Quantification of cotyledon and flower organs.

The Arabidopsis ENHANCER OF PINOID (ENP) protein and the AGC-kinase PINOID (PID) synergistically impact on polarization and function of the auxin transporter PIN-FORMED1 (PIN1) required for plant leaf and flower organ development. ENP offers a PID-independent input for PIN-function since enp pid double mutants lead to cotyledon- and flower-less plants in contrast to pid single mutants, which develop cotyledons and abnormal albeit fertile flowers. This indicates that ENP, which depicts a similar polar localization as PIN1, is a potential interactor of PINs including PIN1. Here we show that the modular structure of ENP predicted by AlphaFold separates the capability for its own cellular polarization and its function linked to polar PIN1 activity. The part of ENP from aa1 to aa470 is subdivided into three structured domains. They are supportive and/or essential for cellular polarity. In contrast, the C-terminus, which is an intrinsically disordered region (IDR), is completely dispensable for polarity but essential for ENP-mediated PIN1-function. FLIM-FRET shows ENP to be closely associated with the plasma membrane and its IDR to significantly interact with PINs. Moreover, the modification status of two prominent phosphorylation sites in the IDR determines ENPs stability and its capability in supporting PIN1. Our results show ENP to be an element in the assumed PIN-multiprotein complex and explain its impact on PID-independent PIN1 activity.本研究聚焦拟南芥PINOID增强子（ENHANCER OF PINOID, ENP）蛋白与AGC激酶PINOID（PID），二者可协同调控生长素转运蛋白PIN-FORMED1（PIN1）的极性定位与功能，而该过程是植物叶片及花器官正常发育的必要前提。ENP能够为PIN蛋白功能提供不依赖PID的调控通路：与仅产生具正常子叶但花器官异常且可育的单突变体pid相比，enp pid双突变体无法形成子叶与花器官。上述结果提示，与PIN1具有相似极性定位特征的ENP，是包括PIN1在内的PIN家族蛋白的潜在互作因子。本研究证实，AlphaFold预测的ENP模块化结构可将其自身细胞极性形成能力与PIN1极性活性相关的功能相互分离。ENP的氨基酸1至470区段可划分为三个结构化结构域，该区域对细胞极性的建立具有支持或必需作用；与之相反，作为内在无序区域（intrinsically disordered region, IDR）的C端区域虽对ENP的细胞极性形成完全非必需，但却是ENP介导PIN1功能所必不可少的组分。荧光寿命成像-荧光共振能量转移（FLIM-FRET）实验显示，ENP与质膜紧密结合，且其IDR区域可与PIN家族蛋白发生显著相互作用。此外，IDR区域内两个关键磷酸化位点的修饰状态，决定了ENP的稳定性及其辅助PIN1发挥功能的能力。本研究结果表明，ENP是推测的PIN多蛋白复合物的组成元件之一，并阐明了其对不依赖PID的PIN1活性的调控机制。