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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer
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Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer

机译：通过磷酸化和结合14-3-3蛋白激活植物质膜H + -ATPase，将二聚体转化为六聚体

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摘要

Plant plasma membrane H+-ATPases (PMAs) can be activated by phosphorylation of their penultimate residue (a Thr) and the subsequent binding of regulatory 14-3-3 proteins. Although 14-3-3 proteins usually exist as dimers and can bind two targets, the in vivo effects of their binding on the quaternary structure of H+-ATPases have never been examined. To address this question, we used a Nicotiana tabacum cell line expressing the Nicotiana plumbaginifolia PMA2 isoform with a 6-His tag. The purified PMA2 was mainly nonphosphorylated and 14-3-3-free, and it was shown by blue native gel electrophoresis and chemical cross-linking to exist as a dimer. Fusicoccin treatment of the cells resulted in a dramatic increase in Thr phosphorylation, 14-3-3 binding, and in vivo and in vitro ATPase activity, as well as in the conversion of the dimer into a larger, possibly hexameric, complex. PMA2 phosphorylation and 14-3-3 binding were observed also when cells in stationary growth phase were metabolically activated by transfer to fresh medium. When expressed in yeast, PMA2 was also phosphorylated and formed a complex with 14-3-3 proteins without requiring fusicoccin; no complex was observed when phosphorylation was prevented by mutagenesis. Single-particle analysis by cryoelectron microscopy showed that the PMA2-14-3-3 complex is a wheel-like structure with a 6-fold symmetry, suggesting that the activated complex consists of six H+-ATPase molecules and six 14-3-3 molecules.
机译：植物质膜H + -ATPase（PMA）可以通过其倒数第二个残基（a Thr）的磷酸化和随后调控14-3-3蛋白的结合而被激活。尽管14-3-3蛋白通常以二聚体形式存在并且可以结合两个靶标，但从未研究过它们结合对H + -ATPase的四级结构的体内作用。为了解决这个问题，我们使用了一种烟草细胞系，该细胞表达带有6-His标签的Nicotiana plumbaginifolia PMA2亚型。纯化的PMA2主要是未磷酸化的和不含14-3-3-的，并且通过蓝色天然凝胶电泳和化学交联显示为二聚体。细胞的Fusicoccin处理导致Thr磷酸化，14-3-3结合以及体内和体外ATPase活性显着增加，以及二聚体转化为更大的可能的六聚体复合物。当静止生长阶段的细胞通过转移到新鲜培养基中被代谢激活时，也观察到PMA2磷酸化和14-3-3结合。当在酵母中表达时，PMA2也会被磷酸化，并与14-3-3蛋白形成复合物，而无需使用泛霉素。当通过诱变防止磷酸化时，未观察到复合物。冷冻电子显微镜的单颗粒分析表明，PMA2-14-3-3复合物是具有6倍对称性的轮状结构，表明活化的复合物由六个H + -ATPase分子和六个14-3-3组成分子。

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