Many proteins pass through the Sec61 translocon en route to their ultimate subcellular or extracellular destinations. Potentially, ERAD substrates may also pass through this channel in a retrograde fashion en route to proteasomal degradation. Aberrant, translocationally stalled proteins might limit flux through the channel in either direction, and eukaryotes may therefore have evolved mechanisms for eliminating such obstructions. Although Deg1-Sec62 (like many heavily studied model ERAD substrates) is an artificial protein, it may illuminate a previously unappreciated protein quality-control pathway mediated by the Hrd1 ubiquitin ligase and serve as a prototype for proteins that aberrantly engage or occupy the translocon. Indeed, we find that clearance of apoB, a protein previously reported to be cotranslocationally degraded, also depends on Hrd1.
Deg1-Sec62 inserts into the ER membrane in two distinct steps. Initially, the two TM segments of Deg1-Sec62 are probably cotranslationally translocated to yield the same topology as WT Sec62. In a second, Deg1-dependent step, a loop within the normally cytoplasmic N-terminal domain of Deg1-Sec62 penetrates the ER membrane. This rearrangement allows access of previously cytoplasmic residues to the N- and O-glycosylation machinery of the ER lumen. Although glycosylation is not required for Hrd1-mediated degradation, the switch in degradation dependence from Doa10 to Hrd1 tracks very closely with acquisition of these PTMs. Our data strongly suggest that the topological rearrangement across the ER membrane is the crucial event dictating E3 specificity ().
Figure 8. Model for Hrd1-dependent ERAD of Deg1-Sec62. (A) After its initial cotranslational translocation, Deg1-Sec62 aberrantly engages the Sec61 translocon, resulting in PTT of the normally cytoplasmic N-terminal Sec62 tail. After membrane penetration, Deg1 (more ...)
Other work supports the 4-TM model of rearranged topology (). Potential O-mannosylation sites (serines) were systematically introduced throughout Deg1
-Sec62 (Scott and Schekman, 2008
). We inferred that residues that became O-mannosylated reached the ER lumen; nonmodified serine substitutions were judged to be in the membrane-spanning or cytoplasmic segments. O-mannosylation of serines inserted at positions 132 and 170 confirmed that this span is present within the ER lumen (see ). Substitutions at other positions, such as immediately upstream of the first known TM, did not result in O-mannosylation. The most straightforward interpretation of these data is the 4-TM model of Deg1
-stimulated topological rearrangement (), although the failure to observe O-mannosylation of a particular serine does not exclude its luminal residence (Lommel and Strahl, 2009
Topological rearrangement of Deg1
-Sec62 likely occurs via Sec61-mediated PTT. Disrupting contacts of Deg1
-Sec62 with the translocon or impairing PTT by Sec61 mutation prevents domain dislocation and significantly reverts degradation dependence to Doa10 ( and ). Additionally, a cysteine found within the initially cytoplasmic N-terminal domain of Deg1
-Sec62 forms a transient disulfide linkage with a cysteine on the interior of the Sec61 channel (Scott and Schekman, 2008
). This bond appears to facilitate the altered topology of the Sec62 N-terminal domain, as mutation of either participating cysteine delays Deg1
-Sec62 PTM and causes a partial switch to Doa10-dependent degradation ().
Fusion of Deg1
to Sec62 was not anticipated to trigger PTT of the normally cytoplasmic Sec62 N-terminal domain. MATα2, from which Deg1
is derived, is a soluble nuclear protein, and Deg1
fused to the isolated soluble Sec62 N-domain (residues 1–149) does not trigger membrane translocation (Scott and Schekman, 2008
). Rather, Deg1
is degraded in a Doa10-dependent manner, behaving like other characterized soluble Deg1
fusions (Fig. S2 B). Potentially, fusing any protein sequence to the Sec62 N terminus could promote the observed topological rearrangement. This is not the case, however, as Deg1
bearing a 20-residue internal deletion does not promote PTM (or degradation) when fused to Sec62 (Scott and Schekman, 2008
, by virtue of novel protein–protein and/or protein–membrane interactions stimulated by enforced proximity to the translocon, may conformationally alter the N-terminal portion of Sec62 or cause it to linger persistently near the opening of the Sec61 translocon channel. The translocon could respond by attempting to conduct the Sec62 N-terminal tail into the ER lumen. The disulfide linkage between Deg1
-Sec62 and the Sec61 channel may strengthen the interaction of the Sec62 N-domain with a signal sequence-binding site within the channel. Consistent with this, the cysteine cross-link is not strictly necessary for topological rearrangement but significantly accelerates it (; and Fig. S1 D).
Lateral release of true TM segments from the translocon into the plane of the lipid bilayer depends strongly on the hydrophobic character of the TM helices (Shao and Hegde, 2011
; Zimmermann et al., 2011
). The N-terminal loop of Deg1
-Sec62 does not include bona fide TM domains; thus, we speculate that the membrane-spanning polypeptide segments flanking the N-glycosylated luminal element resist lateral release from the translocon and persistently occupy the Sec61 channel. We propose that Hrd1 recognizes persistent translocon engagement by Deg1
-Sec62 after the aberrant translocation event. In this respect, Deg1
-Sec62 may resemble a translocationally stalled or arrested protein. Hrd1, by virtue of its reported association with the translocon (Schäfer and Wolf, 2009
), may be ideally poised to target such incompletely translocated proteins for ubiquitin-mediated degradation. It is also possible that the Sec61-dependent topological change in Deg1
-Sec62 generates a permanently misfolded TM protein or misassembled translocon complex that is recognized by Hrd1. Nevertheless, the cofactor requirements for Deg1
-Sec62 differ from other well-characterized Hrd1 substrates (), which is consistent with a unique mechanism for recognition and degradation.
has been found to destabilize other TM proteins in a partially Hrd1-dependent manner. Like Deg1
-Sec62, both Deg1
-Hmg1 and Deg1
-Hmg2 fusion proteins are strongly stabilized in the absence of Ubc7. Deletion of HRD1
partially impairs turnover of these proteasome substrates (Wilhovsky et al., 2000
). Whether these proteins are targeted in a mechanism similar to that targeting Deg1
-Sec62 remains to be determined.
The factors preventing Doa10 from recognizing Deg1-Sec62 remain enigmatic. After topological rearrangement of Deg1-Sec62, Deg1 remains on the cytoplasmic face of the ER membrane (). However, this posttranslationally translocated form of the fusion protein is resistant to Doa10-mediated proteolysis. The translocon-dependent rearrangement might alter the position or conformation of Deg1 such that it is inaccessible to the Doa10 complex. Preventing interaction of Deg1-Sec62 with the translocon causes a strong reversion to Doa10-dependent degradation (). Therefore, Deg1-Sec62 is not inherently unrecognizable by Doa10. Interfering with events downstream of translocon binding (e.g., inhibiting PTT or preventing Deg1-Sec62–Sec61 disulfide formation) also causes a significant switch to Doa10-dependent degradation (). Thus, Doa10 is capable of recognizing Deg1-Sec62 even when conditions allow initial interaction with the translocon. In these cases, Doa10 might target Deg1-Sec62 that is in complex with the translocon but has not yet undergone PTT or might recognize Deg1-Sec62 that has transiently dissociated from Sec61.
The discovery that abnormal translocon engagement precedes Deg1
-Sec62 degradation led us to hypothesize that cotranslocational proteolysis of apoB might occur by a similar mechanism. ApoB uses the translocon as a platform for progressive lipid binding. When lipid binding is inhibited, translocation into the ER lumen is slowed, and the protein is destroyed by the cytoplasmic proteasome (Fisher et al., 1997
; Pariyarath et al., 2001
). Earlier studies implied that degradation of a model variant of apoB occurred via the ERAD system in yeast (Hrizo et al., 2007
). Our results directly demonstrate Hrd1 dependence for apoB29 degradation in yeast (). The cofactor requirements for Deg1
-Sec62 and apoB Hrd1-dependent degradation, however, differed, which suggests that the details of E3-substrate recognition diverge for these substrates.
A previous study had led to the suggestion that Deg1
-Sec62 glycosylation and disulfide formation with Sec61 occur during Sec61-dependent retrotranslocation of a Doa10 substrate (Scott and Schekman, 2008
). Our results, however, strongly suggest that these events occur in the process of Hrd1 substrate generation. Given the general dependence of substrate retrotranslocation upon functional ubiquitin conjugation machinery (Biederer et al., 1997
; de Virgilio et al., 1998
), N-glycosylation of Deg1
-Sec62 in doa10Δ hrd1Δ
cells () indicates that such modification happens upstream of substrate selection. The disulfide cross-link between Deg1
-Sec62 and Sec61 was interpreted as representing a briefly stalled retrotranslocation intermediate, as preventing this disulfide from forming accelerated degradation. However, we found that the increased degradation rate of Deg1
-Sec62 in the absence of the disulfide linkage is caused by a switch from Hrd1-dependent degradation to comparatively rapid Doa10-mediated proteolysis (). Therefore, although our data establish a role for Sec61 in the biogenesis of a Hrd1 substrate, they do not directly implicate Sec61 as a retrotranslocon for Deg1
-Sec62, although this remains possible. It is tempting to speculate that, in a manner mirroring its membrane insertion, retrotranslocation of Deg1
-Sec62 occurs in two steps: extraction of the aberrantly rearranged portion from the Sec61 channel followed by removal of the normal TM segments, potentially also through Sec61. Similarly, the precise mechanism by which apoB is extracted from the ER membrane remains to be elucidated. Translocationally stalled apoB might simply be extruded back into the cytoplasm via retrograde transport through the translocon in which it stalls, or it might be transferred to another retrotranslocating complex.
We speculate that Hrd1 may play an important role in removing proteins that persistently engage the translocon in a process provisionally called ERAD-T (for “translocon associated”). ERAD-T substrates may include proteins that aberrantly stall in the translocon either because of an abnormality in the translocating protein (such as may be the case for Deg1-Sec62) or because of malfunction in the translocation process itself. Such substrates may be difficult to identify experimentally, as they likely represent a small subpopulation of any given translocated gene product. Artificial translocon-occupying substrates, such as Deg1-Sec62, may serve as a model for understanding such mechanisms. Other cellular pathways may have co-opted this quality-control pathway to mediate the regulated destruction of otherwise normal proteins. The low-density and very-low-density lipoprotein biosynthesis pathway provides a likely example of a protein—apoB—whose regulated degradation utilizes a basic cellular quality control mechanism. Future studies will seek to understand the mechanisms by which Hrd1 recognizes this potentially distinct class of substrates that persistently engage the translocon.