ENTH/ANTH proteins and clathrin-mediated membrane budding

Abstract

IntroductionClathrin-mediated membrane budding drives the formation of endocytic vesicles for the internalization of nutrients, receptors and other proteins at the cell surface Conner and Schmid, 2003a). It also operates at the trans-Golgi network (TGN), where it mediates the trafficking of cargo proteins from the TGN to the endosomal/lysosomal system (Brodsky et al., 2001;Hinners and Tooze, 2003). Several studies have revealed the complex molecular machinery underlying the formation and function of clathrin-coated pits (CCPs) and vesicles (CCVs) at the plasma membrane (Slepnev and De Camilli, 2000;Takei and Haucke, 2001). By contrast, the mechanisms of clathrin-mediated events at the TGN remain less clear. Recently, two new and highly related modules, the epsin N-terminal homology (ENTH) and AP180 N-terminal homology (ANTH) domains, have been identified in proteins that participate in clathrin-mediated budding. In this Commentary, we describe recent structural studies that have significantly advanced our understanding of E/ANTH domains. We also discuss how the identification of new E/ANTH proteins at the TGN, notably the ENTH-domain-containing protein enthoprotin, provides evidence that E/ANTH domains are universal elements in the clathrin-budding machinery. Identification of the ENTH domainThe heterotetrameric clathrin adaptor protein-2 (AP-2) complex plays an important role in the formation of endocytic vesicles. AP-2 contributes to the recruitment of clathrin triskelia to the plasma membrane and the assembly of triskelia into clathrin coats, leading to the formation of CCPs that eventually pinch off from the membrane in a dynamindependent manner to form CCVs (Brodsky et al., 2001; Sever, 2002). AP-2 also binds to endocytic cargo, leading to the concentration of cargo in nascent CCPs (Brodsky et al., 2001). Additionally, AP-2 recruits a diverse array of endocytic accessory proteins to sites of CCV formation (Slepnev and De Camilli, 2000;Takei and Haucke, 2001). These proteins bind through short consensus peptide motifs to a globular domain at the C-terminus of the AP-2 α-adaptin subunit termed the α-ear (Owen et al., 1999;Traub et al., 1999;Brett et al., 2002;Ritter et al., 2003).Members of the epsin family of endocytic regulatory proteins are important binding partners for the α-ear (Chen et al., 1998;Yamabhai et al., 1998;Nakashima et al., 1999;Rosenthal et al., 1999;Spradling et al., 2001) (Fig. 1). The best characterized member of this family, epsin 1, is unusual in that the C-terminal two-thirds of the molecule contains essentially no secondary structure (Kalthoff et al., 2002a). Within this extended, unstructured domain are multiple short peptide motifs that mediate interactions with endocytic proteins (Fig. 1). Included are eight copies of the DPW tripeptide that mediates binding to the α-ear (Owen et al., 1999;Traub et al., 1999) and two distinct clathrin-binding motifs, which mediate interactions with the clathrin heavy chain (Hussain et al., 1999;Rosenthal et al., 1999;Drake and Traub, 2001). Epsin ...

EpsinR2 Interacts with Clathrin, Adaptor Protein-3, AtVTI12, and Phosphatidylinositol-3-Phosphate. Implications for EpsinR2 Function in Protein Trafficking in Plant Cells

“…The highly conserved ENTH domain binds to phospholipids (Legendre-Guillemin et al, 2004). In rats, plasma membrane-targeted epsin1 binds to PtdIns(4,5)P2, whereas EpsinR, which localizes to the TGN, binds to PtdIns(4)P (Itoh et al, 2001;Hirst et al, 2003).…”

“…Epsin family proteins bind to AP complexes (Legendre-Guillemin et al, 2004). Epsin1 has multiple a-adaptin-binding motifs (Chen, et al, 1998), whereas EpsinR has multiple g-adaptin-binding motifs (Mills et al, 2003).…”

“…Epsins have been shown to bind to a number of proteins, including clathrin, AP complexes, and vesicle-associated SNARE (Rosenthal et al, 1999;Drake et al, 2000;Chidambaram et al, 2004;Song et al, 2006). Interestingly, rat epsin1, and yeast Ent1p and Ent2p, which participate in endocytosis, have binding motifs for AP-2, the hetrotetrameric adaptor complex for endocytosis (Chen et al, 1998;Legendre-Guillemin et al, 2004). Animal EpsinR/ enthoprotin/clint, yeast Ent3p, and Arabidopsis EPSIN1, which are involved in vacuolar/lysosomal trafficking, have binding motifs for AP-1, the hetrotetrameric adaptor for lysosomal/vacuolar trafficking (Duncan et al, 2003;Mills et al, 2003;Chidambaram et al, 2004;Song et al, 2006).…”

pH-dependent Binding of the Epsin ENTH Domain and the AP180 ANTH Domain to PI(4,5)P2-containing Bilayers

“…The proteins were concentrated using Millipore concentrators (Millipore) and purified by FPLC. The buffer was exchanged into 20 mM Tris or perdeuterated (d) 11 -Tris, 150 mM KCl, 1 mM d 10 -dithiothreitol, 50 μM 4-amidinophenylmethane sulfonyl fluoride and 7% 2 H 2 O. The purity of the proteins was determined by SDS-PAGE and 1 H NMR.…”

Crystal Structure at 2.8 Å of the DLLRKN-containing Coiled-coil Domain of Huntingtin-interacting Protein 1 (HIP1) Reveals a Surface Suitable for Clathrin Light Chain Binding

“…HIP1 and HIP12/1R interact with membrane phospholipids via an ANTH (AP180 Nterminal homology) domain at its N-terminus (see Fig. 1a) 3,6 . HIP1 and HIP12/1R also have a C-terminal THATCH domain, but only HIP12/1R has a C-terminal latch that activates its THATCH domain to tether CCVs to the cytoskeleton 7 .…”