Sorting of ubiquitinated-protein cargo into ILVs
Ubiquitination plays major roles in trafficking plasma membrane proteins into the endosomal system (Schwihla and Korbei, 2020). Endosomal ubiquitin modification is critical for ESCRT recognition and sorting of ubiquitin-modified protein cargo into ILVs (Williams and Urbé, 2007; Villarroya-Beltri et al., 2014). This endosomal modification facilitates ubiquitinated proteins to lysosomes through MVBs (Williams and Urbé, 2007). Ubiquitination also serves as essential signal for proteins lacking signal peptides recruited to the ESCRT-dependent pathway during MVB sorting (Piper and Luzio, 2001; Agrawal et al., 2010)
Early endosomes, which originate from the trans -Golgi network (TGN) as part of the endocytic membrane transport pathway, are major sorting stations that accept molecules from the extracellular environment. On the other hand, MVBs are prelysosomal organelles acting during endocytosis to regulate incoming and outgoing traffic through homotypic and heterotypic fusion events (Luzio et al., 2003; Luzio et al., 2007; Huotari and Helenius, 2011). Although, homotypic fusion of MVBs occurs with lysosomes and autophagosomes, heterotypic fusion is different in that it can occur with the plasma membrane. But in both cases, fusion results in the delivery of endocytosed cargo. In the case where MVBs fuse with the plasma membrane, ILVs are released into the extracellular space as exosomes, and this has been implicated in cell-cell and trans -kingdom communication including plant-fungal interaction (Cai et al., 2018).
The sorting of MVB is important for a series of biological events, including endosomal sorting, organelle biogenesis, and vesicular transportation (Colombo et al., 2014; Yáñez-Mó et al., 2015; Hyka et al., 2017). Assembly of these molecular cargos takes place inside membrane microdomains of the MVBs and on the plasma membrane (Kuipers et al., 2018). Several ESCRT proteins have been implicated as constituents enriched in EVs, particularly 30-150 nm cup-shaped vesicles (i.e., Tsg101, a protein associated with ESCRT-I, and Alix, an accessory protein associated with ESRCT-III, both of which make up components of the EVs) and are frequently used as biomarkers for them (Juan and Fürthauer, 2018; Anand et al., 2019).
Prior to being sorted into ILVs, protein cargo transverse between membrane-bound protein complexes that recognize and bind to the ubiquitin chains attached to them (Paez Valencia et al., 2016). Equipped with the ubiquitin-interacting domains essential for cargo sorting, the ESCRT complexes -0, -I and -II interact stringently with ubiquitinated cargos and traffic them to the endosomal MVB pathway (Fig 3(B) ). These sub-complexes can recognize and retain endosomal ubiquitinated cargos, and transport them into MVBs (Anand et al., 2019; Mossesso et al., 2019). In addition, endocytosis modulators such as epsins (e.g., Epsin15) possess ubiquitin-interacting motifs (UIM) that function in ubiquitin recognition (Mayers and Audhya, 2012). The yeast homologs of epsin, Ent1 and Ent2, are reportedly essential for endocytosis of ubiquitin-dependent α-factor receptor that requires ESCRT-0 Vps27 for sorting (Shih et al., 2002; Raiborg et al., 2003). Finally, membrane scission of the ECRT complex is promoted by the recruitment of the ESCRT-III assembly together with its accessory proteins including Bro1 or Vps60 (Moreno-Gonzalo et al, 2014; Stoorvogel, 2015; Fig 2).