Ubiquitin detachment (de-ubiquitination) and protein cargo packaging
The process of ubiquitination can be reversed or eliminated by de-ubiquitination enzymes (DUBs). These enzymes represent a large family of proteases that are responsible for cleaving ubiquitin from proteins (Schwihla and Korbei, 2020). In fungi and mammals, deubiquitination appears to neutralize and maintain the amount of ubiquitinated membrane proteins trafficked for degradation through recycling cargo back onto the plasma membrane (McCullough et al., 2004; Schwihla and Korbei, 2020). De-ubiquitination also represents a crucial step during the formation of MVBs.
As MVBs form, prior to incorporation of the endocytic cargo into ILVs, DUBs remove their attached ubiquitin (Raiborg et al., 2009). For instance, the ESCRT-III Snf7 attached to Bro1 activates de-ubiquitination by recruiting Doa4 (d egradation o fa lpha-4 ) to the endosome before incorporation of ubiquitin modified cargos into MVBs (Amerik et al., 2000b; Katzmann et al., 2001; Odorizzi et al., 2003; Luhtala and Odorizzi 2004; Wemmer et al., 2011). However, the de-ubiquitination process does not preclude certain ubiquitinated proteins from remaining in the ILVs. It has been documented that certain EV constituents contained polyubiquitinated non-integral membrane proteins (Buschow et al., 2005). Though this phenomenon is not entirely understood, ubiquitinated proteins in EVs are believed to may have escaped de-ubiquitination and, as such, they may indicate microautophagy uptake or the cytoplasmic cargo destined to degradation in lysosomes (Buschow et al., 2005).
DUBs actively link with the ESCRT components (e.g., SnF7-Bro1) to modify and control the status and fate of ubiquitinated cargo (Wright et al., 2011; Que et al., 2019). The ESCRT-III sub-complex does not have a ubiquitin recognizing domain, yet it can actively recruit DUBs such as Ubp7 (ub iquitin-specific processing p rotease 7 ) and Doa4 that are regulated by ESCRT-III adaptor protein Bro1 in yeasts (Williams and Urbé, 2007; Roxrud et al., 2010; Que et al., 2020). InS . cerevisiae Doa4 plays a major role in ubiquitin homeostasis and ubiquitin-dependent proteolysis (Amerik et al., 2000b; Que et al., 2020). In addition, Doa4 apparently also recovers ubiquitin from ubiquitinated cargo en-route to MVBs (Que et al., 2020). Nevertheless, biological functions of most DUBs are not known in pathogenic plant pathogenic fungi (Wang et al., 2018; Que et al., 2020).