Proteomics analysis of endosomal compartments in Arabidopsis

植物细胞与其邻居及其生物和非生物环境的界面/质膜/经历蛋白质组成的动态变化。质膜定位蛋白包括参与细胞-细胞通讯或植物-病原菌相互作用的受体蛋白和小分子(如糖、激素、离子)转运蛋白或促进剂。一些PM蛋白已被证明在PM和内膜室之间循环(S)，而其他PM蛋白被内化并被定位到液泡进行降解。因此，PM蛋白的动态决定了细胞的行为，并影响植物的表现。考虑到质膜蛋白动力学的基本重要性，我们目前对植物潜在机制的了解几乎是不存在的。在动物中，早期/分选内含体是受体信号传递的重要部位。虽然这可能也适用于植物，但没有标记来区分植物的早期/分选和回收内体。同样，泛素在真菌和动物细胞中作为内吞作用的分类信号，但这种功能在植物中尚未被描述。植物PM蛋白被分选为降解或回收到PM的内体隔间在形态和功能上定义不清，它们的居留蛋白和货运蛋白的组成基本上是未知的。该项目由来自3个资助国的4个小组和西班牙的2个相关小组提出，旨在对拟南芥中受体介导的内吞作用和受体标记的内体隔间进行基于蛋白质组学的分析。利用两种模型受体，即特征良好的人转铁蛋白受体(HTfR)和植物油菜素内酯受体复合体(BRI/SERK)，我们设定了以下目标：1)建立内吞过程的时间进程；2)识别早期/分选、再循环和晚期内体隔室的结构；3)基于受体动力学和免疫分离来纯化内体隔室；4)根据纯化的组分鉴定可获得的内体蛋白体，并为早期/分选和回收内体寻找分子标记；5)分离和鉴定内吞吞过程中的蛋白质复合体；6)确定泛素化在内吞过程中的作用；7)确定在配体-受体系统中识别的蛋白质和标志物的作用。我们预计，我们的结果将定义内吞途径和内体隔间，涉及受体内化、信号传递、分类和降解或再循环。这将为进一步分析受体在植物发育和病原菌反应中的活性奠定基础。

The interface of plant cells with their neighbours and their biotic and abiotic environments / the plasma membrane / undergoes dynamic changes in protein composition. Plasma membrane (PM)-localised proteins include receptor kinases, that are involved in cell-cell communication or plant-pathogen interaction and small-molecule (e.g. sugar, hormone, ion) transporters or facilitators. Several PM proteins have been shown to cycle between the PM and endomembrane compartment(s), whereas other PM proteins are internalised and targeted to the vacuole for degradation. PM protein dynamics thus determines cell behaviour and affects plant performance. Considering the fundamental importance of PM protein dynamics, our current knowledge of the underlying mechanisms in plants is virtually non-existent. In animals, early/sorting endosomes are important sites for receptor signaling. Although this may also apply to plants, there are no markers to distinguish plant early/sorting from recycling endosomes. Likewise, ubiquitin serves as a sorting signal for endocytosis in fungal and animal cells, but such a function has not yet been described in plants. The endosomal compartments in which plant PM proteins are sorted for degradation or recycling to the PM are morphologically and functionally ill-defined, and their composition in terms of resident and cargo proteins is essentially unknown. This project is proposed by 4 groups from 3 financially contributing countries and 2 associated groups from Spain, and aims to carry out a proteomics-based analysis of receptor-mediated endocytosis and receptor-labeled endosomal compartments in Arabidopsis. Using two model receptors, the well-characterised human transferrin receptor (hTfR) and the plant brassinolide receptor complex (BRI/SERK), we set the following objectives: 1) to establish a time course of the endocytic process; 2) to identify the architecture of early/sorting, recycling and late endosomal compartments; 3) to purify endosomal compartments based on receptor kinetics and immuno-isolation; 4) to identify the accessible endosomal proteomes on the basis of the purified fractions and to identify molecular markers for early/sorting and recycling endosomes; 5) to isolate and identify complexes of proteins undergoing endocytosis; 6) to determine the role of ubiquitination in the endocytic process; 7) to determine the role of the proteins and markers identified in the ligand-receptor systems. We anticipate that our results will define endocytic pathways and endosomal compartments involved in receptor internalisation, signaling, sorting and degradation or recycling. This will provide a foundation for future analyses of receptor activity in plant development and pathogen response.