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/经历了蛋白质组成的动态变化。质膜（PM）定位蛋白包括受体激酶，它们参与细胞-细胞通讯或植物-病原体相互作用，以及小分子（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和膜室之间循环，而其他PM蛋白则被内化并靶向液泡降解。因此，PM蛋白动力学决定细胞行为并影响植物性能。考虑到PM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不存在。在动物中，早期/分选核内体是受体信号传导的重要位点。虽然这可能也适用于植物，但没有标记来区分植物早期/分选和循环内体。同样，在真菌和动物细胞中，泛素作为内吞作用的分选信号，但这种功能尚未在植物中被描述。植物PM蛋白被分类降解或再循环到PM的内体室在形态和功能上都不明确，它们的常驻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个研究小组和来自西班牙的2个相关研究小组提出，旨在对拟南芥中受体介导的内吞作用和受体标记的内体区室进行基于蛋白质组学的分析。利用两种模型受体，即特性良好的人转铁蛋白受体（hTfR）和植物油菜素内酯受体复合物（BRI/SERK），我们设定了以下目标：1)建立内吞过程的时间过程；2)确定早期/分类、再循环和晚期内体区室的结构；3)基于受体动力学和免疫分离纯化内体室；4)基于纯化的蛋白质组鉴定可获得的内体蛋白质组，鉴定早期/分选和回收内体的分子标记；5)分离和鉴定内吞作用的蛋白质复合物；6)确定泛素化在内吞过程中的作用；7)确定在配体-受体系统中鉴定的蛋白质和标记物的作用。我们预计我们的结果将定义参与受体内化，信号传导，分类和降解或回收的内吞途径和内体室室。这将为进一步分析植物发育过程中受体活性和病原菌反应提供基础。

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.