title: Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules.
abstract: Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.
authors.affiliation: Section of Molecular Cell and Developmental Biology, Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Xuanhua
authors.initials: X
authors.lastname: Xie
authors.forename: Bomsoo
authors.initials: B
authors.lastname: Cho
authors.forename: Janice A
authors.initials: JA
authors.lastname: Fischer
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017353:Gene Deletion; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051880:Receptors, Notch / Q000378:metabolism*; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; D002966:Clathrin; D029721:Drosophila Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; C000606278:Ser protein, Drosophila; D000072099:Serrate-Jagged Proteins; D025801:Ubiquitin; C064045:delta protein; C114154:epsin