Because the fusion was likely not the limiting factor in the fusion system, the question remains as to why encapsulated substrates were not efficiently retrotranslocated by Hrd1. This could have been caused by multiple factors, the most important of which are listed below:
1. Hrd1 was inactive or not in the correct oligomeric state.
2. The substrates were aggregated, oxidized, or not in a state conducive for retro-translocation.
3. Retrotranslocation occurred but was very inefficient.
4. Hrd1 may be insufficient for retrotranslocation of luminal substrates.
These possibilities are discussed in detail in the following sections.
4.3.1 Was Hrd1 inactive or in the incorrect oligomeric state?
It is unlikely that Hrd1 activity was the problem because Hrd1 autoubiquitinated to acceptable levels (approximately 60%) and the efficiency was independent of fusion (Figures 3.12, 3.24). Neither fusion nor the presence of SNAREs inhibited Hrd1 ac-tivity. Hrd1 autoubiquitination reached higher efficiencies when reconstituted with DMNG compared to DM, which was used in the fusion system (Figure 3.42A com-pared to Figure 3.12). However, when Hrd1 was reconstituted directly into CPY*
liposomes with DMNG, its autoubiquitination efficiency was very high, yet CPY* was inefficiently ubiquitinated (Figure 3.26).
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It is possible that Hrd1 was not in the correct oligomeric state for retrotranslocation.
In normal conditions, ERAD-L requires Usa1, which causes Hrd1 oligomerization and recruits Der1 (Horn et al., 2009; Carvalho et al., 2010). It is not clear if ERAD-L requires a higher oligomeric state of Hrd1 for retrotranslocation. However, it has been shown that the ability for Hrd1 to compensate for the deletion of the other components of the complex depends on its overexpression levels. When Hrd1 is very highly overexpressed from a Gal1 promoter, CPY* is degraded with kinetics similar to the wild-type strain (Carvalho et al., 2010). When Hrd1 is only modestly overexpressed, it shows an intermediate compensation for Hrd3 deletion (Plemper et al., 1999; Denic et al., 2006; Vashistha et al., 2016). It may be that spontaneous Hrd1 oligomerization requires a higher concentration in the absence of Usa1. In vivo, this effect could be due to other factors, such as the decreased affinity for substrates in the absence of Hrd3. In any case, Hrd1 is already in a heterogenous oligomeric state after purification in DMNG (Stein et al., 2014). I used a protein to lipid ratio of 1:2000 in retrotranslocation experiments, identical to what was used in the study by Baldridge and Rapoport (2016). I did not use higher Hrd1 concentrations because of leakage during fusion, which appeared to be dependent on the concentration of transmembrane domains in the liposomes (Figure 3.21).
4.3.2 Substrate aggregation
It appears likely that CPY* and the other ERAD-L substrates used in the fusion system irreversibly aggregated during reconstitution. First, it is important to keep in mind that ERAD does not efficiently degrade aggregated proteins, highlighting the importance of chaperones in keeping misfolded proteins in a soluble state before retrotranslocation (Needham et al., 2019). The main indication of aggregation is that outside-bound CPY* was inefficiently ubiquitinated by Hrd1 during fusion (Figure 3.27). After the first step of the encapsulation protocol, 70% of CPY* was accessible to proteinase K, but only roughly 30% was ubiquitinated by Hrd1. In contrast, when CPY* and PrA*
were added to the outside of Hrd1 liposomes, approximately 90% was ubiquitinated (Figure 3.34).
Experience in our lab indicates that CPY* aggregates within a couple of hours after dilution from 2.5 M urea. For this reason, encapsulation was always performed in the presence of 1 or 2 M urea. Reconstitution was performed as quickly as possible. How-ever, because the reconstitution protocol for optimal ΔN complex activity required dialysis for detergent removal, the time-factor could not be considerably shortened.
Chapter 4 Discussion
Even with reconstitution in the presence of urea, CPY* was inefficiently ubiquitinated (Figures 3.16, 3.18). I attempted to resolubilize aggregated luminal CPY* by includ-ing 3 M urea in the Nycodenz flotation (Figure 3.28). However, I did not observe any retrotranslocation in this case. It is not clear if the urea successfully penetrated into the lumen of liposomes or whether higher concentrations were required. Because PrA* behaved similarly to CPY* during purification, it likely also aggregated during encapsulation. The substrate H14-SUMO-sCPY*-GFP was apparently not in an ag-gregated state, because it could be cleaved by Ulp1 (Figure 3.15). However, it also was not ubiquitinated by Hrd1, despite the fact that it leaked to the outside during fusion (Figure 3.19). The tightly-folded SUMO and GFP flanking the sCPY* domain likely preserved solubility, but the substrate may have aggregated after cleavage of the SUMO tag by Ulp1.
It is possible that in the presence of liposomes, the exposed hydrophobic regions in CPY* and PrA* insert like an amphipathic helix into the bilayer. The may bury the degron that is likely required for initiation of retrotranslocation and ubiquitina-tion. Alternatively, the high protein concentration in the liposome lumen may cause irreversible aggregation. These factors may have contributed to why I was unable to encapsulate transmembrane domain-containing versions of CPY* (section 3.5.8).
4.3.3 Ideas for overcoming substrate aggregation
How can this be solved? Chemical chaperones such as glycerol or sucrose, or harsher denaturing agents like guanidine hydrochloride may help reduce aggregation. Addi-tionally, chaperones such as the Hsp70 Kar2 and its Hsp40 cofactors, which have been shown to be required for CPY* degradation in vivo (Nishikawa et al., 2001), may keep substrates in a soluble state. One issue with Hsp70 chaperones is that their affinity for substrates is relatively low, withKD values in the micromolar range (Bukau et al., 2000). This may be overcome by using high concentrations of chaperone. Alternatively, it may be possible to co-encapsulate a His-tagged chaperone along with the substrate.
Another possibility is to use inducible misfolded substrates. The advantage would be that encapsulation and fusion can be performed with a well-folded, soluble protein.
After fusion, the substrate would be induced to misfold in the lumen, where hopefully Hrd1 can capture it and initiate retrotranslocation before it has the chance to aggregate.
An elegant system of inducible misfolding was developed by the Crews lab, using the modified bacterial dehalogenase protein (HaloTag), which forms a covalent bond with chloroalkane ligands (Los et al., 2008). They synthesized a ligand that causes
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proteasomal degradation of HaloTag fusion proteins in vivo, and induces the UPR when fused to an ER-localized protein (Neklesa et al., 2011; Raina et al., 2014). Most importantly, the ligand is membrane-permeable, which would allow it to access the lumen of liposomes. Of course, a prerequisite would be that the HaloTag has to become a Hrd1 substrate upon ligand binding.
As an alternative, temperature-sensitive mutants of soluble cytosolic proteins have been developed, which are degraded by the UPS upon shifting to non-permissive tem-peratures (Schneider et al., 2018). The temperature-sensitive form of Ubc9 (Ubc9ts), has been shown to misfold at temperatures above 30 °C (Betting and Seufert, 1996;
Kaganovich et al., 2008). Interestingly, Hrd1 and Doa10 are involved in its degradation in the cytosol upon misfolding (Samant et al., 2018). Because temperature shifting is very straight-forward and rapid, it could be applied to induce misfolding of encapsu-lated Ubc9ts. It would have to be first tested whether ER-imported Ubc9ts is a Hrd1 substrate in vivo.
4.3.4 Retrotranslocation may have occurred but was very inefficient
Retrotranslocation experiments showed that approximately 25% of CPY*-H14 was ubiquitinated after fusion, while 18% was accessible to proteinase K before fusion (Fig-ures 3.17 and 3.18). Only 15% of CPY*-H14 was ubiquitinated in the inhibited fusion control. This means that 7-10% of CPY*-H14 was potentially retrotranslocated. It is possible that retrotranslocation is very inefficient after taking into account potential substrate aggregation and the lack of other components such as Der1. Further repeti-tions are required for more definitive conclusions. However, between experiments with slightly different reconstitution conditions, the amount of CPY* protected from pro-teinase K ranged between 80-90%, while ubiquitination after fusion was also variable, ranging between 10-20%. Therefore, I focused my attention on improving retrotranslo-cation efficiency. Because I was interested in understanding mechanistic details about the process, I required a more efficient reaction.
4.3.5 Is Hrd1 is insufficient for retrotranslocation?
The final possibility is that Hrd1 is insufficient for efficient retrotranslocation of luminal substrates. Perhaps other components in the Hrd1 complex are required to efficiently insert substrates into the retrotranslocon. One strong candidate for this role is Der1,
Chapter 4 Discussion
which interacts with luminal substrates with its transmembrane domains their way to Hrd1 (Carvalho et al., 2010; Mehnert et al., 2013). As Der1 is recruited to Hrd1 by Usa1 (Carvalho et al., 2006; Horn et al., 2009), a subcomplex of Hrd1/Usa1/Der1 would need to be reconstituted into the fusion system to test the effect of Der1 on retrotranslocation.