Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo inS. cerevisiae

Abstract

# Equally contributing authorsRunning Title: Lys-Ubiquitination and a Ser/Thr-phosphorylation are required for internalization of a yeast epsin-specific cargo.Endocytosis is a vital cellular process required for multiple cellular functions including nutrient uptake and control of cell membrane composition. This process involves the selective retention of cargo proteins into nascent endocytic sites by the endocytic machinery through the recognition of sequence-encoded motifs or post-translationally added tags.In addition to its classical role in proteosome-mediated protein degradation, ubiquitin (Ub) plays an important role as post-translational tag for protein sorting into different subcellular compartments. Indeed, the Ub-tag mediates membrane protein internalization and targeting to the lysosomal/vacuolar and recycling compartments (reviewed in (1, 2)).Since Ub constitutes the main trafficking signal in Saccharomyces cerevisiae (reviewed in (3)), studies in yeast have been instrumental in advancing our understanding of the mechanisms operating in Ub-mediated sorting. For example, it has been clearly established that budding yeast utilizes Rsp5, the only yeast HECT domain-containing, E3 ubiquitin ligase, to promote cargo ubiquitination relevant to endocytosis and other vesicle trafficking events (3, 4). Indeed, Rsp5-mediated ubiquitination is required for the internalization of cargoes such as the pheromone receptor Ste2 (5, 6), the uracil transporter Fur4 (7), the general amino acid permease Gap1 (8) and the zinc transporter Zrt1 (9) among others.Although Rsp5 is capable of directly interacting with PPxY motif-containing substrates via its WW domains (10-13), this consensus sequence is not present in all cargoes that undergo 14). In those cases, the PPxY motif-containing, Arrestin-Related Trafficking (ARTs) adaptor family specifically bridges cargo proteins to Following ubiquitination, plasma membrane cargoes are recognized by endocytic adaptors, such as epsin and Eps15 (or Ede1 in S. cerevisiae), which contain ubiquitinbinding elements like the Ub-interacting motifs (UIMs) or Ub-Associated (UBA) domains. As a consequence of these interactions, cargo is incorporated into nascent endocytic sites and trafficked towards degradative vacuolar/lysosomal compartments or recycling routes.Specifically, eps15 is known to be responsible for the internalization of the AMPA receptor (21) and Cx43 (22) among other ubiquitinated cargoes; while epsin it is involved in the uptake of vascular endothelial growth factor receptor 2 (VEGFR2) (23), notch ligands (24-27), and the epithelial sodium channel ENaC (28,29).