Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.
Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.
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DOI：
10.1093/jxb/erv212
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发表时间：
2015-08
影响因子：
6.9
通讯作者：
Fujita N
中科院分区：
文献类型：
--
作者：
Crofts N;Abe N;Oitome NF;Matsushima R;Hayashi M;Tetlow IJ;Emes MJ;Nakamura Y;Fujita N
水稻胚乳中的淀粉生物合成酶相互之间存在物理联系，形成具有酶活性的多种蛋白质-蛋白质复合物，其中一些是谷物所共有的，而另一些则是独特的。支链淀粉是高度支化的、有组织的葡萄糖聚合物簇，并且是大米淀粉的主要组分。支链淀粉的合成需要通过可溶性淀粉酶（SS）使葡萄糖聚合物伸长、通过支化酶（BE）产生分支以及通过脱支酶（DBE）去除错位的分支之间的精细协调。在支链淀粉生物合成中起作用的各种同工酶中，已经证明有限数量的SS和BE同工酶通过玉米和小麦淀粉质体中的蛋白质-蛋白质相互作用而相互作用。本研究调查了蛋白质-蛋白质相互作用是否也存在于水稻胚乳中，以及探索物种之间的差异。凝胶渗透色谱法的发展水稻胚乳提取物显示，所有10个淀粉生物合成酶的分析是存在于较大的分子量比各自的单体大小。SSIIa、SSIIIa、SSIVb、BEI、BEIIb和PUL在质量大小> 700 kDa处共洗脱，而SSI、SSIIa、BEIIb、ISA 1、PUL和Pho 1在200- 400 kDa处共洗脱。酶谱分析表明，SSI，SSIIIa，BEI，BEIIa，BEIIb，ISA 1，PUL，和Pho 1洗脱在高分子量馏分是活性的。全面的免疫共沉淀分析揭示了SSs-BE之间的关联，以及BE同工酶、BEIIa-Pho 1和支链淀粉酶型DBE-BEI之间的相互作用。蓝色-天然-PAGE酶谱分析证实了蛋白复合物的葡聚糖合成活性。这些结果表明，一些水稻淀粉生物合成同工酶是物理相互关联，并形成活性蛋白质复合物。对这些复合物的详细分析将有助于阐明支链淀粉独特的分支和簇结构的控制机制以及淀粉的理化性质。
Starch biosynthetic enzymes in rice endosperm are physically associated with each other and form enzymatically active multiple protein–protein complexes, several of which were common to cereals while others were unique. Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein–protein interactions in maize and wheat amyloplasts. This study investigated whether protein–protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200–400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs–BEs, and, among BE isozymes, BEIIa–Pho1, and pullulanase-type DBE–BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch.