Protein biogenesis is a remarkably imperfect process. About one third of all newly synthesized proteins are presumably defective. Functional proteins that were damaged by heat, oxidizing conditions or toxic agents further increase the pool of aberrant polypeptides. Defective proteins are toxic to cells and must be properly taken care of. Accordingly, cellular chaperones identify and repair deviant proteins. When salvage is not possible, the ubiquitin-proteasome pathway (Fig. 1) eliminates the faulty elements. These so-called protein quality control (PQC) pathways are found in most cellular compartments. A major PQC pathway is found in the Endoplasmic Reticulum (ER) and prevents the accumulation of malfolded or unassembled proteins in the secretory pathway. Dysfunctions in this system lead to severe diseases and, in addition, some viruses highjack this system to establish themselves in the infected cell.


The Ubiquitin Conjugation System


Fig. 1: The ubiquitin proteasome pathway (UPS). Conjugation of ubiquitin (ub) to substrate proteins involves three classes of enzymes: Ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligases (E3). E1 activates ub in an ATP-dependent manner. E2’s transfer ub to lysine residues of substrate proteins. This occurs with the help of E3’s that possess substrate specificity and are the most heterogeneous class of enzymes in this pathway. Ub conjugation may occur in different modes (poly-ub or mono-ub). The type of ub-modification is recognized by ub-binding proteins and determines the fate of the substrate protein.


ER-associated protein degradation (ERAD) is an important part of the PQC system of the ER. It can be divided mechanistically into separate steps: First, misfolded proteins are detected within the ER-lumen, a step that most likely requires molecular chaperones as well as lectins that recognize N-linked glycans. Second, proteolytic substrates are targeted to and inserted into an aqueous transport channel, which remains to be identified. Third, substrates are transported back into the cytosol in a process termed protein dislocation. Fourth, a polyubiquitin chain is synthesized on the dislocated substrates. This step requires the action of membrane-bound components of the ubiquitin system. In yeast these are the ubiquitin-conjugating enzymes Ubc1, Ubc6 and Cue1 assembled Ubc7 and the ubiquitin ligases Hrd1/Der3 and Doa10. Fifth, the AAA-ATPase Cdc48/p97 mobilizes ubiquitin-conjugated substrates. Finally, ub-binding proteins help to transfer the substrates to the cytosolic 26S-proteasome for digestion.

Central to ERAD are ubiquitin ligases that are embedded in the ER-membrane. Recently, we have characterized two central ligase complexes on a molecular level and identified novel components with unexpected new functions. In particular, we have identified an ER-anchoring factor for the AAA-ATPase (Cdc48/p97/VCP). This Cdc48 receptor in the ER-membrane is called Ubx2 and associates with Hrd1/Der3 and Doa10. Moreover, we were able to show that the HRD-ligase integrates protein quality control on the luminal site of the ER-membrane with ubiquitin-conjugation and proteolysis by the 26S proteasome on the cytosolic surface of this compartment. Here, we have identified a novel factor, Yos9, that seems to recognize specific oligosaccharide structures on secretory proteins. Our current model of the function of the HRD-ligase is depicted in Fig. 2.


Ubx2 links the Cdc48/p97-Complex to Endoplasmic Reticulum Associated Protein Degradation

We and others have demonstrated that the AAA-ATPase Cdc48/p97 plays a crucial role in protein dislocation. However, the precise role in this transport step is not well characterized. In addition, it remains to be clarified how Cdc48/p97 is recruited to the ER-membrane. Using biochemical approaches we were able to demonstrate that the integral membrane protein Ubx2 mediates interaction of the Cdc48/p97-complex with the ub-ligases Hrd1/Der3 and Doa10. Ubx2 contains an UBX-domain that interacts with Cdc48/p97 and an additional UBA-domain. Both domains are located on the cytoplasmic surface of the ER and are separated by two transmembrane segments. In cells lacking Ubx2, the interaction of Cdc48/p97 with the ligase complexes is abrogated and in turn breakdown of ER-proteins is affected. Thus, protein complexes comprising the AAA-ATPase, the recruitment factor Ubx2, and one of the known ERAD ubiquitin ligases play central roles in ER-associated proteolysis. Furthermore, degradation of a cytosolic/nuclear protein, which is ubiquitinated by Doa10, is disturbed in absence of Ubx2. This demonstrates that different Cdc48/p97 dependent pathways converge at the ER-surface.


The Hrd1 ligase complex - a linchpin between ER-luminal substrate selection and cytosolic Cdc48 recruitment

A central ERAD component is the ubiquitin ligase Hrd1/Der3. We have recently developed methods to study extensively the interactions of yeast ER-membrane proteins by co-immunoprecipitation and co-purification. Using this assay, we were able to describe a complex of Hrd1/Der1 and its partner protein Hrd3 with the ER-membrane protein Der1. Our data imply that Hrd3 is the major substrate receptor of this heterogenic ligase complex in the ER-lumen. Although Hrd3 and Der1 bind to soluble substrate proteins independently, both proteins are essential to trigger substrate dislocation. At the cytosolic face of the ER the Hrd1-complex associates with the AAA-ATPase Cdc48/p97. Cdc48p binding depends, as expected, on Ubx2, but most importantly also on substrate processing by the Hrd1-complex, suggesting that ubiquitination precedes substrate mobilization by the Cdc48/p97-complex.


A complex of Yos9 and the Hrd1-ligase integrates ER-quality control into the degradation machinery

How are newly synthesized proteins, which are in the process of folding distinguished from terminally misfolded proteins? One answer to this question involves N-linked glycans. These sugar structures are attached to newly synthesized proteins en bloc. After the covalent linkage of these sugar moieties, they are ‘trimmed’ by glucosidases and mannosidases. This trimming is a slow process and proteins with incompletely trimmed glycans are protected from degradation. Thus, the slow trimming event provides a time window in which newly synthesized proteins can fold. Although key lectins that may recognize the terminal glycan structures have been identified, a connection that links them to the ERAD pathway remained elusive. Recently we identified an association between the ER quality control lectin Yos9 and Hrd3. This interaction ties both pathways together. We identified designated regions in the luminal domain of Hrd3 that interact with Yos9 and the ubiquitin ligase Hrd1. Binding of misfolded proteins occurs via Hrd3, suggesting that Hrd3 recognizes proteins which deviate from their native conformation while Yos9 ensures that only terminally misfolded polypeptides are degraded.


The HRD Ubiquitin Ligase Complex


Fig. 2: Current model of the function of the HRD-ligase. Substrates are bound to Hrd3 at the luminal surface of the ER-membrane (upper part). Yos9 checks for correct oligosaccharide trimming and only completely trimmed substrates are dislocated from the ER for ub-conjugation by Hrd1 (lower part).