ER-associated degradation: Protein quality control and beyond

ERAD, linking ER quality control to cytoplasmic protein degradation

The earliest evidence for protein quality control at the ER came from observations that unassembled subunits of the T cell receptor were rapidly degraded in the cells (Lippincott-Schwartz et al., 1988). This degradation occurred independently of lysosomal proteases, leading to the proposal that the ER itself would house some uncharacterized proteolytic activity toward misfolded proteins. Then a landmark study in yeast showed that the degradation of a short-lived misfolded ER membrane protein was blocked in cells lacking Ubc6, a component of the ubiquitin conjugation machinery (Sommer and Jentsch, 1993). The ubiquitin system mediates the covalent attachment of ubiquitin, a small 76–amino acid protein, to target proteins in the cytoplasm by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes (Pickart, 2001). Ubiquitin-modified proteins are then recognized and degraded by the proteasome. The involvement of the ubiquitin–proteasome system in ER protein quality control was confirmed by studies on the degradation of mutant and wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a large membrane protein with a complicated folding process (Jensen et al., 1995; Ward et al., 1995). Inhibition of proteasome function led to accumulation of CFTR molecules, and interestingly, a significant fraction of these was detected as ubiquitin conjugates (Jensen et al., 1995; Ward et al., 1995). Soon after, it became clear that a similar mechanism could also account for the degradation of luminal misfolded proteins such as CPY*, a mutant version of the yeast vacuolar carboxypeptidase Y and a prototype ERAD substrate (Hiller et al., 1996). Together, these papers demonstrated that aberrant proteins in the lumen and membrane of the ER are degraded in the cytoplasm where the components of the ubiquitin–proteasome system reside.

Ubiquitin ligase complexes: The hubs in ERAD

Subsequent genetic and biochemical studies, primarily in budding yeast but also in mammalian cells, identified many ERAD components and led to a general understanding of the organization of the pathway. An important realization was that the “one-size-fits-all” model does not apply to ERAD and that this pathway encompasses multiple branches with distinct specificity for different classes of misfolded proteins (Taxis et al., 2003; Vashist and Ng, 2004; Carvalho et al., 2006; Bernasconi et al., 2010; Christianson et al., 2012). However, irrespective of the branch, the same sequence of events leads to the degradation of all ERAD substrates (Fig. 1 A). The first step is the recognition of a substrate in the crowded ER environment. Then the substrate is transported across the ER lipid bilayer back into the cytoplasm, a step known as retrotranslocation. On the cytosolic side of the ER membrane, the substrate is ubiquitinated by a membrane-associated ubiquitin ligase (or E3 ligase). Subsequently, the ubiquitinated substrate is extracted from the membrane in an ATP-dependent manner and released in the cytoplasm for degradation by the proteasome. The execution of these steps is coordinated by a membrane-embedded protein complex named after the E3 ligase at its core. The canonical E3 ligases involved in ERAD are themselves multispanning membrane proteins, in which the RING (really interesting new gene) domain responsible for the ligase activity is in the cytoplasm. These E3 ligase complexes are best characterized in yeast (Fig. 1 B and Table 1) where Doa10 (Swanson et al., 2001) and Hrd1 (Bordallo et al., 1998; Bays et al., 2001a) assemble into the Doa10 and the Hrd1 complexes, respectively, each responsible for the degradation of a class of ERAD substrates (Carvalho et al., 2006).

Based on the analysis of a few model substrates, the E3 ligase complex specificity appears to be determined by the location of the misfolded lesion on a substrate relative to the ER membrane: proteins with misfolded domains in the cytoplasmic side of the membrane (ERAD-C substrates) are degraded via the Doa10 complex; proteins with luminal (ERAD-L substrates) or intramembrane (ERAD-M substrates) misfolded domains are targeted to the Hrd1 complex (Fig. 1 B and Table 1; Taxis et al., 2003; Vashist and Ng, 2004; Carvalho et al., 2006). Factors involved in substrate recognition are unique to the E3 ligase complex and likely determine the substrate specificity of each ERAD branch. On the other hand, the components that act at late steps of ERAD, such as the Cdc48 ATPase complex (p97 in mammals) required for membrane extraction of ubiquitinated substrates (Bays et al., 2001b; Ye et al., 2001; Jarosch et al., 2002; Rabinovich et al., 2002), are common to both E3 ligase complexes.

In mammalian cells the best-studied E3 ligases are Hrd1 and Gp78 (Table 1). They are both homologous to yeast Hrd1 but assemble distinct E3 ligase complexes that preferably target different substrates (Schulze et al., 2005; Mueller et al., 2008; Bernasconi et al., 2010; Christianson et al., 2012; Burr et al., 2013). Several more E3 ligases have been implicated in ERAD in mammalian cells (such as Rma1/Rnf5, Trc8, Rfp2, Rnf170, and Rnf185) but these are still poorly characterized. Only few substrates are known for each ligase and a preference for particular ERAD substrate classes has been difficult to infer (Claessen et al., 2012).

How are ERAD substrates recognized?

Shipping out the trash: Substrate retrotranslocation and cytoplasmic events in ERAD

After being selected, ERAD substrates are retrotranslocated across the ER membrane back into the cytoplasm. In the case of misfolded luminal proteins the complete polypeptide needs to be retrotranslocated, whereas for membrane substrates this step requires the transport of only certain domains. As a consequence, ubiquitination of luminal substrates only occurs at late stages of retrotranslocation, once a portion of the substrate has been exposed to the cytoplasm. In contrast, ubiquitination of most, but not all (Burr et al., 2013), membrane substrates is coupled to their retrotranslocation.

In analogy to the transport of newly synthesized proteins into the ER or mitochondria, it has been postulated that retrotranslocation occurs through a protein-conducting channel. However, the identity of the retrotranslocation channel has been at the center of an intense debate that is almost as old as the research in this field. Over the years, several channel candidates have been proposed but it has been difficult to gather definitive evidence in support of any of them. The Sec61 translocon used for protein import into the ER was the first proposed retrotranslocation channel (Wiertz et al., 1996b). Sec61 was found to interact with ERAD substrates both in yeast and in mammalian cells as well as with the yeast proteasome (Wiertz et al., 1996b; Kalies et al., 2005; Scott and Schekman, 2008). However, the significance of these associations is not clear. Recent work showed that proteins engaging the Sec61 translocon aberrantly or persistently in their way into the ER become substrates of the Hrd1 ligase complex, which might explain the interaction between Sec61 and some ERAD substrates (Rubenstein et al., 2012). In addition, certain yeast sec61 mutants displayed defects in degrading model ERAD substrates, even under conditions in which general “forward” translocation appeared not to be dramatically affected (Pilon et al., 1997; Plemper et al., 1997; Willer et al., 2008). It remains to be determined whether this phenotype is caused by a specific impairment in retrotranslocation.

The E3 ligase complexes interact with ERAD substrates immediately before and after their retrotranslocation, indicating this step occurs in their immediate vicinity. Therefore, multispanning membrane proteins within the E3 ligase complexes have also been seen as good candidates to mediate retrotranslocation (Ye et al., 2001; Lilley and Ploegh, 2004; Kreft et al., 2006; Horn et al., 2009; Carvalho et al., 2010; Mehnert et al., 2014). These include the E3 ligases themselves as well as proteins of the Derlin family (Der1 in yeast), which are essential for the degradation of all luminal ERAD substrates but whose function has remained elusive. In vitro experiments using mammalian-derived microsomes loaded with the yeast ERAD substrate mutant pro-α-factor indicate that Derlin might be involved in retrotranslocation (Wahlman et al., 2007). In this simplified system, in which synthesis and retrotranslocation were uncoupled, the substrate cross-linked to Derlin but not to Sec61. Moreover, its retrotranslocation was blocked by anti-Derlin antibodies whereas antibodies directed to Sec61 had no effect (Wahlman et al., 2007). It should be noted that mutant pro-α-factor is a noncanonical substrate because its retrotranslocation does not require ubiquitination. Interactions between the yeast Der1 and the prototype ERAD-L substrate CPY* were also detected by site-specific photocrosslinking in yeast (Carvalho et al., 2010; Stanley et al., 2011; Mehnert et al., 2014). These cross-links were seen even in cells lacking the substrate recognition factors Hrd3 and Yos9, and were increased if conserved polar residues in the membrane domain of Der1 were mutated. An interpretation of these results is that Der1 mediates the transfer of substrates from the recognition factors Hrd3/Yos9 to the Hrd1 ligase inside the membrane (Mehnert et al., 2014).

A compelling piece of evidence for a function during substrate retrotranslocation was reported for the E3 ligase Hrd1 (Fig. 3; Carvalho et al., 2010). Although commonly working in the context of the Hrd1 complex, overexpressed Hrd1 can mediate the degradation of ERAD-L substrates even in the absence of its membrane partners Hrd3, Der1, and Usa1 (Plemper et al., 1999; Denic et al., 2006; Carvalho et al., 2010). Under these conditions Hrd1 selectivity for misfolded proteins is lost, suggesting that the other subunits of the complex are critical to control Hrd1 activity and substrate specificity (Denic et al., 2006). Hrd1 interacts with a sizeable region of a modified version of CPY* during the early stages of retrotranslocation, as assayed by site-specific photocrosslinking (Carvalho et al., 2010). Importantly, the interaction likely occurs inside of the ER bilayer because it is lost if substrate recognition is blocked or if the transmembrane segments of Hrd1 are mutated. Hrd1 contains only six transmembrane segments; therefore, ERAD-L substrate retrotranslocation requires Hrd1 oligomerization, which normally is facilitated by Usa1 but can occur spontaneously upon Hrd1 overexpression (Horn et al., 2009; Carvalho et al., 2010). All these data make a strong case for a direct role of Hrd1 in the retrotranslocation of ERAD-L substrates, but the possibility that it works with some partner(s), such as Der1, Sec61, or other unknown factors cannot be excluded at this point. Moreover, it is not known whether this is a unique feature of Hrd1 or whether the membrane domains of other E3 ligases also participate in the retrotranslocation of other classes of ERAD substrates.

In most of these cross-linking experiments the retrotranslocation of CPY* was dampened by fusing it to a very tightly folded domain, indicating that ERAD-L substrates need to be unfolded before this step (Bhamidipati et al., 2005; Carvalho et al., 2010). Whether unfolding is also a prerequisite for the retrotranslocation of other classes of ERAD substrates is not settled yet (Fiebiger et al., 2002; Tirosh et al., 2003).

At the cytoplasmic side of the ER membrane, substrates are ubiquitinated, a modification that allows their recognition by the Cdc48/p97 ATPase complex composed of a homohexamer of Cdc48/p97 and by the cofactors Ufd1 and Npl4 (Bays et al., 2001b; Ye et al., 2001, 2003; Jarosch et al., 2002; Rabinovich et al., 2002). The recruitment of this ATPase complex to the ER membrane is facilitated by ubiquitin regulatory X (UBX) domain–containing proteins, Ubx2 in yeast and UBXD8 in mammals (Neuber et al., 2005; Schuberth and Buchberger, 2005; Mueller et al., 2008). The ATPase activity of the Cdc48/p97 complex provides the driving force to move ubiquitinated substrates out of the membrane into the cytosol (Ye et al., 2003). Although the role of Cdc48/p97 in this process is well established, the mechanism that couples ATP hydrolysis to membrane extraction of the substrate is still not understood. In addition to the Cdc48/p97 complex, the ATPase subunits of the proteasome regulatory particle were also shown to play a role in retrotranslocation of some ERAD substrates (Lipson et al., 2008). The driving force for the retrotranslocation of the few non-ubiquitinated substrates, like the cholera toxin or pro-α-factor, is not known (Kothe et al., 2005; Moore et al., 2013).

The Cdc48/p97 complex also serves as a platform for other ubiquitin-modifying enzymes such as de-ubiquitinating enzymes (DUBs; Rumpf and Jentsch, 2006; Jentsch and Rumpf, 2007; Ernst et al., 2009). Although the role of some of these Cdc48/p97-binding factors is not clear yet, it was shown that interfering with the DUBs YOD1 and ataxin-3 affected substrate retrotranslocation (Wang et al., 2006; Ernst et al., 2009). The requirement for DUB activity during retrotranslocation suggests that the process involves cycles of ubiquitination and de-ubiquitination. In some cases these cycles might be important to increase the specificity of substrate recognition, as was shown for the Vpu-mediated degradation of CD4 (Zhang et al., 2013). Interestingly, the retrotranslocation of some noncanonical substrates like cholera toxin, which are not ubiquitinated, is also affected by manipulation of both E3 ligase and DUB activities (Hassink et al., 2006; Bernardi et al., 2013). These results suggest that retrotranslocation requires the ubiquitination of some factors other than the substrates. Future studies should test some obvious candidates such as the components of the E3 ligase complexes themselves.

There is some recent evidence that the yeast Cdc48 complex might be acting also much earlier, during the initial stages of retrotranslocation of an ERAD-L substrate, before it is exposed to the cytoplasm (Fig. 3). Using a cross-linking strategy, it was shown that the interaction between Hrd1 and an early retrotranslocation intermediate was lost in mutants of the Cdc48 complex (Carvalho et al., 2010). Interestingly, a similar defect was detected in Hrd1 mutants impaired for E3 ligase activity (Carvalho et al., 2010). Based on these observations it was proposed that in early stages of ERAD-L substrate retrotranslocation, Hrd1 induces the ubiquitination of an ERAD component, perhaps Hrd1 itself. This ubiquitination signals the recruitment of the Cdc48 complex, which upon ATP hydrolysis would induce changes in the conformation or in the oligomeric status of Hrd1, resulting in substrate retrotranslocation. This model is consistent with the well-established role of Cdc48/p97 in the disassembly and remodeling of protein complexes (Jentsch and Rumpf, 2007). Once the substrate emerges on the cytosolic side and is ubiquitinated by Hrd1, the ATPase complex binds to it and promotes the final stages of its retrotranslocation. Although many aspects of this model still wait for experimental support, it would provide a unifying role for the Cdc48/p97 ATPase as the driving force for substrate retrotranslocation in ERAD (Fig. 3).

After being released from the membrane, substrates are kept soluble and transferred to the proteasome by cytosolic chaperones such as the BAG6 complex (Claessen and Ploegh, 2011; Wang et al., 2011; Xu et al., 2012) or shuttle factors like Rad23 and Dsk2 (Medicherla et al., 2004). The long journey of ERAD substrates ends with their degradation by the proteasome.

Conclusions and future perspectives

In recent years there has been tremendous progress in understanding ERAD. The identification of most of the components involved in this process and how these are pieced together and organized in the different ERAD branches were important achievements. A major challenge for the future is to reveal the mechanistic aspects of the pathway. Such developments should, for example, help in discerning the basis by which misfolded proteins are recognized in each of the different ERAD branches.

The mechanisms of substrate retrotranslocation and the roles played by the different components, such as the E3 ligases and the Cdc48/p97 complex, will certainly be another interesting area to follow. However, progress on these topics might require the development of new approaches, such as in vitro systems with purified components recapitulating individual ERAD steps.

In early days, ERAD was perceived as a process mainly dedicated to ER protein quality control. The picture now emerging places ERAD closer to other ubiquitination systems in the cytoplasm and nucleus, which control the turnover of specific proteins to achieve a certain physiological state. Therefore, another major challenge for the coming years is the detailed characterization of the roles of ERAD beyond quality control. Although the central role of ERAD in sterol homeostasis is unequivocal, it will be important to clarify whether ERAD has a more general role in the regulated degradation of folded ER proteins and in that way modulates other ER-related functions. A systematic and rigorous identification of regulated ERAD substrates should help in addressing these issues. Uncovering the intersections of ERAD with other cellular pathways will provide important insights into the mechanisms of ER and cellular homeostasis.