ER associated protein degradation (ERAD)

Proteins which are damaged or cannot reach their native conformation or fail to get post-translationally modified are selected by the ER quality control system, which sorts them for ER associated protein degradation (ERAD). Unassembled subunits of multimeric protein complexes are selected for this pathway as well (Vembar and Brodsky, 2008). Initially, proteolysis was thought to occur in the ER lumen. However, orphan subunits of the heptameric T-cell receptor complex (TCR), which were not assembled in heterooligomeric complexes before ER export, were found to be degraded in a pre-Golgi compartment but not in the lysosomes (Lippincott-Schwartz et al., 1988). Later, it was found that defective proteins such as mutated Sec61, mutated cystic fibrosis transmembrane conductance regulator (CFTR) and mutated yeast Carboxypeptidase Y (CYP*) are transported from the ER back to the cytoplasm to be degraded by the 26S proteasome (Hiller et al., 1996; Sommer and Jentsch, 1993; Ward et al., 1995). Thus, with exceptions, the majority of misfolded proteins is translocated back to the cytosol for degradation (Schmitz and Herzog, 2004).

Substrates traverse this ERAD pathway in four consecutive steps (Figure 6):

(1) Recognition, selection and targeting to the retrotranslocation machinery (2) Retrotranslocation and ubiquitination

(3) Extraction from the ER membrane

(4) Transfer to the 26S proteasome and degradation

Recognition and selection of ERAD substrates

Molecular chaperones, which recognise ERAD substrates, can differentiate between misfolded and nascent proteins. Although a common biophysical property of ERAD substrates has not been identified, some features were found to make proteins prone to degradation.

An example for that are hydrophobic patches which are normally buried within the protein. If a protein is unfolded, these patches might be exposed to the ER lumen. There, Hsp70 chaperones such as Grp78 bind to these potential substrates to maintain their solubility, which is a prerequisite for later retrotranslocation (Nishikawa et al., 2001). Accordingly, membrane proteins, harbouring defects in their cytoplasmatic domains, are recognised by cytoplasmatic Hsp70 or Hsp90 chaperones, as shown for CFTR which binds to Hdj-2/Hsc70 (Meacham et al., 1999). As the Grp78 orthologue in yeast, Kar2p, is associated with ubiquitination mediating complexes, this chaperone was suggested to have a role in the recruitment of ERAD substrates to these complexes (Denic et al., 2006).

ER lumen

Figure 6: Illustration of the single steps of the ER associated protein degradation (ERAD). (1) Misfolded proteins are recognised with the help of Grp78 (=BiP) and transferred to the ER membrane. (2) E3 ligase containing multiprotein complexes mediate ubiquitination and retrotranslocation of these substrates. (3) The p97 complex extracts polyubiquitinated substrates from the ER membrane. (4) Finally, the substrates are transferred to the 26S proteasome, possibly with the help of a shuttle protein, where they are finally degraded.

During synthesis, nearly all secretory proteins are modified by a preformed N-linked oligosaccharide structure. Trimming of this sugar moiety by glucosidases and folding facilitated by the lectin-like chaperones calnexin and calreticulin results in a glycoprotein which is ready for secretion. Defects in glycoproteins lead to their re-glycosylation by UDP-glucose:glycoprotein-glucosyltransferase (UGGT) so that they can re-enter the calnexin cycle for their correction (Caramelo and Parodi, 2008). However, irreparably misfolded glycoproteins cannot cycle endlessly between calnexin and the UGGT. Therefore, an ER mannosidase acts as a kind of timer and cleaves the mannose residues off the N-glycans.

This cleavage leads to a reduced calnexin/calreticulin re-binding and an enhanced binding to ER degradation enhancing alpha-mannosidase-like protein (EDEM) which results in the degradation of these substrates (Hosokawa et al., 2003; Oda et al., 2003).

Another type of chaperones, protein disulfide isomerases (PDIs), involved in redox state dependent protein maturation, have been shown to interact with Grp78 and to be important for ERAD substrate recognition (Molinari and Helenius, 2000). Recently, the yeast E3 ligase Hrd1p has been shown to directly recognise misfolded membranous proteins, dependent on its own transmembrane regions (Sato et al., 2009).

Retrotranslocation and ubiquitination

Since degradation of ERAD substrates takes place in the cytoplasm, ER derived proteins have to pass the ER membrane in the opposite direction as protein synthesis occurs. This process is termed retrotranslocation. In addition, the process of ubiquitination is also located at the cytoplasmatic site of the ER. This means that for their degradation, ERAD substrates at least have to gain access to this compartment. Starting retrotranslocation is a prerequisite for ubiquitination. All ER lumen and most ER membrane derived proteins are thought to be retrotranslocated presumably by a protein-conducting channel.

A few proteins have been suggested to form this retrotranslocation pore. The mammalian ER transmembrane protein Derlin-1 was found in a complex with cytoplasmatic and membrane-resident ERAD mediating proteins as well as with ERAD substrates (Katiyar et al., 2005;

Lilley and Ploegh, 2004; Schulze et al., 2005). A depletion of Derlin1 results in the induction of the UPR and in retarded degradation of selected substrates (Ye et al., 2005). Also E3 ligases are assumed to mediate retrotranslocation and ubiquitination. In yeast, the RING E3 ligases Hrd1p and Doa10 are integral components of distinct multiprotein complexes consisting of ER luminal, ER membrane and cytoplasmatic proteins. While Hrd1p mediates the degradation of substrates that harbour misfolded domains within the ER lumen (ERAD-L), Doa10 is required for proteins with a folding error in their cytosolic domain (ERAD-C) (Carvalho et al., 2006; Vashist and Ng, 2004).

In mammalia, the membrane-multispanning E3 ligase Gp78, an orthologue of yeast Hrd1p, was suggested to mediate retrotranslocation (Zhong et al., 2004). A different hypothesis implies that retrotranslocation of protein complexes or of large proteins requires the formation of so called lipid droplets from the ER membrane (Ploegh, 2007).

ERAD substrates have to be ubiquitinated prior to extraction and proteasomal targeting and ubiquitination requires the sequential action of E1, E2 and E3 enzymes (see 1.1.3). The mammalian ER-resident E3 ligases Gp78 and Hrd1 (also called Synoviolin), for instance, together with the E2 enzyme Ube2g2 mediate the degradation of CD3-δ and TCR-α, respectively (Fang et al., 2001; Kikkert et al., 2004).

Cytosolic ubiquitin ligases may also be implicated in the ERAD process, as this was reported for the RING E3 parkin which ubiquitinates the parkin-associated endothelin receptor-like receptor (Pael-R) (Imai et al., 2001). Since Synoviolin was described to also ubiquitinate Pael-R, the efficient turnover of substrates may depend on more than one E3 ligase (Omura et al., 2006). This was also shown for the ERAD substrate CFTR which is processed by the cooperation of two E3 ligases, Gp78 and RMA1 (Morito et al., 2008).

Membrane extraction of ERAD substrates

Polyubiquitination of ERAD substrates is not only a signal for proteasomal degradation but also for the extraction of substrates from the ER membrane (Kikkert et al., 2001). To start this process, a minimal length of the polyubiquitin chain bound to the substrate is decisive (Jarosch et al., 2002). The polyubiquitin moiety is recognised by a cytosolic molecular chaperone complex, consisting of the AAA-ATPase p97 (also called VCP for Valosin containing protein or Cdc48 in yeast), which forms a homohexamer, and the associated proteins Ufd1 and Npl4. In yeast, the Cdc48-Ufd1-Npl4 complex was shown to bind ubiquitin conjugates and to be required for the ATP dependent extraction of various substrates such as MHC class I molecules (Dai and Li, 2001; Rape et al., 2001; Ye et al., 2001).

P97 was suggested to act as a motor, actively pulling the substrate out of the ER membrane in an ATP-consuming process and dependent on the binding of p97 to the ER membrane (Ye et al., 2003). The recruitment of the p97 complex to the ER membrane was proposed to be mediated by ER-resident proteins. Among these proteins are the VCP interacting membrane protein (VIMP) and ubiquitin regulator-X (UBX) domain proteins such as Ubxd2 (Liang et al., 2006; Ye et al., 2004). Also Ubxd8 was suggested as a p97 recruitment factor in mammalia (Mueller et al., 2008).

Transfer of ERAD substrates to the 26S proteasome

Since it has been found to interact with 26S subunits, p97 was initially assumed to escort the extracted ERAD substrate to the 26S proteasome for degradation (Dai et al., 1998). Later, a variety of proteins exhibiting a UBA as well as a UBL domain were described to execute this shuttle function. Harbouring both domains allows these proteins to interact with ubiquitinated substrates and the 26 proteasome concomitantly. The most prominent UBL and UBA domain containing proteins are Rad23 and Dsk2 required for ERAD in yeast (Medicherla et al., 2004). In humans, two homologues of Rad23, hHR23A and hHR23B interact with the proteasomal 19S subunit Rpn10 (S5a) via their UBL domain (Hiyama et al., 1999).