Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts
Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts
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DOI：
10.1104/pp.109.135293
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发表时间：
2009-03-01
期刊：
影响因子：
7.4
通讯作者：
Myers, Alan M.
中科院分区：
文献类型：
--
作者：
Hennen-Bierwagen, Tracie A.;Lin, Qiaohui;Myers, Alan M.
来自玉米（Zea mays）和小麦（Triticum aestivum）造粉体的淀粉生物合成酶以高分子量复合物的形式存在于细胞提取物中;然而，这些组装体的性质仍有待确定。本研究测试了玉米淀粉合成酶IIa（SSIIa），SSIII，淀粉分支酶IIb（SBEIIb）和SBEIIa组装成多亚基复合物的相互依赖性。消除其中任何一种蛋白质的突变也会阻止其他蛋白质组装成约670 kD的高分子量形式，因此SSIII、SSIIa、SBEIIa和SBEIIb很可能都存在于同一复合物中。SSIIa，SBEIIb，SBEIIa，但不是SSIII，也是相互依赖的组装成一个复杂的约300 kD。SSIII、SSIIa、SBEIIa和SBEIIb通过连续层析步骤共纯化，SBEIIa、SBEIIb和SSIIa以磷酸化依赖性方式与SSIII共免疫沉淀。SBEIIa和SBEIIb也保留在亲和柱上，该亲和柱具有位于SS催化结构域外部的SSIII的特异性保守片段。在多种生物化学方法中与SSIII共纯化的其他蛋白质包括丙酮酸正磷酸二激酶（PPDK）的两种已知亚型、ADP-葡萄糖焦磷酸化酶的大小亚基和蔗糖合成酶亚型SUS-SH 1。PPDK和SUS-SH 1需要SSIII、SSIIa、SBEIIa和SBEIIb组装成670-kD复合物。这些复合物可能在发育种子的代谢途径之间的碳分配的全局调节中起作用。
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.