Protein disulfide isomerase family proteins involved in soybean protein biogenesis.
Protein disulfide isomerase family proteins are known to 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) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.
Wadahama
Hiroyuki
H
Graduate School of Agriculture, Kyoto University, Uji, Japan.
Kamauchi
Shinya
S
Ishimoto
Masao
M
Kawada
Teruo
T
Urade
Reiko
R
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018548:Cotyledon / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020869:Gene Expression Profiling; D005916:Globulins / Q000378:metabolism; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D030262:Soybean Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013025:Glycine max / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D018076:DNA, Complementary; D005916:Globulins; D007527:Isoenzymes; D011498:Protein Precursors; D011994:Recombinant Proteins; D030262:Soybean Proteins; C011474:glycinin; D019704:Protein Disulfide-Isomerases