Eps 15 membrane-binding and-bending activity acts redundantly with Fcho 1 during clathrin-mediated endocytosis

Clathrin coat assembly on membranes requires cytosolic adaptors and accessory proteins, which bridge triskeleons with the lipid bilayer and stabilize lattice architecture throughout the process of vesicle formation. In Caenorhabditis elegans, the prototypical AP-2 adaptor complex, which is activated by the accessory factor Fcho1 at the plasma membrane, is dispensable during embryogenesis, enabling us to define alternative mechanisms that facilitate clathrin-mediated endocytosis. Here we uncover a synthetic genetic interaction between C. elegans Fcho1 (FCHO-1) and Eps15 (EHS-1), suggesting that they function in a parallel and potentially redundant manner. Consistent with this idea, we find that the FCHO-1 EFC/F-BAR domain and the EHS-1 EH domains exhibit highly similar membrane-binding and -bending characteristics in vitro. Furthermore, we demonstrate a critical role for EHS-1 when FCHO-1 membrane-binding and -bending activity is specifically eliminated in vivo. Taken together, our data highlight Eps15 as an important membrane-remodeling factor, which acts in a partially redundant manner with Fcho proteins during the earliest stages of clathrin-mediated endocytosis. INTRODUCTION How particular subdomains of the plasma membrane are selected for clathrin-mediated endocytosis remains elusive. Nevertheless, numerous components have been implicated in the process, including the lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and a large set of protein cofactors, several of which can bind and alter membrane topology while simultaneously recruiting clathrin and integral membrane substrates destined for internalization (Kaksonen et al., 2005; Traub, 2009; Taylor et al., 2011; Kirchhausen et al., 2014). Perhaps the most central and best-characterized clathrin adaptor is AP-2, a heterotetrameric protein complex, which is regulated by members of the muniscin protein family (Fcho1, Fcho2, and SGIP1; Jackson et al., 2010; Cocucci et al., 2012; Umasankar et al., 2014; Hollopeter et al., 2014). Although the production of SGIP1 is largely restricted to neuronal cells, Fcho proteins are more ubiquitously expressed, and orthologues have been identified in most eukaryotic species (Katoh and Katoh, 2004; Trevaskis et al., 2005; Reider et al., 2009). In metazoans, Fcho proteins harbor three distinct regions: an amino-terminal EFC/F-BAR domain, which associates with PI4,5P2-enriched lipid bilayers and induces membrane curvature; a central, unstructured linker motif, which allosterically promotes AP-2 activity; and a carboxyl-terminal mu homology domain (mu-HD), which functions as a protein interaction hub (Henne et al., 2010; Umasankar et al., 2014; Hollopeter et al., 2014). In particular, the mu-HD binds avidly to the carboxyl terminus of Eps15, another endocytic accessory factor, which was identified originally as a substrate of epidermal growth factor receptor (EGFR) signaling and subsequently shown to be an important binding partner of the AP-2 α-adaptin subunit (Fazioli et al., 1993; Monitoring Editor Sandra Lemmon University of Miami Received: Mar 8, 2016 Revised: Jun 7, 2016 Accepted: Jun 29, 2016 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E16-03-0151) on July 6, 2016. The authors declare no conflicts of interest. *Address correspondence to: Anjon Audhya (audhya@wisc.edu). L.W. , A.J., M.H. , and A.A. conceived and designed the experiments. L.W. , A.J., M.H. , and A.A. performed experiments and analyzed data. L.W. , A.J., M.H. , and A.A. contributed reagents, materials, and analysis tools. L.W. and A.A. wrote the manuscript. © 2016 Wang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: dsRNA, double-stranded RNA; EFC, extended FC; EGFR, epidermal growth factor receptor; EH, Eps15 homology; Fcho, Eps15 and intersectin, FEI; HA, hemagglutinin; MALS, multi-angle light scattering; mu-HD, mu homology domain; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; PS, phosphatidylserine; Rhod-PE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); RNAi, RNA interference; SEC, size exclusion chromatography; SUPER, supported bilayers with excess membrane reservoir; TALEN, transcription activator-like effector nuclease.