Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Nathalie Berger,Vincent Demolombe,Sonia Hem,Amandine Crabos,Véronique Santoni,Gabriel Krouk,Valérie Rofidal,Laura Steinmann,Lionel Verdoucq,Philippe Nacry

doi:10.3390/ijms23041956

Abstract

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Highlights

Plants are exposed to different types of abiotic stress conditions such as drought or salinity that result in diminished plant growth and crop productivity [1]
Using gene ontology (GO) analysis of cell component terms, we showed that a majority of GO terms were associated with membrane proteins, even when the extrinsic proteome was exclusively considered (Figure S1)
We examined ubiquitinated peptides arising from the Ub protein itself to gain insight about poly-Ub linkages that occur in a protein sample

Summary

Introduction

Plants are exposed to different types of abiotic stress conditions such as drought or salinity that result in diminished plant growth and crop productivity [1]. Most of these conditions impose osmotic stress on plants by reducing the water potential of the environment. It is linked to either target proteins or itself through the sequential action of three enzyme classes: Ub-activating enzymes (E1s), Ub-conjugating enzymes (E2s), and Ub ligases (E3s) [4]. The activities of these enzymes result in the covalent attachment of Ub to a lysine (K) residue in the target protein. Poly-Ub chains exhibit different topologies and are associated with

Methods

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Discussion

Conclusion