The post-translational modification ubiquitination (Ub) plays a central role in biology. Among the several forms of Ub, K63-type polyUb is of particular interest since involved in several proteasome-independent functions. However, the large number of factors involved in Ub processes in higher eukaryotes, the complexity of specific polyUb chain formation associated to the lack of resolution on specific Ub types by biochemical and imaging strategies has greatly hampered our ability to tackle the roles of K63 polyUb. This proposal will use an unprecedented combination of cutting edge approaches to unravel new roles of K63 polyUb, and get further insights into the cellular and biological roles of such linkage in Ub-mediated endocytosis. To this purpose, I will take advantage of the model plant Arabidopsis thaliana to grasp the complexity of K63 polyUb in higher eukaryotes. The numerous resources available including a high-quality sequenced genomes, a close-to-saturated knock-out mutant collection, an extensive ORF library covering over 50% of the genes and suitable for large-scale Ub as well as protein-protein interaction analyses associated to powerful cell biology and forward genetics indeed establish Arabidopsis as an excellent multicellular model organism to deconstruct K63 polyUb. The first part of this proposal will investigate deeper the general mechanisms of K63 polyUb-dependent endocytosis. To this purpose, we generated a generic plasma membrane ubiquitinated cargo that undergoes constitutive Ub-dependent endocytosis and targeting to the vacuole for degradation. We propose to identify factors involved in the recognition and the sorting of Ub cargos along the endocytic route by forward genetics looking for mutant plants unable to correctly address the generic Ub cargo to the vacuole. The second part of the proposal will explore new roles of K63 polyUb. We will notably use several complementary approaches using K63 polyUb-specific Ub sensor-based imaging and proteomics, as well as genomics and genetics to provide brand new information at an unprecedented scale on factors and pathways requiring K63 polyUb. This will open pristine areas of research in biology. Altogether, this project will help deconstruct the complexity of K63 polyUb using plant as model and will certainly reveal new roles of K63 polyUb in other model organisms including humans.