Our results provide important insight into the mechanism of ERAD-L. We show that the ubiquitin ligase Hrd1p is the key membrane component for moving a misfolded protein across the ER membrane. This conclusion is based on the observation that the overexpression of Hrd1p bypasses the need for its interaction partners Hrd3p, Usa1p, and Der1p, whereas all downstream cytosolic components are still required. To function in ERAD-L, Hrd1p needs to form homo-oligomers, a process that is normally dependent on Usa1p, but can be induced in the absence of Usa1p by the overexpression of Hrd1p. Using a site-specific photocrosslinking approach, we demonstrate that endogenous Hrd1p interacts directly with a substrate undergoing ERAD. This interaction requires the presence of transmembrane segments of Hrd1p and is dependent on the delivery of substrate through other ERAD components. Unexpectedly, substrate interaction with Hrd1 on the luminal side of the ER membrane is also dependent on the ubiquitination activity of Hrd1p and on the cytosolic Cdc48p ATPase complex. As discussed below, these results suggest a model for the mechanism by which a misfolded luminal protein is moved through the membrane.
Our results indicate that Hrd3p, Usa1p, and Der1p are all regulators of Hrd1p function. Hrd3p and Der1p are involved in substrate delivery to Hrd1p, whereas Usa1p serves both to recruit Der1p to Hrd1p and to induce Hrd1p oligomerization (). Usa1p facilitates Hrd1p oligomerization by interacting with Hrd1p through one domain (segment H) and interacting with another Usa1p molecule through another domain (segment U). Our data show that Usa1p does not significantly interact with substrate, consistent with it being a scaffolding protein (Horn et al., 2009
). Taken together with results in the literature (Carvalho et al., 2006
; Denic et al., 2006
; Gardner et al., 2000
; Gauss et al., 2006a
; Gauss et al., 2006b
; Horn et al., 2009
), we have now a fairly comprehensive picture of the domain structure of the Hrd1p complex components and their interactions (), although it is possible that some of these associations are not permanent, but rather induced by substrate or regulated in other ways. Upon overexpression of Hrd1p, none of the regulatory components is required, indicating that Hrd1p can spontaneously oligomerize and bind substrates on its own. Under these conditions, substrate selection is less specific (Denic et al., 2006
In wild type cells, Hrd3p is a crucial component for substrate delivery to Hrd1p. Our photo-crosslinking experiments indicate that the luminal domain of Hrd3p interacts with substrate and that in the absence of Hrd3p there is no transfer of substrate to Hrd1p. Hrd3p appears to collaborate with two alternative components to recruit substrate, either with Yos9p or Der1p, because only the deletion of both components abolishes all Hrd1p-substrate crosslinking. Dual delivery of substrate to Hrd1p was suggested before on the basis of co-immunoprecipitation experiments (Gauss et al., 2006b
). Yos9p recognizes a terminal α1,6-mannose residue on a carbohydrate chain attached to the substrate (Clerc et al., 2009
; Quan et al., 2008
). We found that Der1p directly interacts with substrate, but its precise role remains unclear. Because both Yos9p and Der1p are essential in ERAD-L, they must have non-redundant functions in addition to providing parallel pathways of substrate recruitment.
The photo-crosslinking experiments give us a snapshot of an early translocation intermediate that follows substrate recognition (). The data show that Hrd1 interacts with a ~12 amino acid region of the sCPY*-DHFR-HA substrate. The interacting segment starts about 30 amino acid residues downstream of the glycosylation site in the degradation signal and immediately precedes the DHFR domain (). The polypeptide likely interacts with Hrd1p close to the luminal side of the membrane, because the degradation signal is in contact with the luminal domain of Hrd3p, and the DHFR moiety is also in the ER lumen (Bhamidipati et al., 2005
). Thus, following recognition of the degradation signal and while still bound to Hrd3p, the adjacent C-terminal segment of the polypeptide chain interacts with Hrd1p. Given that the last four transmembrane segments of Hrd1p are required for substrate interaction, it appears that the polypeptide chain is inserted into the membrane-embedded parts of Hrd1p, likely as a loop. However, considering the length of the crosslinking region in the substrate, the polypeptide cannot be inserted deeply into Hrd1p, certainly not completely across the membrane. The N-terminal part of the substrate loop contacts Der1p (), suggesting that Der1p may play a role in inserting the polypeptide into Hrd1p.
Our data would be consistent with the assumption that the substrate interacts with a Hrd1p monomer, because the crosslinking yields were only moderately reduced in the absence of Usa1p, the component required for efficient Hrd1p oligomerization. Since the crosslinking efficiency is rather low, it is also possible that the substrate crosslinks to a small population of spontaneously generated Hrd1p oligomers. However, dissociation of the Hrd1p oligomer upon substrate binding would be consistent with the observation that blocking substrate flux through Hrd1p, either by mutation of its critical cysteine or by deletion of four of its transmembrane segments, makes Hrd1p oligomerization less dependent on the presence of Usa1p. We therefore propose that substrate and Usa1p have opposing effects on the oligomerization of Hrd1p.
Surprisingly, we found that the ubiquitin ligase activity of Hrd1p is required for an early interaction of substrate with Hrd1p. Because no part of the substrate has yet emerged on the cytoplasmic side of the membrane to become accessible to the ubiquitination machinery, these results suggest that Hrd1p modifies a target that is different from the substrate. The simplest possibility is that Hrd1p ubiquitinates another Hrd1p molecule, although this modification would not be expected to result in poly-ubiquitination since Hrd1p is stable in wild type cells. We do not have direct evidence for self-ubiquitination of endogenous Hrd1p, but it is well established that Hrd1p can modify itself upon overexpression or in the absence of Hrd3p (Bays et al., 2001a
; Carroll and Hampton, 2009
). Furthermore, the retro-translocation of two substrates that are not ubiquitinated themselves still requires the ubiquitination activity of the ligase (Bernardi et al., 2010
; Hassink et al., 2006
Our results also show that the activity of the Cdc48p ATPase complex is involved at an early stage of retro-translocation, likely the same that is dependent on Hrd1p ligase activity, given that the Cdc48p complex is generally recruited to ubiquitinated targets (Ye, 2006
). An attractive possibility is that the Cdc48p ATPase remodels the Hrd1p complex following self-ubiquitination, either changing its conformation or its oligomeric state, a model that would be consistent with known activities of Cdc48p in other processes (Ramadan et al., 2007
; Rape et al., 2001
; Ye, 2006
Based on our results, we propose a simple model for how a polypeptide is moved through the membrane. Because Hrd1p is the crucial component of the Hrd1p complex and needs to form oligomers, it may surround a polypeptide chain during its movement through the membrane. The polypeptide loop that is inserted into Hrd1p at the beginning of retro-translocation might simply be extended, with the transmembrane segments of the Hrd1p oligomer offering transient binding sites for the substrate inside the membrane. Because substrate and Usa1p have opposing effects on the oligomerization of Hrd1p, Hrd1p oligomers appear to be destabilized by substrate binding. The self-ubiquitination of Hrd1p and subsequent Cdc48p ATPase activity may be required for conformational changes of the Hrd1p-substrate complex. One possibility is that Hrd1p undergoes repeated cycles of Cdc48p- and ATP-dependent dissociation and Usa1p-dependent association, which may be coupled to cycles of substrate binding and release. Once the substrate loop has emerged on the cytosolic side of the membrane, it can be poly-ubiquitinated by Hrd1p and pulled out of the membrane by the Cdc48p ATPase complex. According to this model, Hrd1p alone would provide the conduit for a polypeptide through the ER membrane. Our photo-crosslinking data argue against the proposed role for the Sec61 channel in retro-translocation, but the participation of this or other components can only be formally excluded upon reconstitution of the process with purified proteins.
The proposed model could also apply to the degradation of proteins that have their misfolded domains inside the ER membrane (ERAD-M). Because Usa1p is not required for all ERAD-M substrates (Carroll and Hampton; Carvalho et al., 2006
; Horn et al., 2009
), one might assume that the binding of Hrd1p to different regions of the membrane-embedded substrate would promote Hrd1p oligomerization. Although many of the details of ERAD-L and -M remain to be elucidated, it is now clear that future work has to concentrate on Hrd1p and its regulation by the Cdc48p ATPase complex.