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First published online February 7, 2007
doi: 10.1242/10.1242/jcs.03385

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Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs

¹ Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261 USA
² Program in Cell and Lung Biology, Hospital for Sick Children Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada

View larger version (49K): [in this window] [in a new window]   Fig. 1. Ubiquitin, isopeptide linkage and chain conformation variations. (A) Ribbon diagram of the main chain of the ubiquitin molecule (Protein Data Bank ID: 1UBQ) with the seven lysine side chains and Ile44 shown in stick representation. Ubiquitin is composed of a single antiparallel -sheet curved over an adjacent -helix. Solvent-exposed Ile44 demarcates the chief contact surface of the ubiquitin molecule (Hicke et al., 2005; Hurley et al., 2006), and the majority of the ubiquitin lysine residues are located in a circumferential band relative to this key residue. Lys63, however, is positioned opposite the extended C terminus and Gly76 necessary for isopeptide linkage to a target lysine side chain. (B) Schematic representation of monoubiquitylation, Lys48-linked or Lys63-linked polyubiquitylation of target lysine residues. Note that the differential positioning of the lysine within the acceptor ubiquitin dictates different polyubiquitin chain conformations and geometries. We use the term oligoubiquitylation to indicate the presence of either multiple monoubiquitins, a polyubiquitin chain(s), or a combination of these modifications. (C) Examples of different ubiquitin configurations in plasma membrane proteins internalized in mammalian cells. (a) multiple monoubiquitylation (dimeric EGF receptors, for example), (b) polyubiquitylation with Lys63 linkage (for example, dimeric NGF receptor TrkA), (c) monoubiquitylation of subunits of homooligomeric complexes (for example, ROMK1), (d) polyubiquitylation of heterooligomeric complexes (for example, ENaC), (e) oligoubiquitylation in trans (for example, -arrestin 1/2 in complex with 2 adrenergic receptor or insulin-like growth factor-1). Given the available evidence, we believe monoubiquitylation to be an inefficient endocytic signal in higher eukaryotes and therefore do not include it.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Ubiquitin-binding CLASPs. Schematic illustration of the domain organization of eps15/15R and epsin 1 from human (Hs), S. cerevisiae (Sc) and Drosophila (Dm). The ubiquitin-interacting motif (UIM), ubiquitin associated (UBA) domain, AP-2 binding Asp-Pro-Trp/Phe (DP[WF]) sequences, clathrin-binding (CB) sequences and EH-domain-binding Asn-Pro-Phe (NPF) motifs are indicated. The epsin N-terminal homology (ENTH) binds to PtdIns(4,5)P2.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Putative modulatory mechanisms of ubiquitylated cargo recognition by endocytic adaptors harboring UIM domains. (A) Ubiquitylation of both cargo and relevant UIM-containing endocytic adaptors (e.g. eps15/eps15R and epsin) may influence the internalization efficiency. (B) Adaptor autoactivation and formation of a signal relay network by adaptor ubiquitylation. Ubiquitin allosterically activates the UIMs. In addition, increased local concentration may facilitate the assembly of a polyvalent adaptor network at the cell surface through intermolecular ubiquitin-UIM interactions, manifesting in higher avidity for oligoubiquitylated cargo. (C) Adaptor autoinhibition by ubiquitylation. Monoubiquitylation-induced intramolecular interaction between ubiquitin and UIM attenuates adaptor affinity for ubiquitylated cargo. (D) Substrate-induced stabilization of heterooligomeric ubiquitin-binding adaptors. Peptide interactions may facilitate the association of ubiquitin-adaptors and synchronous presentation of multiple UIMs, increasing the avidity towards oligoubiquitylated substrate.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Endocytic regulation of asymmetric cell division and cell fate determination in Drosophila. (A) Lineage of the four cell types, which together form the functional Drosophila sensory bristle, all derived from a single pI SOP precursor. One daughter from pIIb mitotic division undergoes apoptosis. (B) Notch receptor signal transduction in adjacent pII cells. The transmembrane Notch ligand, Delta, is presented at the surface of the signal-sending pIIb cell. During trafficking along the biosynthetic pathway, the extracellular domain of Notch is cleaved at the S1 site by furin. At the pIIa cell surface, ligand (Delta) engagement triggers the S2 site proteolytic processing event by an ADAM metalloprotease and coupled or subsequent endocytosis of the extracellular portion of Notch still bound to Delta. Uptake of the Delta-Notch complex depends upon Delta ubiquitylation by the E3 ligase Neuralized and packaging into clathrin-coated vesicles assembling at the surface of the pIIb by the CLASP Liquid facets (epsin). In the signal-receiving pIIa, the S2 cleavage promotes S3 site cleavage by -secretase to release the Notch intracytoplasmic domain (NICD), which then translocates to the nucleus and acts as a transcriptional co-activator. (C) Notch deactivation in the signal-sending pIIb. At metaphase, certain proteins, including Neuralized and Numb (PTB domain CLASP related to Dab2), localize to one pole of the cell. Following cytokinesis, only one sister, the pIIb, inherits these endocytic components, whereas both express Notch, Delta and Sanpodo. Numb promotes the clathrin-mediated internalization of Sanpodo, interfering with signal transduction of Notch on the pIIb cell surface, whereas Neuralized and Liquid facets drive clathrin-dependent endocytosis of the Delta-Notch binary complex. The overall effect of this endocytic activity is differential signal transduction in the pIIa and pIIb, leading to different cell fates and morphological characteristics.