The Yeast Epsin Ent1 Is Recruited to Membranes through Multiple Independent Interactions

Abstract

In addition to its well known role in targeting proteins for proteasomal degradation, ubiquitin (Ub) is also involved in promoting internalization of cell surface proteins into the endocytic pathway. Moreover, putative Ub interaction motifs (UIMs) as well as Ub-associated (UBA) domains have been identified in key yeast endocytic proteins (the epsins Ent1 and Ent2, and the Eps15 homolog Ede1). In this study, we characterized the interaction of Ub with the Ede1 UBA domain and with the UIMs of Ent1. Our data suggest that the UIMs and the UBA are involved in binding these proteins to biological membranes. We also show that the Ent1 ENTH domain binds to phosphoinositides in vitro and that Ent1 NPF motifs interact with the EH domain-containing proteins Ede1 and Pan1. Our findings indicate that the ENTH domain interaction with membrane lipids cooperates with the binding of membrane-associated Ub moieties. These events may in turn favor the occurrence of other interactions, for instance EH-NPF recognition, thus stabilizing networks of low affinity binding partners at endocytic sites.Endocytosis is a multistep process in which cells selectively internalize plasma membrane proteins and lipids, as well as extracellular macromolecules such as nutrients and peptide hormones. It is an effective way to control the composition of the plasma membrane, and thus the physiological responses of the cell, so it is a tightly regulated pathway. Many cytosolic proteins are required for the early stages of clathrin-dependent endocytosis, including the adaptor protein complex AP2, the coat protein clathrin, and more recently, a class of proteins called accessory factors (1, 2). Because accessory factors interact with AP2 and are required for endocytosis, but are not enriched in purified clathrin-coated vesicles, they have been suggested to play a regulatory rather than a structural role in clathrin-coated vesicle formation (3). However, the precise functions, interactions, and order of operation of most accessory factors have not yet been determined.One accessory factor of interest to many investigators is the protein epsin, which is conserved from yeast to humans. Rat epsin 1 (Eps15 interactor 1) was first identified in a yeast two-hybrid screen as a protein that binds to another endocytic accessory factor, Eps15 (4). Epsin and eps15 are localized to endocytic sites at the plasma membrane and interact directly through the Asn-Pro-Phe (NPF) tripeptide motifs of epsin binding to the Eps15 homology (EH) 1 domains of Eps15 (4). The yeast epsins Ent1 and Ent2 were similarly identified as binding partners of the EH domain-containing endocytic protein Pan1 (5, 6). Several independent lines of evidence have indicated an important role for epsin in the internalization step of endocytosis. First, overexpressing fragments of epsin in cultured cells inhibits internalization of ligands such as transferrin and epidermal growth factor (4,7,8). Second, mutation of the yeast epsins results in growth defects and reduced internalization of plasma membrane ...

Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b

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Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

“…The function of these UIMs is to bind ubiquitinated cargo Shih et al, 2002;Aguilar et al, 2003). Ent3p and Ent5p are required for ubiquitin-dependent sorting at the MVB, but they do not possess obvious ubiquitin-binding domains (UBC-like, UIM, UBA, or CUE; Hofmann and Bucher, 1996;Hofmann and Falquet, 2001;Polo et al, 2002;Shih et al, 2002).…”

“…For binding to ubiquitin-sepharose, 50 l ubiquitin-sepharose (Sigma) was incubated with 20 g recombinant protein for 4 h, and washed in TBST as described (Aguilar et al, 2003).…”

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

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