Ubiquitin and its relatives as wizards of the endolysosomal system

Abstract

The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.

Keywords: Bacterial infection; Endosomes; Membrane contact sites; Membrane dynamics; Ubiquitin.

Fig. 1.

Ubiquitylation drives lysosomal turnover of the cell surface proteome. (A) Ubiquitin (Ub) modifications direct cell surface proteins for degradation, as exemplified by the epidermal growth factor receptor (EGFR) whose ligand-induced activation sets in motion its journey to lysosomes. Cargoes endocytosed in early endosomes (EEs) are partitioned between the degradative and recycling membrane pathways at the sorting endosome (SE). Ligand binding triggers EGFR phosphorylation and recruitment of the E3 ubiquitin ligase Cbl for receptor ubiquitylation. Whereas receptors lacking Ub marks traffic back to the plasma membrane (PM) via recycling endosomes (REs), ubiquitylated receptors are recognized by endosomal adaptors and incorporated into intralumenal vesicles (ILVs) of multivesicular bodies (MVBs). Subsequent fusion of MVBs with proteolytic lysosomes (LYS) commits ILV cargoes for degradation. The latter fate can, however, be averted by timely action of DUBs at various steps along the endosomal trafficking route. Besides cargo degradation, other ILV pathways exist, including return to the limiting membrane (LM) via retrofusion and release as extracellular vesicles (EVs). The autophagy pathway also feeds into the endolysosomal system via double-membrane autophagosomes (AUT) that form upon sealing of the isolation membrane (IM) around ubiquitylated cytosolic cargoes. Processes labeled B–D are shown in more detail in the other panels. (B) Ub-dependent functions of endosomal sorting adaptors are regulated by reversible ubiquitylation. Monoubiquitylation of EPS15 inhibits recognition of ubiquitylated cargoes, and deubiquitylation by USP9x restores this function. ESCRT-0 function is also positively regulated by its associated DUBs USP8 and AMSH. (C) Sequestration of ubiquitylated cargoes on ILVs is orchestrated by sequential actions of the ESCRT-0, -I, -II, and -III complexes. ESCRT-associated deubiquitylation during ILV formation ensures Ub homeostasis on endosomal membranes and maintains the cellular Ub pool. (D) SUMOylation of the RNA-binding protein hnRNPA facilitates incorporation of the long noncoding RNA ELNAT1 into ILVs for release in EVs.

Fig. 2.

Ubiquitylation regulates retromer-mediated recycling. (A) Membrane recycling from maturing endosomes towards the plasma membrane (PM) and the trans-Golgi network (TGN) is carried out by the retromer complex comprising the cargo-selective subunit VPS35 and its partners VPS29 and VPS26. The retromer associates with the WASH complex for actin filament assembly and membrane-deforming sorting nexins (SNX) proteins. Processes labeled B–D are shown in more detail in the other panels. (B) Retromer assembly and stability are regulated by ubiquitylation. The E3 ligase Parkin ubiquitylates VPS35 and stabilizes other retromer components, however, whether ubiquitylation of VPS35 promotes retromer assembly is unclear. Association of the DUB OTULIN with SNX27 antagonizes its binding to VPS26 and thus inhibits retromer-mediated recycling to the PM. (C) Ubiquitylation of the WASH complex, mediated by the retromer-associated E3 ligase MAGE-L2 in conjunction with its substrate adaptor TRIM27 and conjugating E2 enzyme UBE2O, activates localized actin polymerization and promotes recycling from endosomes to the PM. (D) Polyubiquitylation of the SNARE VAMP3 by the E3 ligase RNF167 enhances proteasomal destruction of VAMP3 and inhibits fusion of TGN-derived recycling compartments with the PM.

Fig. 3.

Ub and Ubls in endosomal membrane identity transitions. (A) Regulated switches between small GTPases determine endosome identity, transport and fusion through selective effector recruitment. During maturation, late endosome (LE)-associated Rab7 displaces early endosome (EE)-associated Rab5. Rab7 engages the effector RILP and the dynein motor to direct transport of late endosomes (LEs) to the perinuclear region. Here, HOPS-mediated tethering and fusion with lysosomes carrying Rab7 and/or Arl8b in complex with the effector PLEKHM1 takes place. On the other hand, displacement of Rab7 by Arl8b in complex with effector SKIP allows for kinesin motor-dependent transport to the cell periphery. Polyubiquitylation of Arl8b by E3 ligase RNF167 leads to its degradation by the proteasome. Other small GTPases including Rab8a are also turned over by the proteasome in a manner dependent on the cytosolic chaperone BAG6. Processes labeled B–D are shown in more detail in the other panels. (B) Ub and Ubl conjugation regulates Rab-mediated recycling. Endocytosed β2-adregergic receptor recruits the E3 ligase HACE1 to ubiquitylate and activate Rab11 for recycling to the plasma membrane (PM). Also, SUMOylation of Rab17 promotes SNARE syntaxin 2-mediated recycling to the apical surface. (C) Key consequences of GTPase ubiquitylation include altered GTP hydrolysis rate and effector interactions. This is illustrated by Rab5 whose monoubiquitylation on different lysine residues affects distinct aspects of its functional cycle. (D) Left, reversible ubiquitylation of Rab7 by the E3 ligase Parkin and DUB USP32 regulates effector preference. Monoubiquitylation of Rab7 disfavors interaction with RILP and promotes association with the retromer complex. Right, loss of USP32 inhibits recycling tubule resolution and blocks endosomes-to-TGN traffic. Acceptor lysine residues are indicated within some shapes.

Fig. 4.

Reversible ubiquitylation at membrane contact sites. (A) ER membrane contact sites (MCSs) regulate endocytic traffic by directing endosome transport, specifying location and timing of endosome fission, and positioning endosomes (and lysosomes) in the perinuclear space. ER MCSs are also involved in autophagosome biogenesis. Processes labeled B and C are shown in more detail in the other panels. (B) Ub-dependent reversible ER-endosome MCS formation. The membrane-embedded UBE2J1–RNF26 E2–E3 pair ubiquitylates SQSTM1 on K435 to position endosomes at the perinuclear ER. Deubiquitylation by USP15 in turn dissolves this MCS, releasing endosomes for fast transport. (C) Regulation of early steps in autophagosome biogenesis by the ER. The ULK1 complex localizes at omegasome regions of the ER membrane, where it stimulates formation of the isolation membrane (IM). Timing of IM elongation is controlled by the E3 ligase RNF31 of the LUBAC complex, which mediates linear polyubiquitylation of ATG13. Once the autophagosomal double-membrane is sealed, deubiquitylation of ATG13 by OTULIN promotes autophagosome maturation.

Fig. 5.

Hijacking of Ub and Ubls by intracellular bacteria to reprogram the endolysosomal system of the host. (A) Intracellular bacteria exploit reversible Ub and Ubl conjugation to reprogram the host endolysosomal system in ways benefitting their infection cycle. Both Legionella and Salmonella establish vacuolar compartments that mature into replication permissive niches while avoiding lysosomal degradation. IAM, infection associated macropinosome; EE, early endosome; LE, late endosome; LYS, lysosome. Processes labeled B and C are shown in more detail in the other panels. (B) Following bacterial entry, Legionella exploits the host ubiquitylation machinery, as well as secreting its own unique repertoire of enzymes that enable the Legionella-containing vacuole (LCV) membrane to acquire ER-like characteristics and evade the endolysosomal system. This strategy includes ubiquitylation of GTPases Rab1 and Rab10, temporal regulation of membrane expansion by reversible ubiquitylation of the SNARE Sec22b, and promotion of LCV maturation through noncanonical modifications of host factors with ADP-ribosylated Ub. (C) Ubiquitylation of the Salmonella effector SopB directs it to the Salmonella-containing vacuole (SCV), where it regulates membrane identity dynamics at the SCV. Conversely, the non-ubiquitylated form of SopB localizes predominantly to the PM and instigates activation of Akt signaling. Salmonella infection also induces a global SUMOylation blockade in the host cell through downregulation of the SUMO-conjugating enzyme UBC9. This stabilizes the key endolysosomal Rab7 that promotes maturation of the SCV. Acceptor lysine residues are indicated within some shapes.