Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem
Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem
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DOI：
10.1073/pnas.1703258114
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发表时间：
2017-07-11
影响因子：
11.1
通讯作者：
Hardtke, Christian S.
中科院分区：
文献类型：
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作者：
Breda, Alice S.;Hazak, Ora;Hardtke, Christian S.
蛋白质的极性细胞定位通常与其功能和活性相关。在植物中，相对较少的极性定位因子已被描述。其中，拟南芥质膜相关蛋白OCTOPUS（OPS）和BREVIS RADIX（BRX）在发育中的根原韧皮部细胞中分别显示出芽端和根端极性定位。ops和brx缺失突变体在原韧皮部分化中均表现出缺陷。在这里，我们表明，在这个过程中，OPS和BRX的作用在遗传上是平行的，虽然OPS剂量的增加，修复缺陷所造成的BRX功能丧失。OPS蛋白的功能是古老的，在大多数基础被子植物中是保守的，然而，许多高度保守的结构特征并不是OPS功能的严格要求。它们包括一个油菜甾醇不敏感2（BIN 2）相互作用结构域，据推测该结构域介导通过异位OPS过表达获得的功能获得效应。然而，在关键的磷酸化位点S318中设计越来越多的正电荷，逐渐放大OPS活性。这种过度活跃的OPS版本甚至可以补充brx ops双突变体的严重表型，并且最活跃的变体最终触发功能获得性表型。最后，BRX-OPS以及OPS-BRX融合蛋白定位于发育中的原生韧皮部细胞的根端，但互补OPS突变体与根端定位的OPS一样有效。因此，我们的研究结果表明，S318磷酸化状态，而不是一个主要的shootoplasty极性定位，是OPS活性的主要决定因素。
Polar cellular localization of proteins is often associated with their function and activity. In plants, relatively few polar-localized factors have been described. Among them, the plasma membrane-associated Arabidopsis proteins OCTOPUS (OPS) and BREVIS RADIX (BRX) display shootward and rootward polar localization, respectively, in developing root protophloem cells. Both ops and brx null mutants exhibit defects in protophloem differentiation. Here we show that OPS and BRX act genetically in parallel in this process, although OPS dosage increase mends defects caused by brx loss-of-function. OPS protein function is ancient and conserved in the most basal angiosperms; however, many highly conserved structural OPS features are not strictly required for OPS function. They include a BRASSINOSTEROID INSENSITIVE 2 (BIN2) interaction domain, which supposedly mediates gain-of-function effects obtained through ectopic OPS overexpression. However, engineering an increasingly positive charge in a critical phosphorylation site, S318, progressively amplifies OPS activity. Such hyperactive OPS versions can even complement the severe phenotype of brx ops double mutants, and the most active variants eventually trigger gain-of-function phenotypes. Finally, BRX-OPS as well as OPS-BRX fusion proteins localize to the rootward end of developing protophloem cells, but complement ops mutants as efficiently as shootward localized OPS. Thus, our results suggest that S318 phosphorylation status, rather than a predominantly shootward polar localization, is a primary determinant of OPS activity.