Components and assembly of the ESCRT-dependent pathway in fungi
The ESCRT-0 heterodimer is comprised of two subunits, Vps27 and Hse1 (Fig 2), found in S . cerevisiae (Williams and Urbé, 2007). They are orthologous to the human protein Hrs (h epatocyte growth factor r egulated tyrosine kinase s ubstrate) and STAM (s ignal t ransducing a daptor m olecule), respectively (Raiborg and Stenmark, 2009; Henne et al., 2011; Mosesso et al., 2019; Xie et al., 2019a). The ubiquitin-interacting motifs (UIMs) of both subunits comprise of an N -terminal VHS (named after the proteins V ps27, H rs and S TAM) domain (Ren and Hurley, 2010). Although both subunits display some structural resemblance, the Vps27/Hrs structure is slightly different in that it encompasses a FYVE (named after the proteins F ab1, Y OTB,V ac1 and E EA1) zinc finger domain (Katzmann et al., 2003; Henne et al., 2011). The cysteine-rich FYVE binds to an endosomal lipid, phosphatidylinositol (Ptdlns) 3-phosphate (PI3P), that is crucial for MVB formation and endocytic trafficking and required for the Vps27/Hse1 complex functions and localization (Gillooly et al., 2001; Raiborg et al., 2003; Katzmann et al., 2003; Xie et al., 2016). InDrosophila melanogaster , inhibition of PI3P alters the recruitment of Hrs and consequently obstruct MVB formation (Lloyd et al., 2002; Raiborg et al., 2003). Moreover, defects in the Vps27 UIM do not impact vesicle budding and delivery onto the MVB lumen, but hinder sorting of ubiquitinated cargo protein into MVBs (Katzmann et al., 2001, 2002; Piper and Katzmann, 2007; Conibear, 2010).
The ESCRT-I complex is comprised of four subunits, namely Mvb12, Vps23, Vps37 and Vps28 (Fig 2). However, only Vps23 and Vps28 (Table 1) are present in F. graminearum and interact with each other (Xie et al., 2019a). Vps23 and Vps28 also interact with both ESCRT-0 and ESCRT-II at opposite ends of the complexes (Katzmann et al., 2001; Chu et al., 2006; Curtiss et al., 2007; Henne et al., 2011). In mammals, there are multiple isoforms of these ESCRT-I subunits, which may mirror their variations based on tissue-specificity (Henne et al., 2011). TheN -terminal ubiquitin E2 variant (UEV) domain of the Vps23 subunit propels to the ESCRT-I stalk, while the UEV binds to the PTAP-like motifs (P ro-T hr/Ser-A la-P ro) of Vps27, an upstream MVB sorting component, then recruits the ESCRT complex protein to the endosomal membrane (Katzmann et al., 2003; Xie et al., 2019a).
The ESCRT-II protein sub-complex is comprised of three subunits, namely Vps22, Vps36, and Vps25 (Babst et al., 2002b). Through the interaction between Vps25 and Vps20, ESCRT-II recruits downstream ESCRT-III, thus, in turn activating this protein sub-complex (Xie et al., 2019a). The interaction of ESCRT-II with ESCRT-I involves provision of the endosomal localization, the GLUE (G RAM-l ike u biquitin-binding in E ap45) domain of Vps36 and Vps28 subunits that bind to ubiquitin PI3P, while Vps25 binds Vps20 and attach to ESCRT-III, thus exhibiting the crucial role of ESCRT-II in activating the formation of ESCRT-III complex (Teo et al., 2006; Hanson et al., 2009; Henne et al., 2011).
The ESCRT-III sub-complex is comprised of four major subunits, Vps20, Snf7/Vps32, Vps24, and Vps2 (Fig 2). They occur as monomers in the cytosol and cluster rapidly on the endosomal membrane into an active complex (Babst et al., 2002a; Xie et al., 2019b). SnF7 homo-oligomerise post interaction with Vps20 and it is also the most abundant component of ESCRT-III and can self-interact as reported in previous studies (Lin et al., 2005; Teis et al., 2008; Xie et al., 2019b). The major subunits may contain a few accessory or adaptor proteins such as Did2, Bro1, and Vps60 (Table 1, Ahmed et al., 2019). ESCRT-III protein sub-complex is unique from the first three ESCRT complexes because it does not form part of the stable cytoplasmic complex but rather exist in a closed autoinhibited condition in the cytoplasm (Henne et al., 2011; Xie et al., 2019b).
According to Teo et al., (2004), ESCRT-III is triggered by the interaction between Vps25 and Vps20 from which it forms a complex, as it is recruited to the endosome. Prior to the activation of ESCRT-III, ESCRT-I and ESCRT-II complexes link through the interactions between Vps23 and Vps22, Vps23, and Vps36, and finally Vps28 and Vps36, during which the subunits Vps25 and Vps36 of ESCRT-II complex directly interact with ESRCT-III protein complex (Xie et al., 2019b). Despite the differences in the components of some ESCRT complexes, the sequential recruitment and clustering of the ESCRT complexes from interactions among ESCRT protein subunits in F. graminearum has exhibited some consistencies comparable to the yeast and mammalian models (Xie et al., 2019b). This suggests that the main interactions of this pathway are well-conserved in eukaryotes (Martin-serrano et al., 2003; Von Schwedler et al., 2003; Bowers et al., 2004; Teis et al., 2008; Xie et al., 2019b).
Lastly, ESCRT-IV disassembles the ESCRT-III complex when ESCRT-II interacts with Vps20, which recruits SnF7 (Tang et al., 2016). SnF7 has been found to play roles that are vital to the pathogenic process in fungi including cell wall integrity, endocytosis, vesicle trafficking, as well as growth and differentiation (Table 1) (Cheng et al., 2018). SnF7 then recruits Vps24 and Vps2, thus completing the assembly of ESCRT-III, shortly after Vps2 engages Vps4 (Obita et al., 2007; Teis et al., 2008; Henne at al., 2011). For stabilization, SnF7 may also recruit adaptor proteins from ESCRT-III i.e., Bro1 and Doa4 DUBs (Odorizzi et al., 2003; Luhtala and Odorizzi, 2004).
For ESCRT-III to disassemble from the membrane, energy is required and that is usually provided by class I AAA (A TPase a ssociated with various cellular a ctivities) ATPase Vps4 (Fig 1) (Babst et al., 1998). As with SnF7, other ESCRT-III accessory proteins also regulate the interaction between ESCRT-III and Vps4 (i.e., Did2, Vta1 or Vps60) to modulate the functions of Vps4 in several ways, such as facilitating the self-interaction of Vps4, Vps4 interaction with ESCRT-III subunits, or the stimulation of ATP hydrolysis (Ahmed et al., 2019). Furthermore, Vta1 directly interacts with Vps60 of the ESCRT-III complex. A super complex that is stabilized by a flexible Vta1 cap is formed when Vta1 stimulates the ATPase activity of Vps4 (Ahmed et al., 2019).