat dry milk (blocking solution) at 4°C for 16 h. After 546

blocking, the membrane was incubated in 0.2 μM bait protein in blocking solution without 547

nonfat dry milk (TBST) for 3 h at 4°C. The membrane was incubated with anti-bait 548

antiserum and further with horseradish peroxidase-conjugated IgG antiserum (Promega 549

Corporation) as the secondary antibody diluted with blocking solution. Blots were washed 550

four times for 20 min with TBST and developed using the Western Lightning 551

Chemiluminescence Reagent (Perkin Elmer Life Sciences). 552

553

Assay of Mixed Disulfide Complexes 554

A 4-μL aliquot of purified GmPDIL-2 or GmPDIL-2 variants, and GmPDIM or 555

GmPDIM variants, were incubated at 25°C for 60 min and quenched with N-ethyl 556

maleimide. Proteins were subjected to 7.5% SDS-PAGE under nonreducing conditions. 557

Lanes cut from the first SDS-polyacrylamide gel were incubated in SDS-sample buffer with 558

5% 2-mercaptoethanol for 120 min at 37°C. The gel slices were then subjected to 12.5% 559

SDS-PAGE under reducing conditions. The separated proteins were stained with a silver 560

stain II kit (Wako Pure Chemical Industries Ltd.). 561

562

Measurement of Redox Equilibrium Constants of GmPDIM and GmPDIL-2 563

GmPDIM, GmPDIL-2, and their variants (1 μM) were incubated with 0.1 mM GSSG 564

and 0.015–28 mM GSH at 25°C for 1 h in 0.1 M sodium phosphate buffer, pH 7.0, 565

containing 1 mM EDTA and 150 mM NaCl. After incubation under N2 at 25°C for 1 h, 566

further thiol-disulfide exchange was prevented by the addition of 10% TCA. The 567

aggregated proteins were precipitated by centrifugation and washed with 100% acetone. 568

The protein pellet was solubilized and incubated in 0.1 M sodium phosphate buffer, pH 7.0, 569

containing 2% SDS and 3 mM methoxypolyethylene glycol-maleimide (mPEG5000-mal) 570

(Fluka Sigma-Aldrich) at 25°C for 30 min. Proteins were separated by SDS-PAGE and 571

stained with Coomassie Brilliant Blue. Values for the reduced fraction, oxidized fraction, 572



24

and intermediate fraction (in the case of wild-type GmPDIM and GmPDIL-2, which have 573

two active sites) were quantified using ImageJ software (National Institutes of Health). The 574

values for the completely oxidized or reduced states were regarded as 0 or 100, respectively, 575

and all intermediate states were recalibrated. In the case of wild-type GmPDIM and 576

GmPDIL-2, the intensity of the band in which one of the two active centers was oxidized 577

was calibrated as one-half of the reduced form. The sum of the completely reduced form 578

and one-half of the half-reduced form was plotted. The Keq was calculated by fitting the 579

recalibrated fraction of the apparent reduced form to the following equation: R = 580

([GSH]2/[GSSG])/{Keq + ([GSH]2/[GSSG])}, in which R is the relative ratio of the reduced 581

forms. The equilibrium redox potential of proteins was calculated using the Nernst equation 582

[E’0 = E’0(GSH/GSSG) – (RT/nF) * ln Keq] using the glutathione standard potential E’0(GSH/GSSG) 583

of -0.240 V at pH 7.0 and 25°C. 584

585

Statistical Analysis 586

All data are shown as the mean ± SEM from at least three replicates. Welch's t tests 587

(two tailed, unpaired) or Tukey-Kramer test were used for comparisons between more than 588

three experiments. 589

590

591

Sequence data from this article can be found in the DDBJ/EMBL/GenBank databases under 592

accession numbers GmERO1a (AB622257), GmERO1b (AB622258) and GmCNX 593

(AB196933). 594

595

Supplemental Data 596

The following materials are available. 597



25

Supplemental Figure S1. Amino acid sequences of GmERO1a, GmERO1b, and HsEro1α. 598

Supplemental Figure S2. Oxidation of ER oxidoreductases by recombinant GmERO1. 599

Supplemental Figure S3. Refolding of RNase A by multiple soybean ER oxidoreductases. 600

Supplemental Figure S4. Expression, purification, and activity of wild-type GmPDIL-2 601

and its variants. 602

Supplemental Figure S5. Expression and purification of wild-type GmPDIM and its 603

variants. 604

Supplemental Figure S6. Expression and purification of recombinant C-terminal truncated 605

soybean calnexin. 606

607

Supplemental Table S1. List of primers and template plasmids for PCR of variant 608

preparation. 609

Supplemental Materials and Methods. 610

611

List of author contributions 612

R.U. and M.M. designed the study and wrote the paper. M.M. performed the majority of 613

experiments presented. K.K., K.H., T.M. and R.U. cloned GmERO1a cDNA, and 614

established the expression system of recombinant GmERO1a. R.M. helped prepare mutant 615

recombinant proteins. K.I. performed immunoprecipitation experiments. Y.N. performed 616

the confocal microscopy experiments and the N-glycosylation analysis. A.O., K.K., Y.T. 617

and A.H. performed assay of oxidation of oxidoreductases by GmERO1a. A.O. measured 618

equillibrium constants between oxidoreductases and glutathione. 619

620



26

Figure Legends 621

Figure 1. Identification of soybean Ero1. A, Schematic representation of Ero1 orthologs. 622

Numbered circles represent the positions of the cysteine residues. Lines indicate disulfide 623

bonds. B, GmERO1 is ubiquitously expressed in tissues. The proteins extracted from root 624

(4.5 µg protein) (lanes 1 and 5), stem (10 µg protein) (lanes 2 and 6), leaf (30 µg protein) 625

(lanes 3 and 7), and immature cotyledon (100 mg) (30 µg protein) (lanes 4 and 8) were 626

separated under reducing (lanes 1–4) and nonreducing SDS-PAGE (lanes 5–8) and 627

subjected to western blot analysis with an antiserum prepared against recombinant 628

GmERO1. C, GmERO1 is N-glycosylated. GmERO1 in the immature cotyledon (30 mg) 629

was detected by western blot analysis with the anti-GmERO1 serum after treatment with 630

(+) or without (-) endoglycosidase H (Endo H) or endoglycosidase F (Endo F). The 53-kDa 631

band of GmERO1 was shifted to 50 kD after Endo H treatment. D, GmERO1 is a 632

membrane-bound protein. An immature cotyledon (100 mg) was homogenized by 633

sonication. The homogenate was centrifuged at 100,000 × g for 2 h at 4°C in the absence (-) 634

(lanes 1 and 2) or presence (+) of 1% Triton X-100 (lanes 3 and 4). GmERO1 protein in the 635

supernatant (S) (lanes 1 and 3) and pellet (P) (lanes 2 and 4) was detected by western blot 636

analysis with anti-GmERO1 serum, anti-soybean calnexin (GmCNX) serum, or anti-637

GmPDIL-1 serum. E, GmERO1 localizes in the ER. The immature cotyledon (195 mg) was 638

fixed and embedded in resin. The sections were cut with a microtome and immunostained 639

with anti-GmERO1 guinea pig serum and anti-GmPDIS-1 rabbit serum, and observed 640

under a confocal microscope. Bars, 20 μm. F, Expression of GmERO1 in cotyledons during 641

development. The proteins extracted from cotyledons were analyzed by western blot with 642

anti-GmERO1 serum. G, Relative amounts of GmERO1a (black bars) and GmERO1b 643

(white bars) mRNA in cotyledons during development were quantified by real-time PCR. 644

Data are represented as mean ± SEM of n = 3. H, Expression levels of GmERO1a and 645

GmERO1b mRNA are upregulated under ER stress. Young soybean leaves were treated 646

with (+, black bars) or without (-, white bars) 5 μg/mL tunicamycin (TM) for 2 or 5 h. After 647

treatment, GmERO1a and GmERO1b mRNA were quantified by real-time PCR. Data are 648

represented as mean ± SEM of n = 3. 649



27

Figure 2. Expression of recombinant GmERO1. A, Cleavage of recombinant glutathione-650

S-transferase (GST)-fused GmERO1 by PreScission protease. The gray box indicates GST, 651

and the solid black line denotes the PreScission cleavage sequence. The N-terminal 652

sequence GPLGS was cleaved from the fusion protein after protease treatment. B, 653

Expression and purification of recombinant GmERO1. Recombinant GST-fused GmERO1 654

expressed in E. coli (lane 1) was purified by a glutathione Sepharose 4B affinity column 655

chromatography. GmERO1 was cleaved by PreScission protease and eluted from the 656

column (lane 2), followed by gel filtration chromatography (lanes 3 and 4). Proteins in each 657

eluate were separated by reducing SDS-PAGE (lanes 1–3) and nonreducing SDS-PAGE 658

(lane 4), and stained with Coomassie Blue. C, Far-UV circular dichroism spectrum of 659

recombinant GmERO1 in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 660

10% glycerol was obtained using a J-720 spectropolarimeter (JASCO Corp., Tokyo, Japan) 661

in a 1-mm path-length cell with a scan speed of 20 nm/min at 14°C. D, Absorbance 662

spectrum of recombinant GmERO1 was measured in the absence (solid line) or presence 663

(dotted line) of 6 M guanidine hydrochloride. Free FAD spectrum (dashed line) was also 664

measured. Black and white arrows show the maximum absorbance at 454 nm and the 665

shoulder at 485 nm of GmERO1, respectively. E, GmERO1-bound FAD is reduced by DTT. 666

Absorbance changes at 454 nm of 10 μM GmERO1 (circles) and 10 μM free FAD 667

(triangles) were monitored in the presence of 2 mM DTT. DTT was added at zero time. 668

GmERO1 in the absence of DTT was also measured (rectangles). 669

670

Figure 3. GmERO1a shows broad specificity for ER oxidoreductases. A, Schematic 671

representation of soybean ER oxidoreductases. Boxes represent domains. B, Schematic 672

representation of the continuous oxidation of PDI family members by Ero1 in the presence 673

of GSH and the reduction of O2. C, Oxygen consumption rate of GmERO1 (1μM) in the 674

presence of 3 μM each ER oxidoreductase and 10 mM GSH at 25°C. Data are represented 675

as mean ± SEM of n = 3. 676



28

Figure 4. Reconstitution of oxidative protein folding with ER oxidoreductases and 677

GmERO1a in vitro. A, GmERO1a (1μM) without (GmERO1a alone), or with 3 μM of each 678

ER oxidoreductase, was incubated with reduced and denatured RNase A (8 μM), and the 679

recovered RNase A activity was assayed. 'None' shows refolding of RNase A alone. B, Lag 680

time (white bars) and maximum refolding rate (black bars) of reduced and denatured RNase 681

A (8 μM) by 3 μM each ER oxidoreductase in the presence of GmERO1a. Data are 682

represented as mean ± SEM of n = 3. C, Refolding of reduced and denatured RNase A in 683

the absence (-) or presence (+) of GmERO1a and each ER oxidoreductase, quenched with 684

AMS, and was analyzed by nonreducing SDS-PAGE. Dred, reduced and denatured RNase 685

A; Dox, denatured RNase A with non-native disulfides; N, native RNase A. D, Model of 686

refolding of RNase A by ER oxidoreductases in the presence of GmERO1a. Left, GmPDIL-687

1 transfers non-native disulfide bonds to Dred. Dox is promptly folded into the native form 688

accompanied by the isomerization of disulfide bonds. Right, GmPDIM, GmPDIS-1, or 689

GmPDIS-2 transfers non-native disulfide bonds to Dred. Dox accumulates due to rate-690

limiting isomerization activities of GmPDIM, GmPDIS-1, and GmPDIS-2. 691

692

Figure 5. GmPDIL-2 and GmPDIM form a complex and synergistically refold RNase A 693

with using disulfide bonds produced by GmERO1a. A, Detection of ER oxidoreductase 694

complexes. Immunoprecipitation (IP) from the cotyledon extract was carried out with each 695

anti-ER oxidoreductase serum or preimmune serum (PI). Immunoprecipitates and the 696

cotyledon extract (lane 1) were analyzed by western blot with each anti-ER oxidoreductase 697

serum. B, Effects of the addition of GmPDIL-2 to GmPDIM on the refolding of RNase A. 698

Reduced and denatured RNase A (8 μM) was incubated with 3 μM ER oxidoreductases in 699

the presence of 1 μM GmERO1a. C, GmPDIL-2 concentration dependence of the refolding 700

activity in the presence of 1 μM GmERO1a and 1 μM GmPDIM. Data are represented as 701

mean ± SEM of n = 3. D, Far-western blot analysis of the association of the GmPDIL-2 702

(WT) or GmPDIL-2 domain fragment with GmPDIM. Indicated amounts of GmPDIL-2, 703

each GmPDIL-2 domain fragment or bovine serum albumin (BSA) (prey) were dot-blotted 704



29

and incubated with GmPDIM. Bound GmPDIM was immunostained. E, GmPDIM (WT), 705

each GmPDIM domain fragment or BSA (prey) was dot-blotted and incubated with 706

GmPDIL-2. Bound GmPDIL-2 was immunostained. 707

708

Figure 6. . Active centers of GmPDIL-2 play vital roles in the acceleration of both 709

refolding and disulfide bond formation. A, Schematic representation of wild-type GmPDIL-710

2 (WT) and its mutants. “CGHC,” “AGHA,” and “CGHA” represent the amino acid 711

sequences of the active sites of the a and a’ domains. Numbers in the name of the mutants 712

represent cysteine residues mutated to alanine. B, Acceleration of refolding by GmPDIL-2 713

mutants and GmPDIL-2 domain fragments in the GmERO1a/GmPDIM system. Reduced 714

and denatured RNase A (8 μM) was incubated with 1 μM GmPDIM and 4 μM wild type 715

GmPDIL-2 (WT), its mutants or domain fragments in the presence of 1 μM GmERO1a. 716

Data are represented as mean ± SEM of n = 3–5. Bars with the same letters are not 717

significantly different at P < 0.05 by Tukey-Kramer test. C, Disulfide bond formation in 718

reduced and denatured RNase A (8 μM) during cooperative refolding by GmPDIM or 719

GmPDIM(C192/195A) (1 μM) and GmPDIL-2 or GmPDIL-2 mutants (3 μM) in the 720

presence of GmERO1a (1 μM) was analyzed by nonreducing SDS-PAGE. Dred, reduced 721

and denatured RNase A; Dox, denatured RNase A with non-native disulfides; N, native 722

RNase A. D, Disulfide bond formation in RNase A over time. Reduced and denatured 723

RNase A was incubated with indicated combinations of proteins as in C. The amounts of 724

free thiol groups in the reaction mixtures were measured and net disulfide bonds were 725

calculated by subtracting the free disulfide bonds from total cysteine residues. Red and blue 726

dotted lines show the amounts of native disulfide bonds formed in RNase A, which were 727

calculated from the amounts of refolded RNase A. Data are represented as mean ± SEM of 728

n = 3. 729

730



30

Figure 7. GmPDIL-2 accelerates the oxidation of GmPDIM by GmERO1a. A, Oxygen 731

consumption was monitored during incubation of 2 μM GmERO1a and 3 μM GmPDIM 732

without or with 3 μM GmPDIL-2 or its mutants in the presence of 10 mM GSH. Data are 733

represented as mean ± SEM of n = 6. *, P < 0.05; **, P < 0.01 compared with the reaction 734

without GmPDIL-2 (Welch's t test). B, Schematic representation of the coupling reaction of 735

oxidation by ERO1 and the reduction of GSSG by glutathione-disulfide reductase (GR) in 736

the presence of GSH and NADPH. C, NADPH consumption over time in the presence of 1 737

μM GmERO1a and 3 μM GmPDIM, 3 μM GmPDIL-2, or 3 μM GmPDIM and 3 μM 738

GmPDIL-2 in the presence of 3 mM GSH, 120 μM NADPH, and 1 U/mL glutathione-739

disulfide reductase. D, Decrease in NADPH in the presence of indicated combinations of 740

proteins as in C. Data are represented as mean ± SEM of n = 10 (without GmPDIL-2); n = 741

3–4 (with wild-type GmPDIL-2 and its mutants). *, P < 0.05; **, P < 0.01; ***, P < 0.001 742

compared with the reaction without GmPDIL-2 (-) (Welch's t test). E, Schematic 743

representation of wild-type GmPDIM (WT) and its mutants. Numbers in the name of 744

mutants represent cysteine residues mutated to alanine. F, Oxidation of GmPDIM (WT), its 745

mutants, or its domain fragments by GmERO1a without or with GmPDIL-2 was measured 746

as in C. Data are represented as mean ± SEM of n = 3–6. **, P < 0.01; ***, P < 0.001 747

(Weltch's t test). G, Far-western blot analysis of the association of GmPDIM or its domain 748

fragment with GmERO1a. Indicated amounts of GmPDIM (WT), each domain fragment or 749

bovine serum albumin (BSA) (prey) was dot-blotted and incubated with GmERO1a. Bound 750

GmERO1a was immunostained. 751

752

Figure 8. GmPDIM oxidizes the active centers of GmPDIL-2. A, The mixed disulfide of 753

the GmPDIM and GmPDIL-2 mutants. GmPDIM(C67A) (6 μM), 3 μM GmPDIL-754

2(C104/443A) and 1μM GmERO1a were incubated at 25 °C for 60 min and separated by 755

two-dimensional electrophoresis. M1, GmPDIM monomer; L21 GmPDIL-2 monomer; M2, 756

GmPDIM dimer; L2M1 and L2M2, mixed disulfide complex of GmPDIL-2 mutants and 757

GmPDIM(C64A). B–F, Indicated combinations of proteins were incubated and analyzed as 758



31

described in A. G–L, Assay of redox equilibrium constants of GmPDIM, GmPDIL-2, and 759

their mutants. Coomassie Brilliant Blue staining of the SDS-polyacrylamide gels are shown 760

above the redox graphs. Data are represented as mean ± SEM of n=3. 761

762

Figure 9. Models of cooperation of GmPDIM and GmPDIL-2. A, Role-sharing model of 763

GmPDIM and GmPDIL-2. GmPDIM oxidized by GmERO1a introduces transient disulfide 764

bonds in an unfolded substrate protein (i). Transient disulfide bonds in the substrate protein 765

are reduced by the reduced form of GmPDIL-2 (ii). Reduced thiols are rearranged into 766

native disulfide bonds, mainly by GmPDIL-2 (iii), but partially by GmPDIM (iv). B, Model 767

of oxidative relays from GmERO1a to GmPDIM, GmPDIL-2, and substrate. GmERO1a 768

oxidizes the active center in the a’ domain of GmPDIM (i). The formed disulfide bond is 769

transferred sequentially from the a’ domain of GmPDIM to the a domain of GmPDIM (ii), 770

either the a or a’ domain of GmPDIL-2 (iii), and substrate (iv). Transferring of a disulfide 771

bond between the a and a’ domains of GmPDIL-2 is unclear. Both a and a’ domains of 772

GmPDIM and both b’ and a’ domains of GmPDIL-2 are essential for the association of 773

GmPDIM and GmPDIL-2. 774

775

776

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20: 2205–2220 781



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Weight of cotyledon (mg)

0

10

20

30

40 5020 90

120

110

140

160

180

200

220

27070

240

40

Rel

ativ

e am

ount

of

Gm

ER

O1

mR

NA H

―＋＋＋＋0

40

80

120

160

200

2 h 5 h 2 h 5 hGmERO1a GmERO1b

Exp

ress

ion

fold

TM ― ― ―

EGmPDIS-1GmERO1 Marge

Weight of immature cotyledon (mg)

30 40 60 70 90 110 130 150 170 190 210 230 250 270 290 310

F

GmERO1

B

25015010075

50

37

kD

reducing

root

stem

leaf

coty

ledo

n

1 2 3 4

nonreducing

53 kD46 kD

root

stem

leaf

coty

ledo

n

5 6 7 8

D

GmERO1

GmCNX

GmPDIL-1

Triton X-100

- +S P S P

1 2 3 4

C

5350

Endo H ― +kD

+

―

――Endo F

G

Figure 1. Identification of soybean Ero1. A, Schematic representation of Ero1 orthologs. Numbered circles represent the positions of the cysteine residues. Lines indicate disulfide bonds. B, GmERO1 is ubiquitously expressed in tissues. The proteins extracted from root (4.5 µg protein) (lanes 1 and 5), stem (10 µg protein) (lanes 2 and 6), leaf (30 µg protein) (lanes 3 and 7), and immature cotyledon (100 mg) (30 µg protein) (lanes 4 and 8) were separated under reducing (lanes 1–4) and nonreducing SDS-PAGE (lanes 5–8) and subjected to western blot analysis with an antiserum prepared against recombinant GmERO1. C, GmERO1 is N-glycosylated. GmERO1 in the immature cotyledon (30 mg) was detected by western blot analysis with the anti-GmERO1serum after treatment with (+) or without (-) endoglycosidase H (Endo H) or endoglycosidase F (Endo F). The 53-kDa band of GmERO1 was shifted to 50 kD after Endo H treatment. D, GmERO1is a membrane-bound protein. An immature cotyledon (100 mg) was homogenized by sonication. The homogenate was centrifuged at 100,000 × g for 2 h at 4°C in the absence (-) (lanes 1 and 2) or presence (+) of 1% Triton X-100 (lanes 3 and 4). GmERO1 protein in the supernatant (S) (lanes 1 and 3) and pellet (P) (lanes 2 and 4) was detected by western blot analysis with anti-GmERO1 serum, anti-soybean calnexin (GmCNX) serum, or anti-GmPDIL-1 serum. E, GmERO1 localizes in the ER. The immature cotyledon (195 mg) was fixed and embedded in resin. The sections were cut with a microtome and immunostained with anti-GmERO1 guinea pig serum and anti-GmPDIS-1 rabbit serum, and observed under a confocal microscope. Bars, 20 μm. F, Expression of GmERO1 in cotyledons during development. The proteins extracted from cotyledons were analyzed by western blot with anti-GmERO1 serum. G, Relative amounts of GmERO1a(black bars) and GmERO1b (white bars) mRNA in cotyledons during development were quantified by real-time PCR. Data are represented as mean ± SEM of n = 3. H, Expression levels of GmERO1a and GmERO1b mRNA are upregulated under ER stress. Young soybean leaves were treated with (+, black bars) or without (-, white bars) 5 μg/mL tunicamycin (TM) for 2 or 5 h. After treatment, GmERO1a and GmERO1b mRNA were quantified by real-time PCR. Data are represented as mean ± SEM of n = 3.

Figure 1

shuttle cysteine pair

active cysteine pair

regulatory cysteine

transmembrane region

A

Soybean ERO1a

Yeast Ero1p 40 52 90100

105 143 150 166 349 355208 295

49 51 6566

10267

104

113

118

121 146 217 226 332 367370

373104 118 123

11

Human Ero1 35 37 46 48 8594

99 104 131 166 391

394

397208 241

Human Ero1 42 44 8190

95 100 130 165 390

393

396207 240 262

N-glycosylation consensus site

360 399

280 384

383140145122

13021 35 53 342 425458

468491



GST-GmERO1

B

250150100

50

37

25

kD

reducing nonreducing

48 kD

38 kD

1 2 3 4

A

GST GmERO1

cleavage by PreScission protease

GmERO1

E70 G442

E70 G442

GPLGS

190 210 250230Wavelength (nm)

[θ]

(x10

3de

g /

cm2

/ dm

ol) 10

-10

-20

0

C

0.00

0.05

0.10

0.15

0.20

0.25

0.30

350 380 410 440 470 500

Abs

orba

nce

Wavelemgth (nm)

E

GmERO1

FAD + DTT

GmERO1 + DTT

200 400 60000

0.02

0.04

0.06

0.08

0.10

Abs

orba

nce

at 4

54 n

m

Time (sec)

Figure 2

D

Figure 2. Expression of recombinant GmERO1. A, Cleavage of recombinant glutathione-S-transferase (GST)-fused GmERO1 by PreScission protease. The gray box indicates GST, and the solid black line denotes the PreScission cleavage sequence. The N-terminal sequence GPLGS was cleaved from the fusion protein after protease treatment. B, Expression and purification of recombinant GmERO1. Recombinant GST-fused GmERO1 expressed in E. coli (lane 1) was purified by a glutathione Sepharose4B affinity column chromatography. GmERO1 was cleaved by PreScission protease and eluted from the column (lane 2), followed by gel filtration chromatography (lanes 3 and 4). Proteins in each eluate were separated by reducing SDS-PAGE (lanes 1–3) and nonreducing SDS-PAGE (lane 4), and stained with Coomassie Blue. C, Far-UV circular dichroism spectrum of recombinant GmERO1 in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 10% glycerol was obtained using a J-720 spectropolarimeter (JASCO Corp., Tokyo, Japan) in a 1-mm path-length cell with a scan speed of 20 nm/min at 14°C. D, Absorbance spectrum of recombinant GmERO1 was measured in the absence (solid line) or presence (dotted line) of 6 M guanidine hydrochloride. Free FAD spectrum (dashed line) was also measured. Black and white arrows show the maximum absorbance at 454 nm and the shoulder at 485 nm of GmERO1, respectively. E, GmERO1-bound FAD is reduced by DTT. Absorbance changes at 454 nm of 10 μM GmERO1 (circles) and 10 μM free FAD (triangles) were monitored in the presence of 2 mM DTT. DTT was added at zero time. GmERO1 in the absence of DTT was also measured (rectangles).



Figure 3

B O2

H2O2GSH

e- e-

ERO1PDI family

C

5

7

0

3

4

1

2

6

8

O2

cons

umpt

ion

rate

(μ

M /

min

)

GmPDIL-1 GmPDIL-2 GmPDIM GmPDIS-1 GmPDIS-2

GmPDIL-1

GmPDIL-2

GmPDIM

GmPDIS-1

GmPDIS-2

b’ba a’

b’ba a’

baaERp29caa

ERp29caa

A

Figure 3. GmERO1a shows broad specificity for ER oxidoreductases. A, Schematic representation of soybean ER oxidoreductases. Boxes represent domains. B, Schematic representation of the continuous oxidation of PDI family members by ERO1 in the presence of GSH and the reduction of O2. C, Oxygen consumption rate of GmERO1(1μM) in the presence of 3 μM each ER oxidoreductase and 10 mM GSH at 25°C. Data are represented as mean ± SEM of n = 3.



A B

DGmEro1a / GmPDIL-1

oxidation isomerizationfolding

Dred Dox N

GmEro1a / GmPDIM, GmPDIS-1, GmPDIS-2

oxidation isomerizationfolding

Dred Dox N

CGmPDIM

50+ -

2010 4030 400 N

Dred

DoxN

GmPDIL-2

50+ -2010 4030 400 NTime (min)

GmEro1

GmPDIL-1

50+ -2010 4030 400 N

GmPDIS-1

50+ -

2010 4030 400Time (min)

GmEro1N

GmPDIS-2

50+ -2010 4030 400 N

-

+50 40

-

Dred

Dox

0 40

N

Dred

DoxN

Dred

DoxN

Dred

DoxN

Dred

DoxN

GmPDIS-2

GmPDIL-1

20

30

0

10

50

40

Time (min)

Ref

olde

d R

Nas

eA

(%

)

GmPDIL-2GmPDIM

GmPDIS-1

5 10 15 20

0.0 0.5 1.0 1.5

5

GmPDIL-1

GmEro1 (μM)

10

0

200

0

100

300

Lag

time

(min

)

Maxim

um refolding rate

(mm

olRN

aseA

/ molP

DI/ m

in)

0.0 0.5 1.0 1.5

5

GmPDIM

GmEro1 (μM)

10

0

200

0

100

300

Lag

time

(min

)

Maxim

um refolding rate

(mm

olRN

aseA

/ molP

DI/ m

in)

0.0 0.5 1.0 1.5

GmPDIS-1

GmEro1 (μM)

10

0

5

200

0

100

300

Lag

time

(min

)

Maxim

um refolding rate

(mm

olRN

aseA

/ molP

DI/ m

in)

20010

0

5

0.0 0.5 1.0 1.5

GmPDIS-2

GmEro1 (μM)

0

100

300

Lag

time

(min

)

Maxim

um refolding rate

(mm

olRN

aseA

/ molP

DI/ m

in)

Figure 4

Figure 4. Reconstitution of oxidative protein folding with ER oxidoreductases and GmEro1a in vitro. A, GmERO1a(1μM) without (GmERO1a alone), or with 3 μM of each ER oxidoreductase, was incubated with reduced and denatured RNase A (8 μM), and the recovered RNase A activity was assayed. 'None' shows refolding of RNase A alone B, Lag time and maximum refolding rate of reduced and denatured RNase A (8 μM) by 3 μM each ER oxidoreductase in the presence of GmEro1a. Data are represented as mean ± SEM of n = 3. C, Refolding of reduced and denatured RNase A in the absence (-) or presence (+) of GmEro1a and each ER oxidoreductase, quenched with AMS, and was analyzed by nonreducing SDS-PAGE. Dred, reduced and denatured RNase A; Dox, denatured RNase A with non-native disulfides; N, native RNase A. D, Model of refolding of RNase A by ER oxidoreductases in the presence of GmEro1a. Top, GmPDIL-1 transfers non-native disulfide bonds to Dred. Dox is promptly folded into the native form accompanied by the isomerization of disulfide bonds. Bottom, GmPDIM, GmPDIS-1, or GmPDIS-2 transfers non-native disulfide bonds to Dred. Dox accumulates due to rate-limiting isomerization activities of GmPDIM, GmPDIS-1, and GmPDIS-2.

0

none

GmERO1a alone



A CB

Ref

olde

d R

Nas

eA

(%

) 50

30

20

10

0

40

60

0 155 10Time (min)

20

IP

PIInput

GmPDIS-1

GmPDIS-2

GmPDIL-1

GmPDIM

GmPDIL-2

1 2 3 4 5 6 7

E

a’-b (A151-L438) CGHC

b (T270-L438)

a’ (A151-E269) CGHC

a (L29-K150) CGHC

a-a’ (L29-E269) CGHC CGHC

(pmol)80 40 520 10 2.5

WT (L29-L438)

a ba’

CGHC CGHC

prey

D (pmol)

80 40 520 10 2.5

b-b’ (I176-V412)

a’ (E392-L551) CGHC

b’-a’ (I300-L551) CGHC

a (D24-K175) CGHC

CGHCa-b-b’ (D24-V412)

WT (D24-L551)a b b’ a’

CGHC

a-b (D24-K301) CGHC

CGHC

prey

GmPDIM + GmPDIL-2

GmPDIL-2

GmPDIM

0

100

200

300

400

500

600

700

0 1 2 3 4 5 6Act

ivity

(m

mol

RN

aseA

/ m

olP

DIM

/ m

in)

Concentration of GmPDIL-2 (μM)

Figure 5

Figure 5. GmPDIL-2 and GmPDIM form a complex and synergistically refold RNase A with using disulfide bonds produced by GmERO1a. A, Detection of ER oxidoreductase complexes. Immunoprecipitation (IP) from the cotyledon extract was carried out with each anti-ER oxidoreductase serum or preimmune serum (PI). Immunoprecipitates and the cotyledon extract (lane 1) were analyzed by western blot with each anti-ER oxidoreductase serum. B, Effects of the addition of GmPDIL-2 to GmPDIM on the refolding of RNase A. Reduced and denatured RNase A (8 μM) was incubated with 3 μM ER oxidoreductases in the presence of 1 μM GmERO1a. C, GmPDIL-2 concentration dependence of the refolding activity in the presence of 1 μM GmERO1a and 1 μMGmPDIM. Data are represented as mean ± SEM of n = 3. D, Far-western blot analysis of the association of the GmPDIL-2 (WT) or GmPDIL-2 domain fragment with GmPDIM. Indicated amounts of GmPDIL-2, each GmPDIL-2 domain fragment or bovine serum albumin (BSA) (prey) were dot-blotted and incubated with GmPDIM. Bound GmPDIM was immunostained. E, GmPDIM (WT), each GmPDIM domain fragment or BSA (prey) was dot-blotted and incubated with GmPDIL-2. Bound GmPDIL-2 was immunostained.

BSA BSA

BSA



C101/104A AGHA CGHC

C101/104/440/443A AGHA AGHA

AWT

a b b’ a’CGHC CGHC

C440/443A CGHC AGHA

C443A CGHC CGHA

C104A CGHA CGHC

C104/443A CGHA CGHA

B

0

100

200

300

400

500

600

Act

ivity

(m

mol

RN

aseA

/ m

olP

DIM

/ m

in)

-

Figure 6

Figure 6. Active centers of GmPDIL-2 play vital roles in the acceleration of both refolding and disulfide bond formation. A, Schematic representation of wild-type GmPDIL-2 (WT) and its mutants. “CGHC,” “AGHA,” and “CGHA” represent the amino acid sequences of the active sites of the a and a’ domains. Numbers in the name of the mutants represent cysteine residues mutated to alanine. B, Acceleration of refolding by GmPDIL-2 mutants and GmPDIL-2 domain fragments in the GmERO1a/GmPDIM system. Reduced and denatured RNase A (8 μM) was incubated with 1 μM GmPDIM and 4 μM wild type GmPDIL-2 (WT), its mutants or domain fragments in the presence of 1 μM GmERO1a. Data are represented as mean ± SEM of n = 3–5. Bars with the same letters are not significantly different at P < 0.05 by Tukey-Kramer test. C, Disulfide bond formation in reduced and denatured RNase A (8 μM) during cooperative refolding by GmPDIM or GmPDIM mutant (1 μM) and GmPDIL-2 or GmPDIL-2 mutants (3 μM) in the presence of GmERO1a (1 μM) was analyzed by nonreducing SDS-PAGE. Dred, reduced and denatured RNase A; Dox, denatured RNase A with non-native disulfides; N, native RNase A. D, Disulfide bond formation in RNase A over time. Reduced and denatured RNase A was incubated with indicated combinations of proteins as in C. The amounts of free thiol groups in the reaction mixtures were measured and net disulfide bonds were calculated by subtracting the free disulfide bonds from total cysteine residues. Red and blue dotted lines show the amounts of native disulfide bonds formed in RNase A, which were calculated from the amounts of refolded RNase A. Data are represented as mean ± SEM of n = 3.

D

g

a

efg

c

b

d

eefg efg efg efg

efgef

fg

0

10

20

30

0 10 20 30 400

10

20

30

0 10 20 30 40

Time (min)

Net

Dis

ulfid

e bo

nd (

µM

) WTGmPDIM

GmPDIM + C101/104A

GmPDIM + WT

GmPDIM + C101/104A/440/443A

GmPDIM + C440/443A

GmPDIM(C192/195A) + WT

C none

50+ -

2010 6040 600Time (min)GmERO1

N

Dred

DoxN

C101/104/440/443A

50+ -2010 6040 600 NTime (min)

GmERO1

Dred

DoxN

C101/104A

50+ -

2010 6040 600 N

Dred

Dox

Time (min)GmERO1

N

C440/443A

5+ -

2010 6040 600 N

DredDoxN

Time (min)GmERO1

WT

50+ -2010 6040 600 N

Dred

Dox

N

Time (min)GmERO1

WT

50+ -

2010 6040 600 N

Dred

Dox

Time (min)GmERO1

N

GmPDIM +

GmPDIM +

GmPDIM +

GmPDIM +

GmPDIM +

GmPDIM(C192/195A) +



A

G

a’-b (A151-L438)

b (T270-L438)

a’ (A151-E269)

a (L29-K150)

a-a’ (L29-E269)

WT (L29-L438)

prey(pmol)

80 40 520 10 2.5EWT (L29-L438)

a ba’

CGHC CGHC

C192/195A CGHC AGHA

C64/67A AGHA CGHC

C67A CGHA CGHC

CGHC CGHAC195A

GRNADPH GSSG

GSH

ERO1PDI family

NADP

B

F

GmPDIL-2

GmPDIM WT

- +

C192/195A

+-

C64/67A

+-+-

C195A

+-

C67A a’-b

+-

a-a’

+-

a’

+-

a

+-

Dec

reas

e of

NA

DP

H(μ

M/

min

)

6

0

4

5

2

3

1

+

-

C

80

40

20

Time (min)

NA

DP

H c

onsu

mpt

ion

(M

)

160 GmPDIL-2

GmPDIM + GmPDIL-2

GmPDIM

none

03020100

120

D

WT ’a-b-b’- ’b-b’-a’ ’b’-a’ ’a’a-ba ’b-b’

Dec

reas

e of

NA

DP

H(μ

M/

min

)

5

1

3

6

2

4

0

**

**

** ***

**

* * * *** * * ***

**

0

1

2

3

4

5

6

7

0

3

4

5

1

2

6

7

O2

cons

umpt

ion

rate

(μ

M /

min

)

*

**

** *

Figure 7

Figure 7. GmPDIL-2 accelerates the oxidation of GmPDIM by GmERO1a. A, Oxygen consumption was monitored during incubation of 2 μM GmERO1a and 3 μM GmPDIM without or with 3 μMGmPDIL-2 or its mutants in the presence of 10 mM GSH. Data are represented as mean ± SEM of n = 6. *, P < 0.05; **, P < 0.01 compared with the reaction without GmPDIL-2 (Welch's t test). B, Schematic representation of the coupling reaction of oxidation by ERO1 and the reduction of GSSG by glutathione-disulfide reductase (GR) in the presence of GSH and NADPH. C, NADPH consumption over time in the presence of 1 μM GmERO1a and 3 μM GmPDIM, 3 μM GmPDIL-2, or 3 μM GmPDIM and 3 μM GmPDIL-2 in the presence of 3 mM GSH, 120 μM NADPH, and 1 U/mL glutathione-disulfide reductase. D, Decrease in NADPH in the presence of indicated combinations of proteins as in C. Data are represented as mean ± SEM of n = 10 (without GmPDIL-2); n = 3–4 (with wild-type GmPDIL-2 and its mutants). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the reaction without GmPDIL-2 (-) (Welch's t test). E, Schematic representation of wild-type GmPDIM (WT) and its mutants. Numbers in the name of mutants represent cysteine residues mutated to alanine. F, Oxidation of GmPDIM (WT), its mutants, or its domain fragments by GmERO1a without or with GmPDIL-2 was measured as in C. Data are represented as mean ± SEM of n = 3–6. **, P < 0.01; ***, P < 0.001 (Weltch's t test). G, Far-western blot analysis of the association of GmPDIM or its domain fragment with GmERO1a. Indicated amounts of GmPDIM (WT), each domain fragment or bovine serum albumin (BSA) (prey) was dot-blotted and incubated with GmERO1a. Bound GmERO1a was immunostained.

WT-

*

**

** **

**

**

***

***

***

BSA



GmPDIL-2

Rat

io o

f re

duce

d pr

otei

n (%

)

[GSH]2/[GSSG] (mM)

J

Keq = 3.38 ± 0.21

0.001 0.01 1 10 100 10000.1 10000

1.0

0.8

0.6

0.4

0.2

0

R

Ox

71

45

32

kD136 95 71 45 32kD

M2 M1

L21

ERO1L2-M2

L2-M1

AGmPDIM(C67A): GmPDIL2(C104/443A): GmERO1

71

45

32

kD136 95 71 45 32kD

M1

L21

ERO1

GmPDIM (WT): GmPDIL2(C104/443A): GmERO1

E

GmPDIM(C64A): GmPDIL2(C443A): GmERO1

71

45

32

kD136 95 71 45 32kD

M2M1

L21

ERO1L2-M1

CGmPDIM(C67A): GmPDIL2(C104A): GmERO1

71

45

32

kD136 95 71 45 32kD

M2M1

L21

ERO1L2-M2

B

GmPDIM(C67A): GmPDIL2(C104/443A)

71

45

32

kD136 95 71 45 32kD

M1

L21

DGmPDIM(C195A): GmPDIL2(C104/443A): GmERO1

71

45

32

kD136 95 71 45 32kD

M1

L21

ERO1

F

G

Rat

io o

f re

duce

d pr

otei

n (%

)

[GSH]2/[GSSG] (mM)

Keq = 2.02 ± 0.06

0.001 0.01 1 10 100 10000.1 10000

1.0

0.8

0.6

0.4

0.2

0

R

Ox

GmPDIM

[GSH]2/[GSSG] (mM)

Rat

io o

f re

duce

d pr

otei

n (%

) Keq = 2.66 ± 0.38

L

0.001 0.01 1 10 100 10000.1 10000

1.0

0.8

0.6

0.4

0.2

0

R

Ox

GmPDIL-2 (C101/104A)

Rat

io o

f re

duce

d pr

otei

n (%

)

[GSH]2/[GSSG] (mM)

Keq = 4.33 ± 0.81

K

0.001 0.01 1 10 100 10000.1 10000

1.0

0.8

0.6

0.4

0.2

0

R

Ox

GmPDIL-2 (C440/443A)

Rat

io o

f re

duce

d pr

otei

n (%

)

[GSH]2/[GSSG] (mM)

I

0.001 0.01 1 10 100 10000.1 10000

1.0

0.8

0.6

0.4

0.2

0

Keq = 3.53 ± 0.99

R

Ox

GmPDIM (C64/67A)

Rat

io o

f re

duce

d pr

otei

n (%

)

[GSH]2/[GSSG] (mM)

Keq = 0.73 ± 0.17

H

0.0001 1 1000.01 10000

1.0

0.8

0.6

0.4

0.2

0

R

Ox

GmPDIM (C192/195A)

Figure 8

Figure 8. GmPDIM oxidizes the active centers of GmPDIL-2. A, The mixed disulfide of the GmPDIM and GmPDIL-2 mutants. GmPDIM(C67A) (6 μM), 3 μM GmPDIL-2(C104/443A) and 1μM GmERO1a were incubated at 25 °C for 60 min and separated by two-dimensional electrophoresis. M1, GmPDIM monomer; L21 GmPDIL-2 monomer; M2, GmPDIM dimer; L2M1 and L2M2, mixed disulfide complex of GmPDIL-2 mutants and GmPDIM(C64A). B–F, Indicated combinations of proteins were incubated and analyzed as described in A. G–L, Assay of redoxequilibrium constants of GmPDIM, GmPDIL-2, and their mutants. Coomassie Brilliant Blue staining of the SDS-polyacrylamide gels are shown above the redox graphs. Data are represented as mean ± SEM of n = 3.

E0’ = -167 mV

E0’ = -160 mV

E0’ = -163 mVE0’ = -169 mV

E0’ = -166 mVE0’ = -147 mV



B

GmPDIL2

native form

reduced form

A

GmERO1

O2

H2O2

GmPDIM

oxidized intermediate form

i

ii

iiiiv

GmERO1

O2

H2O2

GmPDIL2GmPDIM native formunfolded protein

i iiiii

iv

iv

?

Figure 9

Figure 9. Models of cooperation of GmPDIM and GmPDIL-2. A, Role-sharing model of GmPDIM and GmPDIL-2. GmPDIM oxidized by GmERO1a introduces transient disulfide bonds in an unfolded substrate protein (i). Transient disulfide bonds in the substrate protein are reduced by the reduced form of GmPDIL-2 (ii). Reduced thiols are rearranged into native disulfide bonds, mainly by GmPDIL-2 (iii), but partially by GmPDIM (iv). B, Model of oxidative relays from GmERO1a to GmPDIM, GmPDIL-2, and substrate. GmERO1a oxidizes the active center in the a’ domain of GmPDIM (i). The formed disulfide bond is transferred sequentially from the a’ domain of GmPDIMto the a domain of GmPDIM (ii), either the a or a’ domain of GmPDIL-2 (iii), and substrate (iv). Transferring of a disulfide bond between the a and a’ domains of GmPDIL-2 is unclear. Both a and a’ domains of GmPDIM and both b’ and a’ domains of GmPDIL-2 are essential for the association of GmPDIM and GmPDIL-2.



Parsed CitationsAndème Ondzighi C, Christopher DA, Cho EJ, Chang S-C, Staehelin LA (2008) Arabidopsis protein disulfide isomerase-5 inhibitscysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds. PlantCell 20: 2205-2220

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Pubmed: Author and Title www.plantphysiol.orgon February 16, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 20%5BVolume%5D AND 2205%5BPagination%5D AND And�me Ondzighi C%2C Christopher DA%2C Cho EJ%2C Chang S%2DC%2C Staehelin LA%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=And�me Ondzighi C, Christopher DA, Cho EJ, Chang S-C, Staehelin LA (2008) Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds. Plant Cell 20: 2205-2220

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=And�me&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=And�me&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Cell Biol%5BTitle%5D%29 AND 202%5BVolume%5D AND 861%5BPagination%5D AND Araki K%2C Iemura SI%2C Kamiya Y%2C Ron D%2C Kato K%2C Natsume T%2C Nagata K%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Araki K, Iemura SI, Kamiya Y, Ron D, Kato K, Natsume T, Nagata K (2013) Ero1-a and pdis constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases. J Cell Biol 202: 861-874

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Araki&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ero1-a and pdis constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28Antioxid Redox Signal%5BTitle%5D%29 AND 16%5BVolume%5D AND 790%5BPagination%5D AND Araki K%2C Inaba K%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Araki K, Inaba K (2012) Structure, Mechanism, and Evolution of Ero1 Family Enzymes. Antioxid Redox Signal 16: 790-799

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Araki&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Structure, Mechanism, and Evolution of Ero1 Family Enzymes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Structure, Mechanism, and Evolution of Ero1 Family Enzymes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Araki&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Ero1%2DPDI interactions%2C the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum%2E%5BTitle%5D%29 AND Benham AM%2C van Lith M%2C Sitia R%2C Braakman I%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Benham AM, van Lith M, Sitia R, Braakman I (2013) Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum. Philos Trans R Soc Lond B Biol Sci 368: 20110403

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Benham&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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Nguyen VD, Saaranen MJ, Karala AR, Lappi AK, Wang L, Raykhel IB, Alanen HI, Salo KEH, Wang CC, Ruddock LW (2011) Twoendoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation. J Mol Biol406: 503-515


Oh D-H, Kwon C-S, Sano H, Chung W-I, Koizumi N (2003) Conservation between animals and plants of the cis-acting elementinvolved in the unfolded protein response. Biochem Biophys Res Commun 301: 225-230


Onda Y, Kumamaru T, Kawagoe Y (2009) ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation inthe rice endosperm. Proc Natl Acad Sci U S A 106: 14156-14161


Onda Y, Nagamine A, Sakurai M, Kumamaru T, Ogawa M, Kawagoe Y (2011) Distinct roles of protein disulfide isomerase and P5sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice. Plant Cell 23: 210-223


Oka OB, Yeoh HY, Bulleid NJ (2015) Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirementfor an oxidase or reductase for each enzyme. Biochem J 469: 279-288


Pagani M, Fabbri M, Benedetti C, Fassio A, Pilati S, Bulleid NJ, Cabibbo A, Sitia R (2000) Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response. J Biol Chem 275: 23685-23692


Pollard MG, Travers KJ, Weissman JS (1998) Ero1p: a novel and ubiquitous protein with an essential role in oxidative proteinfolding in the endoplasmic reticulum. Mol Cell 1: 171-182


Pu Y, Bassham DC (2013) Links between ER stress and autophagy in plants. Plant Signal Behav 8: e24297Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ramming T, Okumura M, Kanemura S, Baday S, Birk J, Moes S, Spiess M, Jenö P, Bernèche S, Inaba K, et al (2015) A PDI-catalyzedthiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1. Free Radic Biol Med 83: 361-372


Sato Y, Kojima R, Okumura M, Hagiwara M, Masui S, Maegawa K, Saiki M, Horibe T, Suzuki M, Inaba K (2013) Synergisticcooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding. Sci Rep 3: 2456


Satoh-Cruz M, Crofts AJ, Takemoto-Kuno Y, Sugino A, Washida H, Crofts N, Okita TW, Ogawa M, Satoh H, Kumamaru T (2010)Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in riceendosperm. Plant Cell Physiol 51: 1581-1593


Serve O, Kamiya Y, Maeno A, Nakano M, Murakami C, Sasakawa H, Yamaguchi Y, Harada T, Kurimoto E, Yagi-Utsumi M, et al (2010)Redox-dependent domain rearrangement of protein disulfide isomerase coupled with exposure of its substrate-bindinghydrophobic surface. J Mol Biol 396: 361-374


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http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Mol Biol%5BTitle%5D%29 AND 406%5BVolume%5D AND 503%5BPagination%5D AND Nguyen VD%2C Saaranen MJ%2C Karala AR%2C Lappi AK%2C Wang L%2C Raykhel IB%2C Alanen HI%2C Salo KEH%2C Wang CC%2C Ruddock LW%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nguyen&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Biochem Biophys Res Commun%5BTitle%5D%29 AND 301%5BVolume%5D AND 225%5BPagination%5D AND Oh D%2DH%2C Kwon C%2DS%2C Sano H%2C Chung W%2DI%2C Koizumi N%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Oh D-H, Kwon C-S, Sano H, Chung W-I, Koizumi N (2003) Conservation between animals and plants of the cis-acting element involved in the unfolded protein response. Biochem Biophys Res Commun 301: 225-230

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Oh&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Conservation between animals and plants of the cis-acting element involved in the unfolded protein response&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Conservation between animals and plants of the cis-acting element involved in the unfolded protein response&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Oh&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Proc Natl Acad Sci U S A%5BTitle%5D%29 AND 106%5BVolume%5D AND 14156%5BPagination%5D AND Onda Y%2C Kumamaru T%2C Kawagoe Y%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Onda Y, Kumamaru T, Kawagoe Y (2009) ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm. Proc Natl Acad Sci U S A 106: 14156-14161

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Onda&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Onda&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell%5BTitle%5D%29 AND 23%5BVolume%5D AND 210%5BPagination%5D AND Onda Y%2C Nagamine A%2C Sakurai M%2C Kumamaru T%2C Ogawa M%2C Kawagoe Y%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Onda Y, Nagamine A, Sakurai M, Kumamaru T, Ogawa M, Kawagoe Y (2011) Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice. Plant Cell 23: 210-223

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Onda&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Onda&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Biochem J%5BTitle%5D%29 AND 469%5BVolume%5D AND 279%5BPagination%5D AND Oka OB%2C Yeoh HY%2C Bulleid NJ%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Oka OB, Yeoh HY, Bulleid NJ (2015) Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme. Biochem J 469: 279-288

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Oka&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Oka&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Biol Chem%5BTitle%5D%29 AND 275%5BVolume%5D AND 23685%5BPagination%5D AND Pagani M%2C Fabbri M%2C Benedetti C%2C Fassio A%2C Pilati S%2C Bulleid NJ%2C Cabibbo A%2C Sitia R%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pagani M, Fabbri M, Benedetti C, Fassio A, Pilati S, Bulleid NJ, Cabibbo A, Sitia R (2000) Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response. J Biol Chem 275: 23685-23692

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pagani&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pagani&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Mol Cell%5BTitle%5D%29 AND 1%5BVolume%5D AND 171%5BPagination%5D AND Pollard MG%2C Travers KJ%2C Weissman JS%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Ero1p: a novel and ubiquitous protein with an essential role in oxidative protein folding in the endoplasmic reticulum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pollard&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Links between ER stress and autophagy in plants%2E%5BTitle%5D%29 AND Pu Y%2C Bassham DC%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pu Y, Bassham DC (2013) Links between ER stress and autophagy in plants. Plant Signal Behav 8: e24297

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http://scholar.google.com/scholar?as_q=Links between ER stress and autophagy in plants.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Links between ER stress and autophagy in plants.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pu&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Free Radic Biol Med%5BTitle%5D%29 AND 83%5BVolume%5D AND 361%5BPagination%5D AND Ramming T%2C Okumura M%2C Kanemura S%2C Baday S%2C Birk J%2C Moes S%2C Spiess M%2C Jen� P%2C Bern�che S%2C Inaba K%2C et al%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ramming T, Okumura M, Kanemura S, Baday S, Birk J, Moes S, Spiess M, Jen� P, Bern�che S, Inaba K, et al (2015) A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1. Free Radic Biol Med 83: 361-372

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ramming&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ramming&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Synergistic cooperation of PDI family members in peroxiredoxin 4%2Ddriven oxidative protein folding%2E%5BTitle%5D%29 AND Sato Y%2C Kojima R%2C Okumura M%2C Hagiwara M%2C Masui S%2C Maegawa K%2C Saiki M%2C Horibe T%2C Suzuki M%2C Inaba K%5BAuthor%5D&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sato Y, Kojima R, Okumura M, Hagiwara M, Masui S, Maegawa K, Saiki M, Horibe T, Suzuki M, Inaba K (2013) Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding. Sci Rep 3: 2456

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sato&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sato&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28Plant Cell Physiol%5BTitle%5D%29 AND 51%5BVolume%5D AND 1581%5BPagination%5D AND Satoh%2DCruz M%2C Crofts AJ%2C Takemoto%2DKuno Y%2C Sugino A%2C Washida H%2C Crofts N%2C Okita TW%2C Ogawa M%2C Satoh H%2C Kumamaru T%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Satoh-Cruz&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Satoh-Cruz&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=%28J Mol Biol%5BTitle%5D%29 AND 396%5BVolume%5D AND 361%5BPagination%5D AND Serve O%2C Kamiya Y%2C Maeno A%2C Nakano M%2C Murakami C%2C Sasakawa H%2C Yamaguchi Y%2C Harada T%2C Kurimoto E%2C Yagi%2DUtsumi M%2C et al%5BAuthor%5D&dopt=abstract

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334: 1086-1090Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sun L, Yang ZT, Song ZT, Wang MJ, Sun L, Lu SJ, Liu JX (2013) The plant-specific transcription factor gene NAC103 is induced bybZIP60 through a new cis-regulatory element to modulate the unfolded protein response in Arabidopsis. Plant J 76: 274-286


Takemoto Y, Coughlan SJ, Okita TW, Satoh H, Ogawa M, Kumamaru T (2002) The rice mutant esp2 greatly accumulates the glutelinprecursor and deletes the protein disulfide isomerase. Plant Physiol 128: 1212-1222


Tavender TJ, Bulleid NJ (2010) Molecular mechanisms regulating oxidative activity of the Ero1 family in the endoplasmicreticulum. Antioxid Redox Signal 13: 1177-1187


Tien AC, Rajan A, Schulze KL, Hyung DR, Acar M, Steller H, Bellen HJ (2008) Ero1L, a thiol oxidase, is required for Notch signalingthrough cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster. J Cell Biol 182: 1113-1125


Tu BP, Weissman JS (2004) Oxidative protein folding in eukaryotes: Mechanisms and consequences. J Cell Biol 164: 341-346Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Vitu E, Kim S, Sevier CS, Lutzky O, Heldman N, Bentzur M, Unger T, Yona M, Kaiser CA, Fass D (2010) Oxidative activity of yeastEro1p on protein disulfide isomerase and related oxidoreductases of the endoplasmic reticulum. J Biol Chem 285: 18155-18165


Wadahama H, Kamauchi S, Ishimoto M, Kawada T, Urade R (2007) Protein disulfide isomerase family proteins involved in soybeanprotein biogenesis. FEBS J 274: 687-703


Wadahama H, Kamauchi S, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T, Urade R (2008) A novel plant protein disulfideisomerase family homologous to animal P5 - Molecular cloning and characterization as a functional protein for folding of soybeanseed-storage proteins. FEBS J 275: 399-410


Wang C, Li W, Ren J, Fang J, Ke H, Gong W, Feng W, Wang C (2012) Structural Insights into the Redox-Regulated DynamicConformations of Human Protein Disulfide Isomerase. Antioxid Redox Signal 19: 36-45


Wang L, Zhu L, Wang C (2011) The endoplasmic reticulum sulfhydryl oxidase Ero1ß drives efficient oxidative protein folding withloose regulation. Biochem J 434: 113-121


Zito E, Chin K-T, Blais J, Harding HP, Ron D (2010) ERO1-beta, a pancreas-specific disulfide oxidase, promotes insulin biogenesisand glucose homeostasis. J Cell Biol 188: 821-832



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