Proteomics analysis of endosomal compartments in Arabidopsis

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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.