A novel plant protein disulfide isomerase family homologous to animal P5 - molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins.
The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.
Wadahama
Hiroyuki
H
Graduate School of Agriculture, Kyoto University, Uji, Japan.
Kamauchi
Shinya
S
Nakamoto
Yumi
Y
Nishizawa
Keito
K
Ishimoto
Masao
M
Kawada
Teruo
T
Urade
Reiko
R
D000818:Animals; D052179:Antigens, Plant; D015153:Blotting, Western; D003001:Cloning, Molecular; D018548:Cotyledon / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005916:Globulins / Q000378:metabolism; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055314:Seed Storage Proteins; D012639:Seeds / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D030262:Soybean Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics
D052179:Antigens, Plant; D018076:DNA, Complementary; D018744:DNA, Plant; D005916:Globulins; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; D019704:Protein Disulfide-Isomerases