The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER

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Ubiquitination is a highly dynamic process carefully controlled by opposing Ub-conjugating and deconjugating activities. The p97 complex and the 19S cap of the proteasome, the two major Ub-dependent protein unfolding machines in the eukaryotic cytosol, employ both types of activity to regulate the fate of their substrates (Elsasser and Finley, 2005; Jentsch and Rumpf, 2007). In the case of the 26S proteasome, deubiquitination removes the impediment of attached Ub chains to allow the passage of substrates to the interior of the proteolytic core particle, accessible only through a narrow pore (Pickart and Cohen, 2004). Given the similarity between the p97 complex and the 19S cap of the proteasome, we hypothesized that p97 might make use of deubiquitinating activities in most, if not all pathways in which ubiquitinated substrates are engaged by this complex. The involvement of p97 in ER dislocation (Ye et al., 2001) inspired us to look for Ub-specific proteases that might directly interact with it, thus prompting our examination of YOD1, a UBX-domain-containing member of the otubain family of unknown function.

We showed that p97 associates with YOD1 by virtue of its UBX domain and that YOD1 participates in a p97 complex that also contains NPL4 and UFD1 (Fig. 1 and Fig. S1), involved in the dislocation of misfolded proteins from the ER (Ye et al., 2001, 2003). This complex also contains Derlin-1 and UBXD8 (Fig. 7), ER-resident components of the dislocation machinery (Lilley and Ploegh, 2004; Mueller et al., 2008; Ye et al., 2004). Importantly, a deletion variant of YOD1 (DUBX-GFP YOD1) that does not bind p97 still co-precipitated UBXD8 (Fig. 7 B). Thus, binding of UBXD8 is not mediated indirectly via p97 alone and therefore cannot be a mere consequence of YOD1 overexpression and unspecific retrieval of p97 interactors. The specificity of the UBXD8 binding is further supported by the fact that UBXD2, another UBX-domain containing interactor of p97 (Liang et al., 2006), does not interact with YOD1 (Fig. 7 B).

The catalytic role of YOD1 in dislocation is demonstrated by overexpression of a catalytically inactive form (YOD1 C160S), which almost quantitatively stabilized RI₃₃₂, a model substrate for ER dislocation. The magnitude of this dominant negative effect (Fig. 3 A) was comparable to that commonly seen upon complete proteasomal inhibition (Fig. S4). Similarly to RI₃₃₂, NHK and unpaired TCRa-chain, were profoundly stabilized by YOD1 C160S. The dominant negative effect was entirely dependent on the ER localization of the dislocation substrate: we saw no effect on protein stability once the ribophorin substrate was topologically uncoupled from the dislocation pathway.

We may thus place YOD1 in an ER-dislocation pathway, and suggest that the correct positioning of the substrate and of YOD1 itself relative to p97 are important for p97 function. Given its effect on both luminal and membrane-bound substrates, YOD1 must lie at or beyond the point where the relevant dislocation pathways converge, most likely between the dislocon and the proteasome, in agreement with its ability to associate with p97. It is still not known how many distinct dislocation pathways exist in the mammalian cell and whether or not they are assigned to particular substrate classes. Knockdown experiments with YOD1 or ataxin-3, another DUB implicated in protein dislocation from the ER, did not affect the degradation of several dislocation model substrates (Wang et al., 2004; Zhong and Pittman, 2006). At this point, we cannot assess whether the remaining levels of YOD1 that escape RNA silencing are sufficient to promote dislocation, or if p97-associated DUBs have overlapping or redundant functions. Several DUBs contain an UBX domain that could facilitate their binding to p97. The extent to which these other UBX-containing DUBs participate in dislocation remains to be established.

Is the arrest in dislocation imposed by YOD1 C160S indeed attributable to a deubiquitination defect? Cells expressing catalytically inactive YOD1 C160S accumulated polyubiquitinated RI₃₃₂ to a much higher degree than YOD1 WT cells (Fig. 6 A), suggesting that the loss of catalytic activity of YOD1 is responsible for the accumulation of p97-associated RI₃₃₂ in ubiquitinated form. In fact, this interpretation was substantiated by the observation that YOD1 modulates the ubiquitination status of not only RI₃₃₂, but also of other p97-associated substrates (Fig. 6 C). The identity of these p97-associated Ub conjugated adducts was not determined, but likely includes a multitude of different dislocation substrates. YOD1 WT reversed the ubiquitination of dislocation substrates trapped by stable association with an ATPase-deficient p97 mutant (p97 QQ), whereas expression of YOD1 C160S in the same cells stabilized the Ub-adducts even more (Fig. 6 C and Fig. S5 B). We conclude that YOD1 activity is required for the p97-driven dislocation of misfolded proteins, and we attribute the inhibitory effect exerted by YOD1 C160S to a failure to deubiquitinate p97-associated dislocation intermediates en route to the cytosol (Fig. S7).

In our model, p97 engages dislocation substrates by means of the UFD1/NPL4 adaptor as soon as they emerge in the cytosol and undergo E3-catalyzed ubiquitination (Fig. S7 I). We speculate that once the substrate is transferred to the p97 pore and threaded through p97’s axial channel, YOD1 trims the Ub chain on the associated substrate to remove Ub moieties that present a steric impediment to the threading process (Fig. S7 II). Based on the data presented, we do not know whether this mechanism requires the en bloc removal of the entire Ub chain, akin to Rpn11 (Verma et al., 2002), or if a processive trimming-type activity like Ubp6 (Crosas et al., 2006) is sufficient to allow such threading. In the presence of YOD1 C160S, the Ub chain on the dislocation intermediate persists, thus preventing the processive movement of the substrate towards the cytosol (Fig. S7 IIb). In this scenario, a stoichiometric quantity of arrested intermediates is sufficient to titrate a limited number of dislocons, resulting in a pronounced accumulation of misfolded proteins within the ER (Fig. S7 IIIb).

Given that AAA ATPases of the 19S cap can accommodate looped polypeptides (Lee et al., 2002; Liu et al., 2003), and that this capability is conserved even in distantly related prokaryotic AAA+ ATPases endowed with threading activity (Haslberger et al., 2008), single Ub modifications may well be tolerated by the p97 assembly. A mono- or oligo-ubiquitinated substrate that emerges from p97/cdc48 at the other end may then be subjected to Ufd2-like E4 activities known to associate with Cdc48 (Richly et al., 2005).  This would eventually extend the Ub chain to a length that is optimal for the transfer to proteasomal shuttling factors, such as Rad23/Dsk2 (Raasi et al., 2004; Richly et al., 2005). In this scenario, substrate dislocation is processively coupled to proteasomal degradation.  Genetic evidence in S. cerevisiae established that Cdc48 cooperates with numerous cofactors in the following order: NPL4/UFD1®Cdc48®Ufd2®Rad23/Dsk2 (Medicherla et al., 2004; Richly et al., 2005). The proposed threading mechanism is reminiscent of that used by many other pore-forming hexameric AAA+ ATPases (Hinnerwisch et al., 2005; Martin et al., 2008; Schlieker et al., 2004; Weibezahn et al., 2004). The presence of a central channel in p97 (DeLaBarre and Brunger, 2003; Huyton et al., 2003), as well as the observation that mutations in pore-located residues in both AAA domains of p97 severely compromise ER dislocation activity (DeLaBarre et al., 2006) is in excellent agreement with our model. The functional assignment of the Otu1, VCIP135, Ataxin 3, and YOD1 to p97-dependent cellular processes in yeast (Rumpf and Jentsch, 2006) and mammalian cells (Wang et al., 2006; Wang et al., 2004; Zhong and Pittman, 2006) suggests an evolutionary conserved role for DUBs in the context of p97/Cdc48 activity. Future work will be required to probe the proposed mechanism further and to address whether other biological activities that rely on p97 function employ deubiquitinating activities as well.

In conclusion, we have firmly placed YOD1 in the dislocation pathway at a point where several of the distinct mammalian dislocation pathways converge. Given the multiplicity of exit strategies, combined with the near-universal involvement of the UPS in targeting the extracted proteins for degradation, it will be interesting to see how many other deubiquitinating enzymes are involved. While the ER is in sharp focus as a compartment where dislocation occurs or is initiated, we should remain open to the possibility that other intracellular locations might participate as well, each with unique machinery dedicated to the task, including perhaps novel components of the UPS.