Endosomal transport via ubiquitination

Abstract

Cell survival, growth, differentiation and homeostasis rely on exquisite control of the abundance of particular cell-surface membrane proteins. Cell-surface proteins must respond appropriately to environmental and intracellular cues, often undergoing regulated internalization and lysosomal degradation. These proteins also can sustain damage and must be recognized and removed. A unifying mechanism has emerged for the trafficking of damaged and downregulated proteins to the lysosome by their attachment to ubiquitin (Ub), which serves as a sorting signal for clathrin-mediated internalization and sorting into late endosomes. Major questions remain as to how this system is governed, how it is adapted for different proteins, and whether Ub serves as more than a one-way ticket to the lysosome for degradation. Here, we highlight recent insights and the challenges that remain.

Figure 1. Ubiquitination and Deubiquitination for Lysosomal Sorting

Several key enzymes have been found to play major roles in controlling the ubiquitination and deubiquitination of cargo. a) Shows a schematic of Ub ligases. Nedd4 family ligases bind to substrates or substrate adaptors via their WW domains [69]. Nedd4 family ligases use the HECT catalytic domain for Ub transfer, which covalently carries Ub via a thioester bond before transfer to substrates [68]. Other Ub ligases have RING domains or Ubox domains that associate with E2 conjugating enzymes and that position the E2 conjugating enzyme which bears the thioester-linked Ub near the substrate. Many RING-containing MARCH Ub-ligases have transmembrane domains, and may use these domains as means to recognize membrane protein substrates. Other RING ligases such as Cbl and Tripartate Skp1/Cullen/Fbox complexes such as SCFβTrCP can recognize phosphorylated cell surface via a phosphotyrosine binding domain (PTB) or other modules that bind phosphorylated serine/threonine residues [92, 93]. The ligase CHIP uses a Ubox to associate with an E2 conjugating enzyme. CHIP forms a complex with the chaperone Hsp70 to recognize and ubiquitinate unfolded damaged proteins fulfilling an important role in peripheral quality control [94]. b) Mono-Ub (left) has an interaction surface (green) centered around L8, I44, R42, H68, and V70 which is involved in binding the majority of Ub-binding domains [1]. Ub also has 7 lysines (blue: 6,11,27,29,33,48,63) as well as the N-terminus that can act as conjugation sites for other Ub molecules via an isopeptide bond with the C-terminus G76 residue [2]. Structures of di-Ub linked via different lysines show dramatically different conformations and position the tandem interaction surfaces (green) within each Ub moiety in different orientations that might be differentially recognized by Ub-binding domains and/or DUbs. c) shows a schematic of where key E3 Ub-ligases and counteracting DUbs are thought act in the endocytic pathway. Either exerting their control at the cell surface to determine whether a cell surface protein undergoes clathrin mediated internalization or whether it undergoes sorting into intralumenal vesicles of MVB/endosomes.

Figure 2. Endosomal Ub-Sorting Machinery

Overview of the machinery that mediates Ub-dependent endosomal trafficking steps. Eps15 and Epsin are early-acting clathrin associated proteins that are thought to bind and concentrate ubiquitinated cargo in clathrin coated pits. Epsin and Eps15 associate with Ub using their UIM (Ub-interaction motifs). Other Ub-binding proteins that also associate with clathrin may also be present in clathrin-coated pits and could potentially act as additional cargo receptors or instead by regulated by the presence of Ub-cargo or ubiquitinated internalization machinery [1, 50]. Later, ubiquitinated cargo is recognized by the ESCRT-0 subunits Hrs and STAM as well other Ub-binding ESCRT components to usher ubiquitinated cargo into intralumenal vesicles that fill multivesicular bodies/late endosomes [31]. Additional Ub-binding domains are found within ESCRT-I and ESCRT-II, whose activities are also coupled with membrane deformation and scission with the additional ESCRT-III polymer thought to constrict the neck of intralumenal vesicles [30]. Other ESCRT-associated proteins (eg GGAs, TOM1, ALIX not pictured here) also bind Ub [31]. Collectively, these endosomal Ub-binding proteins may fulfill the function of redundant receptors for ubiquitinated-cargo or may be required for sorting of specific types of ubiquitinated cargo. Alternatively, only some of these Ub-binding ESCRT components may be directly involved in gathering ubiquitinated cargo, leaving other UBDs to fulfill other Ub-dependent modes of regulation.