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First published online December 1, 2003
doi: 10.1242/10.1242/jcs.00928

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ENTH/ANTH proteins and clathrin-mediated membrane budding

Valerie Legendre-Guillemin^*, Sylwia Wasiak^*, Natasha K. Hussain, Annie Angers and Peter S. McPherson

Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada

View larger version (24K): [in a new window]   Fig. 1. E/ANTH proteins and their domains. A series of ENTH- and ANTH-bearing proteins from human and yeast are depicted. Within their C-terminal region, these proteins contain short peptide motifs and protein domains that predominantly mediate protein interactions. The I/LWEQ domain, also called the talin-like domain, can bind to actin. The ubiquitin-interacting motif (UIM) is a consensus motif (EDExLxxAxxxSxxE/D) that mediates interactions with ubiquitylated proteins. The methionine-rich domain, found within the C-terminal region of enthoprotin, contains 17% methionine residues but its function is unknown. NPF motifs bind to Eps15 homology (EH) domain-containing proteins. A -ear/GAE-domain-binding motif with the consensus sequence [D/E][G/A](0-1)F[G/A][D/E], where is any hydrophobic amino acid, has been recently identified in enthoprotin and other proteins (Duncan and Payne, 2003). Clathrin-binding motifs encompass multiple sequences that bind to the terminal domain of the clathrin heavy chain including DLL motifs and type I and II clathrin boxes. For HIP1 and HIP12/HIP1R, the C-terminal most clathrin-binding motif represents a putative new binding site for clathrin light chains present within an extended -helical segment of the proteins (V.L.-G. et al., unpublished). The -ear-binding motif includes DPW, DPF and FxDxF motifs that bind to the ear of the -adaptin subunit of AP-2. Hypothetical E/ANTH proteins from S. pombe are denoted by their GenBank accession numbers.

View larger version (27K): [in a new window]   Fig. 2. E/ANTH proteins function in CCP and CCV formation coupled to cycles of PtdIns(4,5)P2 metabolism. (1) PtdIns(4,5)P2 (black) is generated from PtdIns by the sequential actions of PtdIns 4-kinase type II and PtdIns 4-phosphate 5-kinase type I. ARF6-GTP can bind to and activate PtdIns 4-phosphate 5-kinase type I on the plasma membrane. (2) E/ANTH proteins bind to PtdIns(4,5)P2 that may form in localized microdomains within the general pool of membrane phospholipids (light gray). The E/ANTH proteins in turn bind additional endocytic proteins. Other coat components including AP-2 can also bind directly to PtdIns(4,5)P2. Transmembrane receptors, through specific internalization motifs in their cytoplasmic domains, bind to the AP-2 complex. These cooperative interactions lead to the nucleation of clathrin coats on the plasma membrane. (3) Through their C-terminal regions, several E/ANTH proteins stimulate clathrin assembly, leading to the formation of CCPs. -helix 0, formed at the N-terminus of the ENTH domain in response to phospholipid binding, inserts into the plasma membrane, leading to membrane deformation and curvature. E/ANTH proteins may also function to recruit cargo molecules to nascent CCPs through binding to their cytoplasmic tails. (4) CCVs pinch off from the plasma membrane in a dynamin-dependent mechanism. (5) The lipid phosphatase synaptojanin is recruited to CCVs, where it converts PtdIns(4,5)P2 to PtdIns. This leads to destabilization of clathrin coats with Hsc70 and auxilin (not pictured) driving coat disassembly.

View larger version (31K): [in a new window]   Fig. 3. Enthoprotin interactions at the TGN. TGN clathrin adaptors AP-1 and GGA2 are targeted to the membrane through interactions with ARF1-GTP, where they bind peptide motifs present in the cytoplasmic tails of sorting receptors. AP-1 is composed of four distinct subunits: -, ß1-, µ1- and 1-adaptin. - and ß1-adaptin each contains a globular C-terminal region referred to as an ear domain. PtdIns4P binding also contributes to the membrane recruitment of AP-1. GGA2 is a monomeric adaptor containing VHS (Vps27p, Hrs, STAM), GAT (GGA and TOM) and GAE (-adaptin ear) domains. The ear domains are linked to the adaptors via flexible hinge domains. Both adaptors recruit clathrin triskelia to the TGN membrane via clathrin-binding motifs present in the hinge. Enthoprotin is targeted to the TGN through a combination of interactions including ENTH domain binding to PtdIns4P and additional interactions in the C-terminal region. Its unstructured C-terminal tail is involved in interactions with GGA2 and AP-1 through -ear/GAE-domain-binding motifs with the sequence GFADF and DFGDW. Enthoprotin also binds to the terminal domain (TD) of the clathrin heavy chain via clathrin-binding motifs LVDLF and LFDLM and can contribute to clathrin assembly. The ENTH domain could be involved in the production of membrane curvature through membrane insertion.

View larger version (19K): [in a new window]   Fig. 4. Phylogenetic analysis of ENTH domains. Amino acid sequences of ENTH-bearing proteins from human, fly, worm and yeast were retrieved from the GenBank database (Hs-Epsin1, NP_037465; Hs-Epsin2a, AAC78608; Hs-Epsin3, AAG45223; Hs-Enthoprotin, DAA00062; Ce-Liquid facets, NP_510458; Ce-Epsin, NP_509973; Dm-Liquid Facets, AAF05113; Dm-Enthoprotin, AY060606; Sc-Ent1p, NP_010120; Sc-Ent2p, NP_013307; Sc-Ent3p, NP_012659; Sc-Ent4p, NP_013062; Sp-NP_587759; Sp-NP_588237). Sequences of the ENTH domains were aligned using ClustalW. The alignment was then used for phylogenetic comparisons using the PHYLIP package [J. Felsenstein, 1993, PHYLIP (Phylogeny Inference Package) 3.6a3; University of Washington, Seattle]. Analysis was performed with a bootstrap procedure that computes the probability of occurrence of the branches for 100 possible trees. Branching order was determined using the Jones-Taylor-Thornton model included in the PHYLIP package. The tree was rooted by the most divergent sequence, Sc-Ent4p, as the outgroup. Bootstrap values are shown at each node. The tree visualization was performed using TreeView 1.6.6 (Page, 1996). Hs, Homo sapiens; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.