Introduction
Secretory proteins play a crucial role in a wide variety of biological processes. It is therefore not surprising that the eukaryotic proteome contains a considerable number of these proteins, ranging from 10% in yeast to more than 20% in mice and humans (1,2,3). Secretory proteins are typically synthesized by ribosomes associated with the endoplasmic reticulum (ER). Consequently, the ER comprises the initial hub of the secretory pathway. Secretory proteins are translocated into the ER in an unfolded state and are only allowed to leave if they have attained their native, functional conformation (upper panel of figure 1). This means that the ER houses an intricate quality control system made up of different luminal folding factors that jointly monitor the folding of polypeptides and ensure the removal of misfolded or unfolded ones that are typically prone to cytotoxic aggregation. The accumulation of unfolded or misfolded proteins within the ER induces the so-called unfolded protein response (UPR). This is a signal transduction pathway that adapts the cell to ER stress through pausing protein production, degrading misfolded proteins and upregulating the expression of molecular chaperones to protect the cell’s proteome (4). If these steps don’t alleviate the problem, the proteins labelled by the cell as misfolded are transported back from the ER into the cytosol and are subsequently degraded by the ubiquitin-proteasome system (upper panel of figure 1). This process is known as ER-associated degradation (ERAD) and is vital for cellular health (5,6). In fact, in humans, ERAD is currently associated with the pathogenesis of more than 60 diseases, including Alzheimer’s, Parkinson’s, Huntington’s disease and ALS (7). ERAD is, moreover, hijacked by certain viruses and toxins. The ERAD pathway comprises three basic steps, namely: (i) recognition and processing, (ii) retrotranslocation and simultaneous ubiquitination and (iii) extraction and degradation of the polypeptide (lower panel of figure 1 adapted from 5).
Potential ERAD substrates include aberrant soluble and membrane proteins with cytoplasmic, luminal or intramembrane lesions. These are recognized (a) by luminal and cytoplasmic chaperones as well as other folding factors. Subsequently, ERAD substrates are targeted (b) towards the retrotranslocation machinery (c) for export into the cytoplasm. As polypeptides emerge from retrotranslocation machinery they are polyubiquitylated by ubiquitin ligases (d), which drives further retrotranslocation. Within the cytoplasm, the polyubiquitylated polypeptide is recognized by receptors of the proteasome (e). Following removal of the polyubiquitin, the polypeptide is transferred into the proteolytic chamber and degraded into peptide fragments (e). Retrotranslocation into the cytoplasm occurs through a protein-conducting channel, although its exact identity was debated for many years. Recent biochemical evidence, however, strongly suggests that in yeast Hrd1, a protein embedded within the endoplasmic membrane with ubiquitin ligase activity, may form a retrotranslocation channel (6). For example, photocrosslinking experiments showed that Hrd1 is in close contact to a polypeptide during retrotranslocation in yeast, while a crucial role for Hrd1 in retrotranslocation was unequivocally shown by reconstitution this reaction in vitro with purified yeast components, thereby confirming that it is sufficient for retrotranslocation of a model substrate (9,10). To fully understand the mechanism of retrotranslocation, structural insight of the Hrd1 complex is required and it is therefore important to note that recently detailed structures of the Hrd1 ubiquitin ligase complex from yeast were presented (11). Here, I will discuss these structures as well as the current functional understanding.
Endoplasmic reticulum-associated degradation
It was found in the mid 1990’s that aberrant soluble and membrane proteins of the secretory pathway are degraded by the cytosolic ubiquitin-proteasome system (12). This implies that these proteins are rejected by the protein quality control system of the ER and are subsequently shipped back to the cytosol for proteolysis. This process was later termed ERAD and many of the components involved in substrate recognition and degradation were identified through genetic and biochemical studies in the yeast Saccharomyces cerevisiae (5,6). These components are conserved in all eukaryotic organisms and it can therefore be expected that the conclusions from the aforementioned yeast studies are valid for all eukaryotes.
Mechanistically, ERAD is not fully understood, although it is clear that it involves three main steps. ERAD is initiated by the recognition of misfolded or unfolded polypeptides probably by chaperones of the ER protein quality control system. Subsequently, the aberrant polypeptide is transported back across the ER membrane to the cytoplasm, which most likely requires a protein-conducting channel (5,6). At the cytoplasmic face of the ER membrane, the substrate is equipped with polyubiquitin, a 76-residue polypeptide that is conserved in eukaryotes, by ubiquitin ligases. The ubiquitin tag is recognized by a cytoplasmic ATPase complex known as Cdc48 in yeast or p97 in mammals (figure 2 adapted from 13). This ATPase powers the extraction of the polypeptide from the ER membrane into the cytosol at the expense of ATP hydrolysis, thereby enabling its degradation by the proteasome (5,6). The ubiquitin ligases are crucial ERAD components and it is therefore not surprising that these are conserved in all eukaryotes. Ubiquitin ligases are typically oligomeric protein complexes embedded in the ER membrane and form a functional link between the selection of aberrant polypeptides in the ER and their degradation by the proteasome in the cytoplasm. Two distinct ERAD-related ubiquitin ligases have been identified in yeast, which are known as Doa10 and Hrd1 (figure 2). Thus, the two yeast ubiquitin ligases (Doa10 and Hrd1) define three distinct ERAD pathways (figure 2) for substrates with misfolded cytosolic domains (ERAD C), substrates with misfolded luminal domains (ERAD L) or substrates with lesions in TMDs (ERAD M). All three pathways rely on the Cdc48 ATPase complex for membrane extraction of polyubiquitinated substrates into the cytoplasm, enabling their subsequent degradation by the proteasome (14).
Biological features of the yeast Hrd1 ubiquitin ligase complex
Ubiquitin ligases play an essential role in all ERAD pathways, which are typically part of oligomeric complexes made up of different membrane embedded proteins. Ubiquitin ligases contain a variable number of TMDs and a cytosolic RING (really interesting new gene) finger domain that mediates protein-protein interactions (5,6). Functionally, they catalyze ubiquitylation of substrates and associate with different partners forming complexes that orchestrate ERAD at both sides of the ER membrane as well as inside the lumen. The yeast ERAD L pathway, that is used for the degradation of soluble and membrane proteins with luminal lesions, is probably the best characterized one and relies on the Hrd1 ligase complex (5,6). This is shown schematically in figure 3 (adapted from 8) and comprises a membrane-embedded core made up of Hrd1, Hrd3, Usa1 and Der1. Of these, Hrd1 and Der1 are multi-spanning membrane proteins with 8 and 6 TMDs and a mass of 63 and 24 kDa, respectively. Hrd3 and Usa1 are, however, single pass membrane proteins with a mass of 95 and 97 kDa. Hrd3 forms a dimer with Hrd1 in the ER membrane and both are conserved in all eukaryotes. Hrd1 directly interacts with substrates during the early stages of retrotranslocation as shown by site-specific photo crosslinking and in vitro experiments showed that it is sufficient for the retrotranslocation of a model substrate (9,10). Collectively, these results point towards a role of Hrd1 as protein-conducting channel for ERAD substrates (5,6). Hrd3 is equipped with a large luminal domain, which associates with Yos9, a luminal lectin-like protein of 61 kDa. Yos9 recognizes the glycan of misfolded, glycosylated ERAD L substrates, whereas Hrd3 binds to the unfolded polypeptide stretch around the glycan. Thus, Hrd3 serves as a critical component of the Hrd1 complex through regulating substrate binding and the recruitment of luminal factors (5,6). At the cytoplasmic side of the ER membrane, Hrd1 associates with Usa1. It has no significant interaction with substrates but functions as scaffolding protein instead through facilitating oligomerization of Hrd1 and linking Der1 to Hrd1. Yeast Der1 is homologous to mammalian Derlin1, Derlin2 and Derlin3. These belong to the rhomboid family of intramembrane proteases but are enzymatically inactive and are essential for ERAD in humans, while deletion of der1 in yeast blocks the degradation of ERAD L substrates. Based on these results it is conceivable that Der1 functions as an ERAD protein channel in addition to Hrd1 (5,6).
As substrates emerge from the ERAD protein channel at the cytoplasmic face of the ER membrane, they are polyubiquitylated by Hrd1. It was recently found, moreover, that activation of Hrd1 requires auto ubiquitination of its RING finger domain. Substrate ubiquitination requires Ubc7, a ubiquitin-conjugating enzyme of about 19 kDa. Ubc7 and Hrd1 work in concert to catalyze transfer of the ubiquitin tag to a specific lysine (lys48) of the substrate molecule. Polyubiquitination prevents backsliding of the substrate into the ER lumen and, moreover, the polyubiquitin chain is recognized by the cytosolic Cdc48 complex and its cofactors Ufd1 and Npl4. Cdc48 is an ATPase of 92 kDa that belongs to the family of AAA proteins, which typically form homohexameric structures with a central channel. Ufd1 and Npl4 are ubiquitin-binding proteins of 40 and 66 kDa, respectively, that recognize the polyubiquitin chain of the substrate. Next, Cdc48 is thought to pull on the ubiquitin tag of the to extract the substrate from the ER membrane into the cytosol at the expense of ATP hydrolysis. In yeast, Ubx2, a membrane-embedded protein of 67 kDa, associates with Cdc48 via its UBX domain and links it to the Hrd1 ligase complex (5,6). The essential role of Cdc48 and its cofactors is emphasized by the finding that in yeast cells devoid of these components ERAD is blocked (15). Finally, the substrate is delivered at the proteasome for degradation but how this is accomplished is unclear.
Structural features of yeast Hrd1 ubiquitin ligase complex
Despite the improved biochemical understanding of ERAD, the mechanism by which substrates are retrotranslocated from the ER into the cytosol remains poorly understood. This is primarily because there is hardly any structural information on active ubiquitin ligase complexes. Therefore, it is important to note that recently detailed structures of the active Hrd1 complex were reported (11). To obtain the structure of the Hrd1 subcomplex containing all the membrane-embedded subunits, an epitope-tagged Hrd1-Usa1 fusion protein was expressed in yeast cells jointly with untagged Der1 as well as the Hrd3 luminal domain also equipped with an epitope tag. Subsequently, the complex was detergent-solubilized, affinity purified and subjected to size-exclusion chromatography. The structure of the purified Hrd1-Usa1-Der1-Hrd3 subcomplex was assessed next by cryo-EM analysis at a resolution of 4.3 Å. This is shown in figure 4 in both surface (left top panel) and ribbon representation (right top panel), revealing that the complex contains one molecule of each subunit. This therefore suggests that the physiological complex is monomeric, which is consistent with the finding that Hrd1 can function as a monomer in ERAD L pathway in vivo. The 8 TMDs of Hrd1 are well resolved unlike the RING finger domain that is not present owing to its flexibility. Moreover, a lateral gate between TMD3 and 8 is observed when viewed from the lumen (indicated by an asterisk in the bottom left panel) which is in an open state, while a helix between TMD1 and 2 of Hrd1 mediates the interaction between Hrd1 and Hrd3. Usa1 is made up of three helices and serves as a linker between Der1 and Hrd1 through interacting with Hrd1 via its N-terminal cytosolic region and with Der1 through its C-terminal cytosolic region. The structure of Der1 is also well resolved, showing its 6 TMDs and connecting loops.
Moreover, Der1 also contains a lateral gate that is located between TMD2 and 5 (indicated by an asterisk in the bottom left panel). Unlike the lateral gate of Hrd1, the lateral gate of Der1 is in a closed state owing to the contact between TMD5 and 2. The lateral gates of Hrd1 and Der1 face one another and are accessible to the surrounding lipid. Interestingly, molecular dynamics simulations revealed that the lipid bilayer was locally thinned and distorted in the region between the lateral gate of Hrd1 and Der1. The closed lateral gate of Der1 is accompanied by a shallow luminal cavity (right bottom panel) that in other members of the rhomboid family contains the active site, while Hrd1 contains a cytosolic cavity that is formed by five of its TMDs and extends almost to the ER lumen (right bottom panel). Der1 and Hrd1 interact at the luminal side of the ER membrane through TMD2 of Der1 and TMD3 of Hrd1. This interaction ensures that the luminal cavity of Der1 and the cytosolic cavity of Hrd1 are close together. Thus, Hrd1 and Der1 create two so-called half channels with cytosolic and luminal cavities as well as lateral gates that face each other in a locally thinned ER membrane.
To obtain insight into how glycosylated ERAD L substrates are recognized within the ER lumen, the structure of the subcomplex made up of Hrd3 and Yos9 was assessed. Therefore, epitope-tagged Hrd1 was coexpressed in yeast cells with the untagged luminal domain of Hrd3 as well as Yos9 equipped with an epitope tag. The complex was detergent solubilized, affinity purified and subjected to size exclusion chromatography. The structure of the purified complex was subsequently determined by cryo-EM at a resolution of 3.7 Å, thereby focusing on the luminal Hrd3-Yos9 part. This structure is presented in figure 5 in surface (left top panel) and ribbon representation (right top panel), showing that Hrd3 is folded similarly as in the Hrd1-Usa1-Hrd3-Der1 subcomplex. The conformation of Hrd3 is therefore not affected by Yos9 association. Yos9 comprises three domains, namely: dimerization domain (DD), ß-sheet domain and the flexible glycan-binding MRH domain. Yos9 interacts with Hrd3 via its DD and ß-sheet domains.
The luminal domain of Hrd3 is primarily made up of short helices and loops that collectively form N-terminal, middle and C-terminal domains. Overall, Hrd3 adopts an inverted L-like conformation with inner and outer surfaces (16). The inner surface contains a groove (bottom panel) into which the polypeptide backbone of a substrate could bind. The MRH domain partially makes up the wall of this groove and is positioned in such a way to bind the glycan moiety of the substrate. The flexibility of the MRH domain allows substrate entry into the groove of Hrd3 and thereby guiding it into the Der1-Hrd1 complex.
Summarizing conclusion
Secretory proteins comprise about 20% of a typical eukaryotic proteome (1,2.3). These are produced by ribosomes associated with ER membrane, representing the so-called rough ER. Thus, most proteins of the secretory pathway initially transit the ER and are only allowed to leave after they have attained their native structure (5,6). This means that the ER is a major protein folding compartment that houses a suite of molecular chaperones and other folding factors that collectively monitor the folding of polypeptides and ensure the removal of proteins that fail to fold properly. These aberrant proteins are transported back from the ER lumen into the cytosol and are subsequently degraded by the ubiquitin-proteasome system. This process is known as ERAD and is essential for cellular health (5,6). Based on its importance, it is not surprising that, in humans, ERAD is currently associated with the pathogenesis of more than 60 diseases, including Alzheimer’s, Parkinson’s, Huntington’s disease and ALS (7). Moreover, ERAD is hijacked by certain viruses and toxins. Mechanistically, ERAD is not fully understood, although it is clear that it involves three main steps. These are: (i) recognition and processing, (ii) retrotranslocation and simultaneous ubiquitination and (iii) extraction and degradation of the substrate. Ubiquitin ligases play an essential role in all ERAD pathways because they catalyze ubiquitylation of substrates and associate with different partners forming complexes that orchestrate ERAD at both sides of the ER membrane as well as inside the lumen (5,6). Importantly, detailed structures of the yeast Hrd1 ubiquitin ligase complex were reported and based on these a model for the ERAD pathway of luminal substrates (ERAD L) is presented in figure 6 (adapted from 11).
In this model, ERAD L is initiated by binding of the substrate’s glycan to the MRH domain of Yos9 (stage 1), while the polypeptide downstream of the glycan binds into the groove of Hrd3. This requires the polypeptide to be in an extended conformation. The next segment of the polypeptide inserted into the luminal cavity of Der1. This dual recognition ensures that only terminally misfolded proteins are targeted for ERAD L. Conceivably, luminal chaperones may assist in substrate recruitment. Loop insertion (stage 2) would be initiated by the transfer from the Hrd3 groove into the Der1-Hrd1 complex. This presumably occurs as a hairpin with one strand interacting with Der1 and the other with Hrd1. Subsequently, the aforementioned leaves the lateral gate of Der1, thereby attaining a transmembrane orientation (stage 3). Opening of this gate requires reorientation of TMD5. Once inserted as a loop, a suitable lysine emerges into the cytosol and is polyubiquitinated next (stage 4). This serves as recognition signal for the Cdc48 complex, which pulls the substrate into the cytosol. In this model, polypeptide substrates don’t cross the ER membrane via a protein-conducting channel such as the Sec61 translocon. Instead, the substrate is retrotranslocated by two half channels formed by the luminal cavities of Der1 and Hrd1 within a thinned membrane region, which lowers the energy barrier for membrane partitioning.
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