Ubiquitin-dependent sorting in endocytosis

Abstract

When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries.

Figure 1.

The prominent Ub-ligation complexes that work on cell-surface proteins are pictured here. Cullin-Ring ligases, such as the SCF β-TRCP complex, can modify a subset of receptors (such as the growth hormone receptor) at the cell surface. The Cbl family of ligases has a prominent role in binding to tyrosine phosphorylated receptor tyrosine kinases and ubiquitinating them to induce their ligand-stimulated down-regulation. A wide variety of cell-surface proteins are ubiquitinated by members of the Nedd4 family of ligases that possess a HECT Ub-ligase domain. These ligases can bind directly to substrate proteins or work through an intermediary adaptor protein. MARCH ligases are found in multiple domains of the secretory/endocytic system and can modify a wide variety of membrane proteins. Unfolded and damaged membrane proteins can be recognized by some of the same machinery that recognizes damaged proteins in the endoplasmic reticulum (ER). Pictured here is the CHIP ligase, which functions in a complex with Hsp70 and Hsc70 chaperones.

Figure 2.

Ub presents a hydrophobic interaction surface, pictured in green, which is responsible for binding to the vast majority of Ub-binding proteins. (A) Scheme of the types of Ub topologies that can be added to substrate proteins. Mono-Ub is a single Ub attached directly to the substrate. Multiple attached Ubs in a chain can be linked via K63 or by the amino terminus to form a linear chain. These chains have an “open” configuration in which the hydrophobic interface of each Ub moiety is highly exposed. Poly-Ub chains linked through K48 adopt a more closed conformation that makes the hydrophobic interface less accessible. Other linkages position this interaction interface in more varied orientations making it more suitable for binding particular subsets of UBD-containing proteins. (B) Structure of Ub (PDBID:1UBQ) with the hydrophobic interaction surface highlighted in green. (C) Examples of three Ub-interaction modules in a complex with Ub. VHS domain: PDBID:3LDZ; UIM:PDBID:1Q0W; GAT:PDBID:1WR6.

Figure 3.

Cohort of proteins involved in recognition and processing of ubiquitinated membrane proteins at the cell surface and at endosomes. At the cell surface, proteins such as Epsin and Eps15 recognize ubiquitinated proteins and recruit them into clathrin-coated vesicles. Other Ub-binding proteins such as CIN85 and Hrs may have a similar role in helping usher ubiquitinated cargo into vesicles. Once ubiquitinated cargo reaches the endosomes, it is recognized by the ESCRT apparatus, composed of ESCRT-0, -I, -II, and -III. The Hrs subunit of ESCRT-0 has multiple Ub-binding domains making it a critical Ub-sorting receptor, which might pass Ub cargo to other ESCRTs that also bear Ub-binding domains. Other endosomal proteins may serve as alternative ESCRT-0-like receptors that deliver ubiquitinated cargo into the larger ESCRT apparatus. These “Alt 0” proteins include GGAs, TOM1, and the Bro1-family of proteins that includes Bro1, Alix, and HD-PTP.