Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding
Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding
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DOI：
10.1074/jbc.275.16.11829
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发表时间：
2000-04-21
影响因子：
4.8
通讯作者：
Viitanen, PV
中科院分区：
文献类型：
--
作者：
Dickson, R;Weiss, C;Viitanen, PV
与真细菌和线粒体的GroEL同源物不同，高等植物叶绿体伴侣蛋白60 （cpn60）的低聚物制剂由两个不同的亚基- α和β -组成，数量大致相等。这些同工异构体的功能意义、它们组成四分子体的结构组织以及它们与独特的二元叶绿体伴侣蛋白10 （cpn10）的相互作用尚未阐明。为此，我们克隆了豌豆（Pisum sativum） ch-cpn60的α亚基和β亚基，分别在大肠杆菌中表达，并对纯化的单体进行体外重组实验。在没有其他因素的情况下，亚基（单独或结合）都不会自发地组装成更高阶的结构。然而，在MgATP存在的情况下，β亚基在cpn10增强的协同反应中形成四聚体。相比之下，一个亚基只有在β亚基存在的情况下才能组装。尽管β和α / β 14-mers在电子显微镜下无法区分，并且都可以帮助蛋白质折叠，但它们对cpn10的特异性完全不同。与真正的叶绿体蛋白类似，重组的α / β 14-mers在功能上与细菌、线粒体和叶绿体cpn10兼容，相反，重组β 14-mers介导的折叠反应仅与线粒体cpn10有效。体外重建两种功能性低聚物的能力提供了一个独特的工具，这将使我们能够研究这种不寻常的伴侣蛋白系统的机制。
Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, alpha and beta. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the beta subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, a subunits only assemble in the presence of beta subunits, Although beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted alpha/beta 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10, In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.