Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL‐1 and GmPDIL‐2, respectively. Recombinant versions of GmPDIL‐1 and GmPDIL‐2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL‐1 and GmPDIL‐2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon–intron structure across plant species. The promoter sequences of GmPDIL‐1 apparently contain a cis‐acting regulatory element functionally linked to unfolded protein response. GmPDIL‐1, but not GmPDIL‐2, expression was induced under endoplasmic reticulum‐stress conditions. GmPDIL‐1 and GmPDIL‐2 promoters contain some predicted regulatory motifs for seed‐specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL‐1 and GmPDIL‐2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the α′‐subunit of β‐conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or β‐conglycinin, suggesting that GmPDIL‐1 and GmPDIL‐2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.

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Protein disulfide isomerase (PDI) and other PDI family proteins are members of the thioredoxin superfamily and are thought to play important roles in disulfide bond formation and isomerization in the endoplasmic reticulum (ER). The exact functions of PDI family proteins in plants remain unknown. In this study, we cloned two novel PDI family genes from soybean leaf (Glycine max L. Merrill cv. Jack). The cDNAs encode proteins of 520 and 523 amino acids, and have been denoted GmPDIL‐3a and GmPDIL‐3b, respectively. GmPDIL‐3a and GmPDIL‐3b are the first plant ER PDI family proteins reported to contain the nonclassic redox center motif CXXS/C, and both proteins are ubiquitously expressed in the plant body. However, recombinant GmPDIL‐3a and GmPDIL‐3b did not function as oxidoreductases or as molecular chaperones in vitro, although a proportion of each protein formed complexes in both thiol‐dependent and thiol‐independent ways in the ER. Expression of GmPDIL‐3a and GmPDIL‐3b in the cotyledon increased during seed maturation when synthesis of storage proteins was initiated. These results suggest that GmPDIL‐3a and GmPDIL‐3b may play important roles in the maturation of the cotyledon by mechanisms distinct from those of other PDI family proteins. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7137566: Bip (uniprotkb:http://www.ebi.uniprot.org/entry/Q587K1), GmPDIL‐3b (genbank_nucleotide_g:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=Retrieve&dopt=Graphics&list_uids=51848586) and GmPDIL‐3a (genbank_nucleotide_g:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=Retrieve&dopt=Graphics&list_uids=51848584) colocalize (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403) by cosedimentation through density gradients (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0029)

b-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean b-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the a#-and a-subunits of b-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the b-conglycinin complexes containing the disulfide-linked a#/a-subunits were complexes of more than 720 kD. The a#-and a-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between a#/a-subunits residing in different b-conglycinin hexamers, but the binding of P34 to a#-and a-subunits reduces the linkage between b-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when b-conglycinin was expressed under nonreducing conditions. The large majority of seed storage proteins from the leguminous species are globulins with sedimentation coefficients of 7 to 8 S (vicilin) and 11 to 12 S (legumin). The corresponding proteins from soybean (Glycine max) are called b-conglycinin and glycinin, respectively (Casey, 1999). The b-conglycinin from soybean seeds is isolated as a trimer with sedimentation coefficients between 7 and 8 S (Thanh and Shibasaki, 1976;Nielsen and Nam, 1999). The size of b-conglycinin complexes depends on the ionic strength of the solution. b-Conglycinin is a trimer at higher than 0.2 M ionic strength, but at neutral pH, it associates into hexamers at decreasing ionic strength (Thanh and Shibasaki, 1979). The b-conglycinin trimers are mixtures of isoforms consisting of different combinations of the three types of subunits, designated a#, a with M r of 57,000, and b with M r of 42,000 (Thanh and Shibasaki, 1978;Davies et al., 1985;Nielsen and Nam, 1999). The a#-, a-, and b-subunits are synthesized in the endoplasmic reticulum (ER) of the cotyledon cell as polypeptides with a signal peptide and a propeptide (prepro a#-and prepro a-subunits) or as polypeptides with only a signal peptide (pre-b-subunit; Sengupta et al