Sasou Ai, Shigemitsu Takanari, Morita Shigeto, Masumura Takehiro
Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto, 606-8522, Japan.
Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, Kitainayazuma, Seika-cho, Soraku-gun, Kyoto, 619-0244, Japan.
Plant Cell Rep. 2017 Mar;36(3):481-491. doi: 10.1007/s00299-016-2097-5. Epub 2016 Dec 27.
Rice prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of prolamins that must accumulate in PB-Is. Rice seed prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice prolamins, we compared 13a and 13b prolamins because the amino acid sequences of these prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.
水稻醇溶蛋白在内质网衍生的蛋白体中积累，尽管在内质网中保留的保守序列尚未得到证实。我们研究了必须在PB-Is中积累的醇溶蛋白的部分序列。水稻种子醇溶蛋白在内质网衍生的I型蛋白体（PB-I）中积累，但水稻醇溶蛋白多肽中的内质网保留序列尚未得到证实。在这里，我们研究了在PB-Is中积累所需的醇溶蛋白部分序列的长度。在水稻醇溶蛋白中，我们比较了13a和13b醇溶蛋白，因为除了半胱氨酸残基的存在与否外，这些醇溶蛋白的氨基酸序列非常相似。我们还生成并分析了表达几种醇溶蛋白部分序列-GFP融合蛋白的转基因水稻。我们观察到，在13a醇溶蛋白中，当部分序列从起始甲硫氨酸延伸超过第68个氨基酸残基时，醇溶蛋白部分序列-GFP融合蛋白在PB-Is中积累。在13b醇溶蛋白中，当部分序列从起始甲硫氨酸延伸超过第82个氨基酸残基时，醇溶蛋白部分序列-GFP融合蛋白在PB-Is中积累。当在非还原或还原条件下提取这些融合蛋白时，PB-Is中的13a醇溶蛋白部分序列-GFP融合蛋白仅在还原条件下可溶。相比之下，13b醇溶蛋白部分序列-GFP融合蛋白在非还原和还原条件下均为可溶。这些结果表明，13a醇溶蛋白在PB-Is中的积累与13a醇溶蛋白多肽中二硫键的形成和/或疏水性有关，而13b醇溶蛋白在PB-Is中的积累较少参与二硫键的形成。
