Molecular mechanisms of ubiquitin-dependent membrane traffic

Abstract

Over the past 14 years, ubiquitination has emerged as a centrally important mechanism governing the subcellular trafficking of proteins. Ubiquitination, interaction with sorting factors that contain ubiquitin-binding domains, and deubiquitination govern the itineraries of cargo proteins that include yeast carboxypeptidase S, the epithelial sodium channel ENaC, and epidermal growth factor receptor. The molecular structures and mechanisms of the paradigmatic HECT and RING domain ubiquitin ligases, of JAMM- and USP-domain-deubiquitinating enzymes, and of numerous ubiquitin-binding domains involved in these pathways have been worked out in recent years and are described.

Figure 1

Subcellular itineraries of paradigmatic substrates of ubiquitin-dependent sorting pathways. (a) Yeast carboxypeptidase Cps1 is biosynthesized as a transmembrane precursor form (pCps1), which is transported from the biosynthetic pathway to endosomes. Ubiquitination in endosomes, mediated by Rsp5, provides a signal for recognition by the ESCRT machinery. Before the inclusion within the ILVs of MVBs, pCps1 is deubiquitinated by Doa4. When the MVBs fuse with the vacuole, the ILVs and their content are delivered to the vacuole lumen. Here, the ILV membranes are degraded by lipases, whereas pCps1 is processed into the mature form (mCps1) by specific proteases. (b) The Na+ channel ENaC is delivered to the apical plasma membrane of epithelial cells where it functions to channel Na⁺ from the extracellular space into the cells. Residency of ENaC in the apical plasma membrane is controlled by the E3 ubiquitin ligase Nedd4-2, which ubiquitinates ENaC and thereby signals its Epsin-dependent endocytosis from clathrin-coated pits and delivery to an apical early endosome. Deubiquitination by UCH-L3 or USP2-45 favours recycling of ENaC to the plasma membrane via the apical recycling endosome. ENaC molecules that remain ubiquitinated are recognized by the ESCRT machinery and sorted into the MVB pathway for lysosomal degradation. (c) When the epidermal growth factor (EGF) binds to its receptor (EGFR) at the plasma membrane, this induces a conformation change of the receptor that promotes dimerization. This triggers tyrosine phosphorylation, which, in addition to initiating signal cascades, promotes receptor ubiquitination by the E3 ubiquitin ligase Cbl. The activated receptor is endocytosed and delivered to an early endosome. If the ligand dissociates in the early endosome, ubiquitination is no more sustained, and the EGFR becomes deubiquitinated by the DUB AMSH. This promotes recycling to the plasma membrane. If still activated in the endosome, the EGFR remains ubiquitinated and is recognized by GGA3 and the ESCRT machinery. This causes its sorting into ILVs following deubiquitination by the DUB USP8. The receptor and its ligand are degraded when the MVB fuses with a lysosome.

Figure 2

Structure of ubiquitin and selected ubiquitin chains. (a) Structure (1UBQ) and major functional features of ubiquitin. The seven Lys residues are shown in a stick model, and the three hydrophobic residues at the center of the main hydrophobic patches are highlighted in space-filling spheres. (b) Structure of a K48-linked diubiquitin chain (1TBE). (c) Structure of a Lys63-linked tetraubiquitin chain (3HM3). In (b) and (c), the Lys involved in the linkage is shown in a stick model, and Ile44 of each ubiquitin monomer is shown in space-filling spheres. The proximal ubiquitin (the one that would be linked directly to cargo) is colored light orange, while other moieties are colored orange.

Figure 3

Rsp5 and the Nedd4 Family of ubiquitin ligases. (a) Domain architecture of Nedd4 and Rsp5. (b) Structure of the C2 domain of Nedd4 (2NSQ). Ca²⁺ ions and their ligands are shown as salmon-colored spheres and sticks, respectively. (c) Structure of the third WW domain of Nedd4 bound to the PPXY motif of EnaC (1I5H). (d) Structure of the Nedd4-2 HECT domain (3JW0; green, with catalytic Cys highlighted in space-filling spheres) in complex with an UbcH5B (magenta)~Ubiquitin (orange) adduct. The Ub-UbcH5B bond is highlighted with a space-filling sphere. Ile44 and Lys63 are highlighted to orient the viewer.

Figure 4

The Cbl family of ubiquitin ligases. (a) Domain architecture of c-Cbl. (b) Structural model for Cbl-dependent ubiquitination of the EGFR intracellular region. The structure of the TKB and RING portion of Cbl (green) in complex with UbcH7 (magenta) and a pTyr peptide (blue) from ZAP70 (1FBV) is used as a stand-in for the complex with Ube2D1-4 and the Tyr1045 region of EGFR. The catalytic domain of EGFR (3GT8) is colored blue. Lys residues of the EGFR catalytic domain are highlighted in space-filling spheres, even though not all of these Lys have been directly shown to be ubiquitinated. Structural zinc ions of the Cbl RING domain are shown as salmon-colored spheres. (c) Dimeric complex of the Cbl-b UBA domain (two shades of green) with ubiquitin (two shades of orange) (2OOB).

Figure 5

The zinc isopeptidase AMSH. (a) Domain architecture of AMSH. (b) Structural model for the catalytic complex of AMSH-LP bound to a Lys63-Ub₂-modified cargo. The structural model was derived by superimposing the structure of active, zinc-bound, Ub-free form of AMSH-LP (2ZNR) on the structure of the Lys63-Ub₂ complex (2ZVN; two shades of orange for the two moieties) with an inactivated mutant lacking the catalytic zinc ion. To illustrate the positioning of the proximal and distal moieties of the Lys63-Ub, the cargo is modeled as a single pass transmembrane protein with the ubiquitin conjugated close to the transmembrane domain. Zinc ions are shown as salmon-colored space-filling spheres.

Figure 6

The Cys isopeptideases USP8 and Doa4. (a) Domain architecture of USP8 and Doa4. (b) Dimeric structure of the N-terminal domain of USP8 (2A9U). The portions of the two monomers the correspond to the MIT sequence motif are colored red and magenta, and the remainder of the subunits are colored green and cyan, respectively. (c) Structure of the rhodanese domain of USP8 (2GWF; magenta) in complex with the non-catalytic C-terminal domain of the ubiquitin ligase NRDP1 (yellow). (d) A surface representation of the SH3 domain of STAM2 (1UJ0), colored green (hydrophobic residues), blue(basic residues), red (acidic residues), and white (uncharged polar residues). USP8 residues 699-709 are shown in a stick model colored by atom type. (e) Catalytic domain of USP8 in an inactive conformation (2GF0). The non-catalytic (structural) zinc ion is colored salmon. (f) Catalytic domain of HAUSP in covalent complex with Ub-aldehyde (1NBF), as a model for the active form of USP8. The residues of the Cys-His-Asp catalytic triad are shown in space filling spheres and colored by atom type.