Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON, Canada, N1G 2W1.
Biochem J. 2012 Dec 15;448(3):373-87. doi: 10.1042/BJ20120573.
The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.
玉米（Zea mays L.）中的 sugary-2 突变是由于胚乳特异性 SS（淀粉合酶）IIa 同工型的催化活性丧失导致支链淀粉结构发生重大改变的结果。本研究报告了 sugary-2 突变的等位变体的生化和分子分析，该变体表达了一种无催化活性的 SSIIa 形式，并为其在蛋白质-蛋白质相互作用和淀粉颗粒蛋白质组确定中的核心作用提供了新的认识。突变 SSIIa 显示出两个氨基酸取代，其中一个是高度保守的残基（Gly522→Arg），负责丧失催化活性和突变 SSIIa 无法结合淀粉。在 sugary-2 叶绿体中分析蛋白质-蛋白质相互作用时，发现可溶性 SSI、SSIIa 和 SBE（淀粉分支酶）IIb 的三聚体组装与野生型叶绿体相同，但 SSI 和 SBEIIb 的活性大大降低。化学交联研究表明，SSIIa 是该复合物的核心，与 SSI 和 SBEIIb 相互作用，而 SSI 和 SBEIIb 彼此之间没有直接相互作用。尽管 sugary-2 突变淀粉颗粒缺乏支链淀粉合成酶，但 sugary-2 的 SSI 和 SBEIIb 对支链淀粉的亲和力与野生型观察到的相同。数据支持这样一种模型，即参与支链淀粉合成的颗粒结合蛋白由于其在蛋白质复合物中的关联而被分配到淀粉颗粒中，并且 SSIIa 在将 SSI 和 SBEIIb 运输到颗粒基质中起着至关重要的作用。
