Wednesday, 20 June 2012

ERAD: the long road to destruction by Meusser

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) eliminates misfolded or unassembled proteins from the ER. ERAD targets are selected by a quality control system within the ER lumen and are ultimately destroyed by the cytoplasmic ubiquitin–proteasome system (UPS). The spatial separation between substrate selection and degradation in ERAD requires substrate transport from the ER to the cytoplasm by a process termed dislocation. In this review, we will summarize advances in various aspects of ERAD and discuss new findings on how substrate dislocation is achieved.

The ER is the main port of entry into the secretory pathway. Proteins are translocated into the ER in an unfolded state through a narrow channel that is formed by the heterotrimeric Sec61p complex1. Folding of nascent polypeptides commences during translocation and is assisted by various chaperones residing in the ER lumen. In the ER, translocated proteins undergo modifications such as N-linked glycosylation and disulphide-bond formation to support proper folding. However, protein maturation is an imperfect process that produces faulty polypeptides, which must be properly disposed of. The first indication that certain ER proteins are eliminated came from studies in mammalian cells. Orphan subunits of the T-cell receptor, such as TCR-alpha, are retained in the ER and are subsequently degraded2. A mutant cystic fibrosis conductance regulator (CFTR) shares a similar fate. The DeltaF508 mutation in CFTR is the main cause for cystic fibrosis because this mutant is prematurely degraded and thus absent from the surface of epithelial cells3. Because these proteins are destroyed in a pre-Golgi compartment, it was suggested that a protease resides within the ER. Yet, the presence of an aggressive proteolytic apparatus in this compartment is hard to reconcile with the large number of immature proteins in the ER.

The ubiquitin–proteasome system at the endoplasmic reticulum

Two findings provided an unexpected link between the turnover of ER membrane proteins and the cytoplasmic ubiquitin proteasome system (see Fig. 1 for a description of the UPS). First, inhibitor studies showed that degradation of mammalian DeltaF508CFTR is proteasome-dependent4, 5. Second, the yeast ubiquitin-conjugating enzymes Ubc6p and Ubc7p were found to participate in the turnover of mutant Sec61p, a central subunit of the ER translocation channel6, 7. Furthermore, it became evident that the misfolded ER-lumenal protein carboxypeptidase Y (CPY*) is also degraded in a Ubc7p-dependent manner8. Both Ubc6p and Ubc7p localize to the cytoplasmic face of the ER. Whereas Ubc6p is an integral membrane protein6, Ubc7p is recruited to the ER by another membrane protein Cue1p9. The restriction of these Ubc enzymes to the cytoplasmic surface of the ER, and the absence of UPS components from secretory compartments implied that ERAD substrates, lumenal and membrane proteins, must be transported back into the cytoplasm for proteolysis. This process was termed protein dislocation or retrograde transport10 (for a schematic view of ERAD seeFig. 2).
fig1 : ubiquitin-protease system (UPS).  A cascade of enzymatic reactions leads to ubiquitination of lysine residues of the substrate. First, the ubiquitin-activating enzyme (E1) hydrolyses ATP and forms a high-energy thioester linkage between its active site cysteine and the carboxy terminus of ubiquitin (Ub). Activated ubiquitin is then transferred to a member of the family of ubiquitin-conjugating enzymes (Ubc or E2). E2 enzymes together with ubiquitin protein ligases (E3) attach ubiquitin to lysine residues of substrate proteins. In most cases, E3s function as substrate-binding factors that align the substrate and E2 in a way that facilitates ubiquitination. However, one class of E3s containing the HECT (homology to E6AP C terminus) domain is able to form a thioester intermediate with ubiquitin. In this case, direct contact between E2 and substrate (as depicted in this figure) is not necessary. A polyubiquitin chain is formed on the substrate by the successive addition of ubiquitin molecules to lysine residues of the previously attached ubiquitin. Polyubiquitinated proteins are recognized by specific subunits in the 19S capping complexes of the 26S proteasome. The AAA-type ATPases in the 19S cap are required to feed the polypeptide chain into the central chamber of the 20S core particle that harbours the proteolytically active sites. Deubiquitinating enzymes that are associated with the 19S cap cleave ubiquitin from the substrates before terminal digestion.
fig 2: proteasomal degradation of ERAD targets.  Aberrant proteins are recognized within the ER lumen by different quality control mechanisms, which escort terminally misfolded polypeptides to a putative channel that facilitates their export from the ER. Cytoplasmically exposed lysine residues are ubiquitinated by ubiquitin ligases. Dislocation is completed with the help of the Cdc48p/p97 complex and membrane-extracted substrates are conveyed to the proteasome by accessory factors such as Rad23p and Dsk2p.
isfolded proteins are not the only substrates for ERAD. The regulated breakdown of a key enzyme of the mevalonate pathway, HMG CoA (3-hydroxy 3-methylglutaryl coenzyme A) reductase (HMGR), is controlled by this system11. The end products of the mevalonate pathway are sterols and a variety of isoprenoids. When flux through the mevalonate pathway is high, HMGR is rapidly degraded, whereas it is stable when flux is low. In yeast, there are two isozymes of the ER-bound HMGR, Hmg1p and Hmg2p. Hmg2p is degraded by the UPS pathway but Hmg1p is stable. The analysis of Hmg2p turnover led to the identification of a number of ERAD components12 including Hrd1p/Der3p, a multi-spanning membrane protein with RING-finger-type ubiquitin ligase activity exposed to the cytoplasmic ER surface13, 14. Hrd1p/Der3p is also crucial for proteolysis of misfolded CPY* (ref. 15). In vitro, this ligase ubiquitinates denatured proteins suggesting that it functions as a quality control ligase13. The Hrd1p/Der3p ligase functions with Ubc7p and another ubiquitin-conjugating enzyme, Ubc1p13, 16. Another ER membrane protein, Hrd3p, is a cofactor for Hrd1p/Der3p that may target substrates to the site of dislocation via its large lumenal domain17.

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