Protein disulfide isomerase family proteins involved in soybean protein biogenesis
Protein disulfide isomerase family proteins involved in soybean protein biogenesis
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DOI：
10.1111/j.1742-4658.2006.05613.x
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发表时间：
2007-02
期刊：
影响因子：
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通讯作者：
Hiroyuki Wadahama;S. Kamauchi;M. Ishimoto;T. Kawada;R. Urade
中科院分区：
文献类型：
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作者：
Hiroyuki Wadahama;S. Kamauchi;M. Ishimoto;T. Kawada;R. Urade
蛋白质二硫键异构酶家族蛋白在新生多肽的折叠和内质网二硫键的形成中起着重要的作用。本研究从大豆叶片中克隆了两个相似的蛋白质二硫键异构酶家族基因。用从表达序列标签克隆序列设计的正向和反向引物进行RT-PCR。该基因编码364或362个氨基酸的蛋白质，分别命名为GmPDIS-1或GmPDIS-2。GmPDIS-1和GmPDIS-2的核苷酸和氨基酸序列同源性分别为68%和74%。这两种蛋白质都缺乏内质网恢复信号KDEL的C末端。重组蛋白GmPDIS-1和GmPDIS-2在大肠杆菌中以可溶性折叠蛋白的形式表达，显示了变性核糖核酸酶A的氧化复性活性和伴侣活性。通过胰酶或V8酶的肽图分析，它们的结构被鉴定为含有两个硫氧还蛋白样结构域，a和a‘，以及一个ERp29c结构域。在子叶细胞中，共聚焦显微镜显示这两种蛋白质都分布在内质网和蛋白质储存空泡中。来自免疫共沉淀和交联实验的数据表明，GmPDIS-1与种子储存蛋白球蛋白的前体丙球蛋白在子叶中结合。GmPDIS-1的水平在子叶中增加，但GmPDIS-2的水平没有增加，在种子发育过程中球蛋白在子叶中积累。GmPDIS-1，而不是GmPDIS-2在内质网应激条件下被诱导。
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.