Proteomics analysis of endosomal compartments in Arabidopsis

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.

Endocytosis is a vital process common to all eukaryotes that retrieves membrane material and associated cargo from the plasma membrane (PM) for internal utilisation or destruction, or for processing and recycling back to the PM. Thus, endocytosis serves multiple purposes, including cell-cell communication and nutrient uptake. In animal cells, ligand-induced receptor endocytosis modulates sensitivity to ambient ligand concentration. Endocytosed receptors are sorted in early/sorting endosomes for either recycling via the Endocytic Recycling Compartment or further internalisation and eventual degradation in lysosomes. Endocytosis of some receptors such as EGF and PDGF leads to activation of signalling pathways. Plants are unable to escape from adverse environmental conditions and show a remarkable cellular plasticity in response to external cues, which are likely to be mediated by rapid internalisation and recycling of PM proteins. Thus, the process of endocytosis may have undergone evolutionary adaptive changes in the plant lineage. Comparative genomics has revealed that although the Arabidopsis genome encodes homologs of animal or yeast components of the endocytic machinery, the plant family members are often modified such that clear one-by-one orthologues are difficult to identify. In plants, unequivocal proof for receptor internalisation and recycling via clathrin-coated pits at the PM has yet to be demonstrated. There is recent evidence for the internalisation and recycling or degradation of specific PM-localised proteins, such as the boron transporter. Ligand-dependent receptor-mediated endocytosis was recently demonstrated in plant cells for the flagellin receptor, but with no evidence for an interaction between this receptor and the endocytic machinery. Our aim in this project is to define the endocytic pathways and endosomal compartments involved in receptor internalisation, signaling, sorting and degradation or recycling using complementary approaches.