Hul5 was previously shown to be a proteasome-associated ubiquitin ligase which promotes proteasome activity in vivo
and which is able to extend ubiquitin chains on proteasome-associated substrates in vitro
). Here we have shown that the hul5Δ
mutant exhibits accumulation of a partial degradation product of the Ura3-Pcl5 fusion protein. This protein is the product of incomplete proteasomal degradation, as evidenced by the observation that a chemical inhibitor of the proteasome inhibits the generation of the Ura3-Pcl5 partial degradation product and that proteasomal 19S ATPase subunit mutants exhibit increased generation of this partial degradation product.
ATP-dependent proteases in general, and the 26S proteasome in particular, possess an impressive capacity for degrading folded proteins (24
). Nonetheless, some proteins are refractory to complete degradation. By analogy with the few instances of naturally occurring partially processed proteasome substrates (18
) and with degradation of some artificial hybrid substrates (21
), one can surmise that generation of the incomplete proteasomal degradation product of the Ura3-Pcl5 fusion protein is due to the confluence of two factors: (ii) a relatively strongly structured protein domain, in this case Ura3, and (ii) a sequence that impairs effective pulling of the polypeptide into the proteasomal catalytic cavity, in this case a sequence within the N-terminal region of Pcl5. Regarding the first factor, we find that the hard-to-unfold GFP protein (30
), fused N-terminally to Pcl5, elicits incomplete processing even in the HUL5
wild-type background, underscoring the significance of the domain structure in incomplete versus complete degradation by the proteasome. Conversely, replacement of Ura3 with a transcription activation domain, Gcn4(62-202), assumed to have low thermodynamic stability, did allow full degradation of the fusion protein. The importance of the second factor, a sequence in the Pcl5 region adjacent to the stable protein moiety, is revealed by the finding that deletions in the Pcl5 N-terminal region of the GFP-Pcl5 fusion protein either diminish or abrogate the production of a degradation intermediate. This specific sequence at the Pcl5 N terminus might be akin to the GA repeat sequence of EBNA1, which in certain contexts interferes with processive proteasomal degradation (19
), possibly because it prevents the ATPases from exerting sufficient traction on the polypeptide to unravel the adjacent protein domain. There are no apparent simple repeat sequences in the Pcl5 N-terminal domain, but sequences that interfere with polypeptide transfer into the proteasome are not necessarily readily recognizable (45
Hul5's role could be to promote the processivity of the proteasome. Alternatively, given the rapid disappearance of the Ura3-Pcl5(1-53) processing product in the wild-type strain versus its slow turnover in the hul5Δ
mutant, Hul5 could be required for degradation of this processing product independently of the proteasome. This would be analogous to a role recently suggested by Kohlmann et al. for Hul5 in degradation of the partial proteasome degradation product from an artificial endoplasmic reticulum (ER)-associated degradation (ERAD) substrate (23
). However, several observations suggest that Hul5 acts in Pcl5 fusion protein degradation within the context of proteasome function rather than independently of it. First, if the Ura3-Pcl5(1-53) processing intermediate were a Hul5 substrate, then it should be unstable when synthesized by itself; however, the truncated Ura3-Pcl5(1-53) protein synthesized by itself is completely stable even in the presence of Hul5. Second, the Ura3-Pcl5(1-53) processing product is completely stable in the rpt2RF
proteasome mutant in spite of the presence of Hul5, although other proteasomal substrates, and notably the full-length Ura3-Pcl5 protein, are only partly stabilized in the rpt2RF
mutant. If the Ura3-Pcl5(1-53) processing product were a regular proteasome substrate, dependent upon the Hul5 ubiquitin ligase for its degradation, then one would expect to see only partial stabilization of Ura3-Pcl5(1-53) in the rpt2RF
mutant and a cumulative stabilization in the rpt2RF hul5Δ
double mutant, contrary to what was observed. Third, GFP-Pcl5 is partially processed in both wild-type and hul5Δ
mutant cells to a stable processing product but to different extents (~15% processing in the wild type versus ~70% in the hul5Δ
mutant), again indicating that Hul5 plays a direct role in the processivity of proteasomal degradation.
How then does Hul5 affect the processivity of proteasomal degradation? One possibility is that it functions as an ancillary structural component of the 19S “base” subcomplex, whose role it is to unfold the substrate and thread the unfolded polypeptide into the 20S catalytic cavity. Binding of Hul5 could promote activity of the ATPases by allosteric changes. However, the fact that the Hul5 C878A mutant is unable to complement the hul5Δ phenotype argues for a role for Hul5 ubiquitin ligase activity in processive Ura3-Pcl5 degradation.
What is the mechanistic role of the E4 ubiquitin chain elongation activity of Hul5 in proteasome processivity? The simplest possibility is that it is required for tethering a stalled substrate to the proteasome. Conceivably, when the proteasome processively degrades a multidomain protein, including one domain carrying a degradation signal, such as Pcl5, and one hard-to-unfold domain, such as Ura3, then stalled substrate molecules of the Ura3-Pcl5(1-53) type could, with a certain frequency, escape from the proteasome. Since this molecule has lost its degradation signal, once escaped, it would be completely stable. The role of Hul5, a proteasome-associated E4 activity, could then be to ubiquitinate the Ura3 moiety of the stalled substrate, thereby tethering it to the proteasome and enabling additional time to unfold the stable protein domain. Contradicting this simple model, however, is the observation that in the hul5Δ mutant, the processed product is still degraded, albeit with a half-life of about 20 min. In contrast, the directly synthesized Ura3-Pcl5(1-53) protein is completely stable. Similarly, the Ura3-Pcl5(1-53) degradation product generated in the rpt2RF mutant also appears to be completely stable. How then can the degradation of the Ura3-Pcl5(1-53) processing product in the hul5Δ mutant be explained? We cannot exclude the possibility that the processing product garners a molecular modification during its residency at the proteasome, which converts it into a proteasome substrate that is still slowly degraded in the absence of HUL5 but not in the rpt2RF mutant. An alternative possibility is that the Ura3-Pcl5(1-53) processing product remains tethered to the proteasome in the hul5Δ mutant due to the continuous pulling action of the 19S ATPases until the molecule is eventually unfolded and threaded into the catalytic cavity. In the rpt2RF mutant, since the proteasome has a weaker grip on the substrate due to reduced ATPase activity, the stalled partial degradation product could escape the proteasome and therefore be completely stabilized.
If the latter model holds true, then what is the role of Hul5's ubiquitin ligase activity if not to tether the substrate to the proteasome? Since unfolding of the tethered protein domain is one of the challenges confronting the proteasome with this type of substrate, we speculate that polyubiquitination of this domain by Hul5 may facilitate its unfolding. The presence of extended polyubiquitin chains, many times larger than the substrate, linked to different sites on the substrate structure might destabilize this structure by “entropic unfolding” (11
), i.e., subjecting it to physical pulling forces due to the entropy of the attached ubiquitin chains, causing destabilization and transient unfolding of the protein structure. The transiently unfolded polypeptide could then be captured by the 19S ATPases and threaded into the proteasome.
In the other instance of accumulation of a partially degraded processing product in the hul5Δ
mutant, fusion of the ERAD substrates CPY* and Sec61-2L to the Leu2 protein led to transient appearance of a Leu2-containing fragment in wild-type cells and to stable accumulation of this fragment in hul5Δ
mutant cells (23
). The authors' conclusion is that Hul5 contributes to the extraction of the transmembrane substrate from the ER membrane by promoting its interaction with Cdc48. Here we have shown that a cytoplasmic fusion protein substrate exhibits the same requirement for Hul5 and that processivity of that substrate's degradation is dependent on proteasomal ATPases as well. In light of our results, we suggest that the data obtained with ERAD fusion substrates (23
) are also consistent with Hul5-mediated polyubiquitination promoting “entropic pulling” of the membrane protein, similar to the mechanism proposed for Hsp70 function in protein translocation (4
It is striking that Hul5, which was formerly shown to generally promote proteasome activity (3
), was found in the present study and in the work of Kohlmann et al. (23
) to cause partial degradation of multidomain proteins. Of note, these last two studies used artificial fusion protein substrates, raising the question of whether this role of Hul5 also applies to natural multidomain proteins. It is possible that instances of natural multidomain proteins being processed rather than fully degraded in the hul5Δ
mutant await discovery. It is also possible, however, that proteins inherently prone to processing, due to a combination of a highly structured domain with an adjacent “low-grip” polypeptide sequence, are selected against, because polypeptides, such as Ura3-Pcl5, that transiently but consistently “jam” the proteasome even in the wild-type background are expected to be deleterious to the cell. Therefore, the main role of Hul5 may be to help processively degrade cellular proteins that have become hard to unfold due to cross-linking or aggregation rather than to their inherent structural properties.