Servicios Web que soporta la Plataforma:

The machineries of mammalian ERAD are more elaborate than those of yeast (Table 1.3). This reflects the broader array of clients and more complex layers of regulation that both multi-cellularity and developmental pathways demand. Indeed, the key apparatuses that Saccharomyces cerevisiae possesses are all represented and some are expanded. However, in contrast to Saccharomyces cerevisiae, the mammalian cohort of ERAD-implicated E3 ligases is supplemented by a soluble, cytosolic set (reviewed in Nakatsukasa & Brodsky, 2008; Ballinger et al., 1999). The degradation of both the cystic fibrosis transmembrane conductance regulator (CFTR) and apolipoprotein B (ApoB) epitomises the participation of

Page 24

these soluble E3 ligases in ERAD. CFTR is a large, polytopic plasma membrane protein that is avidly degraded by ERAD (Alberti et al., 2004). This degradation occurs to a disease- causing extent in people who are homozygous for the mutant CFTR∆F508. In this version of the protein, the conformation of a cytosolic ATP-binding cassette is disrupted (Alberti et al., 2004; Hirsch et al., 2009). Hsc70 detects the prolonged ER-residency this fault causes (Albert et al. 2004), much as its yeast ortholog, Ssa1, may recognise clients at the outset of the Doa10 pathway (Metzger et al., 2008). Prolonged association of Hsc70 with CFTR∆F508 leads to an increased chance of polyubiquitination. This polyubiquitination is promoted by the soluble, Hsc70-dependent, Hsc70-binding E3 ligase called CHIP (Alberti et al., 2004). CHIP ubiquitinates both client and chaperone, leading to client degradation by localisation of the client-bound complex to the proteasome (Minami et al. 1996; Alberti et al., 2003). This pathway is quite distinctive, in that it delegates the entirety of the recognition and polyubiquitination processes to soluble, cytosolic factors.

This functional contribution of CHIP to the ERAD of CFTR∆F508 highlights the potential significance of another two soluble E3 ligases that have been implicated in ERAD, yet have no yeast correlates. One is FBX2, which recognises and ubiquitinates glycoproteins (Yoshida et al., 2002). The other is Parkin, which is analogous to CHIP in that it selects its substrates in an Hsc70-dependent manner (Pratt et al., 2010). Their exact preference for substrates and prevalence of their involvement in ERAD, however, is not well understood.

Interestingly, a core E3 ligase of yeast, Hrd1, has two homologues in mammals. One is very similar to Hrd1, whilst the other (gp78) has acquired an ubiquitin-binding domain much like yeast‟s Cue1. Morito et al. (2008) showed that this motif gates access of substrates to gp78‟s RING-finger domain, which cannot start ubiquitin chains de novo. They speculate that other E3 ligases work upstream of gp78. Indeed, they show that gp78 co-operates in this manner with another mammal-specific transmembrane E3 ligase, RMA1, in the degradation of CFTR∆F508 (Morito et al., 2008).

The participation of these three complexes – gp78, RMA1 and Hsc70/CHIP – in the degradation of CFTR∆F508 highlights a good example of redundancy between E3 ligases (Morito et al., 2008). Adding to this picture, if CFTR∆F508 is exogenously expressed in yeast, the Doa10 complex facilitates its degradation (Younger et al., 2004). The homolog of Doa10 in mammals, TEB4, has only recently been identified (Kreft et al., 2006; Nakatsukasa and

Page 25

Brodsky, 2008). It therefore remains unclear whether it also handles CFTR∆F508 in the mammalian context.

Table 1.3 - Select factors implicated in both Yeast and Mammalian ERAD

This table is much as printed in Nakatsukasa & Brodsky (2008). Derlin-1 can be immunoprecipitated with those proteins which are underlined (with additions made as noted by Hebert et al. 2010). Additional homologs have also been added, as per Raasi & Wolf (2007).

Mammals Yeast (Saccharomyces cerevisiae)

Cytosol- and membrane-associated

P97-UFD1-NPL4 _{Cdc48-Ufd1-Npl4}

(No obvious correlate) (No obvious correlate) (No obvious correlate) Ufd2 Rad23 Png1 CHIP FBX2 Parkin Soluble E3 ligases ATX3 Rad23 N-glycanase Membrane-associated HRD1-SEL1L gp78

RMA1, TRC8 & RFP2 (Transmembrane E3 ligases). TEB4

Ubc6e Ubc HERP

VCP-interacting membrane protein (VIMP) & ERASIN Derlin-1, -2 and -3

Sec61

(Incorporated into gp78)

Hrd1-Hrd3

(Hrd1-like & Cue1-like domain) (No obvious correlates) Doa10 Ubc6 Ubc7 Usa1 Ubx2 Der1 Sec61 Cue1 ER lumen- and membrane-associated

OS-9 and XTP3-B EDEM-1, -2 and -3 BiP PDI Yos9 Htm1 (now Mns1). Kar2 PDI

Page 26

Apolipoprotein B (ApoB) is the other cited example of a substrate whose ERAD is contributed to by CHIP. However, its ERAD is notable for different reasons as well. ApoB is a core constituent of low-density and very-low density lipoproteins, which are involved in cholesterol transport (Shepherd, 1994). During import into the ER, ApoB is loaded with lipid and cholesterol. However, if either is absent, then the N-terminal domain no longer associates with ApoB-specific lumenal factors that facilitate its co-translational translocation (Gusarova et al., 2001; Brodsky et al., 2007). After dissociation of these factors, translation in the cytosol continues, but the remaining peptide is not passed through the pore, instead distending a misfolded loop into the cytosol. This loop is subsequently recognised by both Hsc70 and Hsp90, which target it for degradation by cooperation with CHIP (Gusarova et al., 2001). The protein is therefore degraded in a co-translational, co-translocational manner in response to regulatory cues from the lumen.

Interestingly, this “aborted” translocation pathway functions more broadly at times of stress (Oyadomari et al., 2006). That is, when the ER is overcome with unfolded protein. This curtails the escalation of the ER‟s quality control burden (Kawaguchi & Ng, 2007). Negative feedback from the lipid content of the lumen also regulates the ERAD of proteins like HMG- CoA reductase (Song et al., 2005). Such feedback appears to be a recurring theme, as there exists an ERAD-implicated E3 ligase that directly senses sterols and down-regulates the proteins responsible for their synthesis, designated TRC8 (Lee et al., 2010; Nakatsukasa & Brodsky, 2008; Brauweiler et al., 2007). Given Ploegh‟s (2007) speculation about the involvement of lipid droplets in the egress of ERAD substrates from the ER, this poorly- characterised category of lipid-sensing factors may hold greater significance for the field of ERAD. For instance, the wider integration of lipid and protein homeostasis. This remains to be determined.