Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes
Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes
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DOI：
10.1104/pp.108.116285
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发表时间：
2008-04-01
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.;Liu, Fushan;Myers, Alan M.
影响特定淀粉生物合成酶的突变通常对同一代谢途径中的其他酶具有多效性效应。这些遗传证据表明淀粉生物合成系统的组分之间的功能关系，包括淀粉酶（SS），淀粉分支酶（BE）和淀粉脱支酶;然而，这些功能相互作用的分子解释是未知的。一种可能性是特定的SS、BE和/或淀粉脱支酶在多亚基复合物中彼此物理关联。为了检验这一假设，本研究试图鉴定来自玉米（Zea mays）造粉体的三种单独的SS多肽（SSI、SSIIa和SSIII）和三种单独的BE多肽（BEI、BEIIa和BEIIb）之间的稳定关联。检测方法包括在酵母（酿酒酵母）细胞核中的体内蛋白质-蛋白质相互作用试验，免疫沉淀，和亲和纯化使用重组蛋白作为固相配体。八个不同的情况下，检测到特定的蛋白质对关联直接或间接在同一个多亚基复合物，直接，成对的相互作用，在酵母的体内试验表明。此外，SSIIa、SSIII、BEIIa和BEIIb在凝胶渗透色谱中均以约600 kD的高分子量形式共迁移，SSIIa、BEIIa和BEIIb也以约300 kD的缺乏SSIII的第二高分子量形式迁移。凝胶渗透色谱法也检测到所有四种蛋白质的MMPs形式。600-和300-kD的复合物在高盐浓度下是稳定的，这表明疏水效应参与亚基之间的关联。
Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/ or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600- and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.