The inside of a cell contains many different compartments called organelles, which are separated by membranes. Each organelle is composed of a unique set of proteins and performs specific roles in the cell. The endoplasmic reticulum, or ER for short, is an organelle where many proteins are produced. Most of these proteins are then released from the cell or sorted to other organelles. The ER has a strict quality control system that ensures any faulty proteins are quickly marked for the cell to destroy. However, the destruction process itself does not happen in the ER, so faulty proteins first need to leave this organelle. This is achieved by a group of proteins known as endoplasmic reticulum-associated protein degradation machinery (or ERAD for short).

To extract a faulty protein from the ER, proteins of the ER and outside the ER cooperate. First, an ERAD protein called Doa10 attaches a small protein tag called ubiquitin to the faulty proteins to mark them for destruction. Then, outside of the ER, a protein called Cdc48 ‘grabs’ the ubiquitin tag and pulls. But that is only part of the story. Many of the proteins made by the ER have tethers that anchor them firmly to the membrane, making them much harder to remove.

To get a better idea of how the extraction works, Schmidt et al. rebuilt the ERAD machinery in a test tube. This involved purifying proteins from yeast and inserting them into artificial membranes, allowing closer study of each part of the process. This revealed that attaching ubiquitin tags to faulty proteins is only one part of Doa10's role; it also participates in the extraction itself. Part of Doa10 resides within the membrane, and this ‘membrane-spanning domain’ can interact with faulty proteins, loosening their membrane anchors. At the same time, Cdc48 pulls from the outside. This pulling force causes the faulty proteins to unfold, allowing them to pass through the membrane.

Given these findings, the next step is to find out exactly how Doa10 works by looking at its three-dimensional structure. This could have implications not only for the study of ERAD, but of similar quality control processes in other organelles too. A build-up of faulty proteins can cause diseases like neurodegeneration, so understanding how cells remove faulty proteins could help future medical research.

The endoplasmic reticulum (ER) is a major site for protein folding and maturation in the endomembrane system of the eukaryotic cell. A conserved quality control pathway called ER-associated protein degradation (ERAD) removes misfolded, unassembled and mistargeted proteins from the ER into the cytosol where they are degraded by the proteasome (Christianson and Ye, 2014; Mehrtash and Hochstrasser, 2019; Ruggiano et al., 2014). ERAD thus contributes to protein homeostasis. Its malfunction results in ER stress (Hwang and Qi, 2018), and it has been linked to several human diseases (Guerriero and Brodsky, 2012; Qi et al., 2017). ERAD is part of the ubiquitin proteasome system. Studies in the yeast Saccharomyces cerevisiae identified two universally conserved membrane-embedded ubiquitin ligases that ubiquitinate ERAD substrates, Hrd1 (SYVN1 in human) (Bordallo et al., 1998; Hampton et al., 1996; Kikkert et al., 2004; Nadav et al., 2003) and Doa10 (TEB-4/MARCH6 in animals, SUD-1 in Arabidopsis thaliana) (Doblas et al., 2013; Hassink et al., 2005; Swanson et al., 2001). In higher eukaryotes, a larger variety of ubiquitin ligases plays a role in ERAD (Olzmann et al., 2013).

Substrates of ERAD can be soluble luminal proteins, or membrane proteins that either need to be moved across or extracted from the ER membrane. This process, termed retrotranslocation or dislocation, requires the AAA protein Cdc48 (VCP or p97 in animals) (Bays et al., 2001; Garza et al., 2009; Jarosch et al., 2002; Nakatsukasa et al., 2008; Rabinovich et al., 2002; Ye et al., 2001). Cdc48 is recruited to substrates by its cofactors Ufd1 and Npl4 which interact with polyubiquitin chains with lysine 48 linkage (Meyer et al., 2002; Ye et al., 2003). The Cdc48 complex is thought to generate a pulling force that drives extraction of polyubiquitinated proteins from the membrane. This notion is based on biochemical experiments with soluble proteins (Bodnar and Rapoport, 2017; Olszewski et al., 2019) and recent cryo-EM structures (Bodnar et al., 2018; Cooney et al., 2019; Twomey et al., 2019), which showed that processive threading of a substrate through the central pore of the Cdc48 hexamer under ATP consumption leads to unfolding.

Apart from the Cdc48 ATPase, membrane proteins of the ERAD machinery are thought to contribute to retrotranslocation. For soluble substrates, Hrd1 forms part of a retrotranslocon pore from the ER lumen to the cytosol (Baldridge and Rapoport, 2016; Carvalho et al., 2010; Stein et al., 2014; Vasic et al., 2020), but other components such as the Derlin Der1 are also involved, as shown by biochemical data and a recent cryo-EM structure of the Hrd1 complex (Mehnert et al., 2014; Wu et al., 2020). Less is known about retrotranslocation of membrane proteins. The machinery that mediates this process needs to be quite versatile, because substrates can exhibit different topologies. They may contain one or multiple transmembrane (TM) segments, stretches of hydrophilic amino acids in luminal loops and tightly folded domains. How these structurally and physicochemically diverse elements move across a phospholipid bilayer during the extraction process is not known. Multipass transmembrane proteins such as members of the Derlin family (Der1 and Dfm1 in yeast; Derlin-1,–2 in animals), Hrd1 and Doa10 have been suggested to act as retrotranslocases for membrane proteins (Carvalho et al., 2010; Hampton and Sommer, 2012; Lilley and Ploegh, 2004; Neal et al., 2018; Swanson et al., 2001; Ye et al., 2004).

Another unresolved but linked question regards the folding state of luminal domains during the retrotranslocation process. It is unclear whether luminal domains are moved across the membrane in a folded state, if unfolding occurs prior to retrotranslocation, potentially by a separate ER luminal machinery, or if unfolding is directly coupled to retrotranslocation (Brodsky, 2012; Shi et al., 2019).

To address these questions, we investigated ERAD mediated by the ubiquitin ligase Doa10 from S. cerevisiae. Doa10 is a 150 kDa protein with 14 TM segments (Kreft et al., 2006). Its substrates include single- and multi-spanning membrane proteins of the ER and inner nuclear membrane, but also soluble proteins of the cyto- and nucleoplasm (Ravid et al., 2006). No completely soluble, luminal substrates of Doa10 have been described. The degrons of Doa10 substrates can be cytoplasmic (Furth et al., 2011; Swanson et al., 2001), or within the TM region (Habeck et al., 2015). Furthermore, Doa10 regulates sterol metabolism in plants, fungi and animals by degrading squalene monooxygenase (Doblas et al., 2013; Foresti et al., 2013). Through degradation of mislocalized membrane proteins, Doa10 has a role in maintaining organelle identity (Dederer et al., 2019; Matsumoto et al., 2019; Ruggiano et al., 2016).

Doa10 works in concert with two ubiquitin conjugating enzymes (E2), the tail-anchored membrane protein Ubc6 and the soluble cytoplasmic protein Ubc7 (Swanson et al., 2001). Ubc7 is anchored to the ER membrane by Cue1 (Biederer et al., 1997). Experiments with soluble cytoplasmic protein fragments showed that Ubc6 and Ubc7 have different roles in the build-up of polyubiquitin chains. Ubc6 initiates ubiquitin chains by transferring the first ubiquitin moiety, whereas Ubc7 extends ubiquitin chains with mainly lysine 48 linkage (Weber et al., 2016). Importantly, Ubc6 is itself an unstable protein and degraded in a Doa10-dependent manner (Swanson et al., 2001; Walter et al., 2001). Similarly, Ubc6 homologues in plants and mammals have been shown to be unstable and are degraded by the proteasome (Lam et al., 2014). Interestingly, in case of the Arabidopsis thaliana Ubc6 homologue Ubc32, and the mammalian Ube2J1, Hrd1 was identified as the ubiquitin ligase involved (Burr et al., 2011; Chen et al., 2016).

Here, we developed a reconstituted system with purified proteins to investigate the role of Doa10 in membrane protein retrotranslocation. This system allowed us to mechanistically investigate membrane protein extraction, without relying on indirect read-outs of downstream reactions such as proteasomal degradation. We show that Doa10 is a membrane protein retrotranslocase. Furthermore, we show how Doa10 cooperates with the Cdc48 ATPase in the extraction of proteins with folded luminal domains.

We reconstituted purified and fluorescently labeled Doa10 and its substrate Ubc6 into separate liposome populations, together with complementary SNARE proteins (Figure 1A and Figure 1—figure supplement 1, A to F). The tail-anchored (TA) membrane protein Ubc6 was chosen as a model substrate, to limit the number of membrane proteins in our system and thus its complexity. To achieve efficient liposome fusion, we employed previously well-characterized engineered versions of rat SNAREs involved in synaptic vesicle exocytosis (Cypionka et al., 2009; Hernandez et al., 2012). Indeed, mixing of the two liposome sets led to SNARE-mediated co-reconstitution of Ubc6 and Doa10 (Figure 1—figure supplement 1G). This approach ensures that Doa10 and Ubc6 only interact in the phospholipid bilayer, avoiding non-native interactions that can occur when membrane proteins are mixed in the presence of detergents for co-reconstitution. Protease protection experiments showed that Doa10 was reconstituted mostly in the correct orientation (Figure 1—figure supplement 1H). For Ubc6, 45% was correctly oriented, another 45% wrong-side out oriented, and a minor fraction not properly membrane inserted (Figure 1—figure supplement 1, I to K).

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This file contains the quantification of fluorescently labeled Ubc6 (Figure 1D) as well as of Rhodamine-labeled lipids (Figure 1—figure supplement 2B).

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This file contains the quantification of the quenched fraction of Ubc6 in samples containing Doa10 compared to samples lacking Doa10, as shown in Figure 1F.

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This file contains the quantification of the TEV-protected fraction of SUMO-Ubc6 and Ubc6 shown in Figure 1H.

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We postulated that retrotranslocase activity of Doa10 facilitates release of a substrate into the aqueous solution, but that such an event should be energetically disfavored unless the membrane-released state was stabilized by chaperones and re-insertion prevented (Figure 1B). To test this hypothesis, we incubated Ubc6 liposomes with Get3, a chaperone for TA proteins (Mateja et al., 2009) that also interacts with Ubc6 (Figure 1—figure supplement 2A). Liposomes were then immobilized to separate soluble and membrane-bound proteins (Figure 1—figure supplement 2B). In the presence of Get3, 43 ± 4% Ubc6 was released from liposomes with co-reconstituted Doa10 (Figure 1C,D and Figure 1—figure supplement 2C). When co-reconstitution of Doa10 with Ubc6 was prevented by inhibiting SNARE-mediated fusion or when Ubc6 liposomes were fused with liposomes lacking Doa10, only 7–9% of Ubc6 were found in the soluble fraction, representing the fraction of Ubc6 sticking to the outside of the liposome surface. In the absence of Get3, or when we used a Get3 mutant defective in TA protein binding (Get3 I193D) (Mateja et al., 2009), we observed no, or drastically reduced release, respectively. Nucleotide hydrolysis was not required for Ubc6 release and an ATPase deficient mutant (Get3 D57N) behaved indistinguishably from wild-type (WT) Get3. This suggests that Doa10 allows for passive movement of its substrate Ubc6 out of the membrane.

Release of Ubc6 from the liposome membrane involves movement of the TM anchor and the luminally encapsulated C-terminus across the lipid bilayer. To directly measure exposure of the C-terminus upon retrotranslocation we used a Ubc6 variant labeled with an AlexaFluor488 (A488) dye at the C-terminus. An anti-A488 antibody quenches A488 fluorescence and reports on accessibility of the C-terminus (Figure 1E). In the absence of Doa10, we observed a sudden decrease in fluorescence by 50% upon antibody addition, corresponding to the fraction of wrong-side out protein that exposes its C-terminus on the outside of liposomes. Upon solubilization of liposomes, the antibody quenches the fluorescence of all A488 epitopes. However, in the presence of Doa10, the sudden decrease in fluorescence was followed by a slower decrease to about 10% of the original fluorescence signal within 30 min. Thus, in the presence of Doa10, the luminally-encapsulated part of Ubc6 becomes accessible to the antibody over time. Ubc6 and Doa10 need to reside in the same membrane as we observed only minor quenching above background when we used a mutant SNARE that only supports liposome docking (Figure 1E,F).

As an alternative read-out for retrotranslocation, we used a protease protection assay. To identify correctly oriented Ubc6 we used an N-terminal SUMO fusion (SUMO-Ubc6) and Ulp1 protease. To monitor retrotranslocation, we introduced a TEV protease cleavage site between the C-terminus of Ubc6 and the fluorescent dye. This cleavage site resides in the liposome lumen and would only become accessible upon retrotranslocation (Figure 1G). Ulp1 incubation resulted in a shift of correctly oriented protein in SDS-PAGE. We then added TEV protease and followed cleavage over time (Figure 1G,H). Wrong-side out SUMO-Ubc6 was completely accessible to TEV protease and was cleaved independently of the presence of Doa10 within 5 min. Strikingly, only in the presence of Doa10, Ulp1-cleaved, and thus right-side out Ubc6 was also accessible to TEV cleavage over longer incubation times, indicating retrotranslocation. In liposomes lacking Doa10, only a small fraction of Ulp1-cleaved Ubc6 was accessible to TEV protease, corresponding to the not properly reconstituted Ubc6. Together, Get3 capture, antibody accessibility, and protease protection assays show that Doa10 facilitates movement of the Ubc6 TM across the membrane into the aqueous phase. Comparison of the fraction of correctly oriented protein and the released fraction shows that retrotranslocation is very efficient in all three assays. Thus, Doa10 is a retrotranslocase.

Importantly, we also tested if another unrelated multipass membrane protein leads to Ubc6 retrotranslocation by destabilizing the lipid bilayer. To this end, we purified the TF_oF₁ ATP synthase from Bacillus PS3, which contains 20 TM segments (Guo et al., 2019). Using the same reconstitution protocol as for Doa10, we co-reconstituted ATP synthase with Ubc6 (Figure 1—figure supplement 3A,B) and then tested for retrotranslocation of Ubc6 using the antibody accessibility assay. Under these conditions, only a minor fraction of Ubc6 becomes accessible to the antibody (Figure 1—figure supplement 3C,D). We conclude that retrotranslocation of Ubc6 is not due to some non-specific perturbation of the membrane caused by any multipass TM protein.

The observation that traps such as Get3 or the antibody are sufficient to drive retrotranslocation to completion suggests that Doa10 allows membrane-inserted and retrotranslocated soluble states of Ubc6 to exist in an equilibrium (Figure 1B). In the absence of Get3 or the anti-A488 antibody, the membrane-embedded state of the substrate is energetically favored. Get3 or the anti-A488 antibody shift the equilibrium towards the soluble state by binding to retrotranslocated Ubc6. Thus, traps bind retrotranslocated Ubc6 and prevent reinsertion. In the cell, retrotranslocation requires Cdc48 activity (Garza et al., 2009; Nakatsukasa et al., 2008; Ye et al., 2001), suggesting that the pulling force generated by Cdc48 provides the directionality.

To test this directly, we next investigated membrane extraction of Ubc6 by Cdc48. As Cdc48 acts on polyubiquitin chains, we first reconstituted Ubc6 polyubiquitination. Degradation of Ubc6 requires its own E2 activity, the E2 Ubc7 and its adapter, the membrane-anchored Cue1 (Biederer et al., 1997; Swanson et al., 2001; Walter et al., 2001). Using the fusion system, we co-reconstituted Doa10 and Cue1 with Ubc6. When we added ubiquitin activating enzyme (E1), ubiquitin, ATP, and Ubc7, we observed robust polyubiquitination of Ubc6 (complete reaction, Figure 2A). In the absence of Ubc7 or Cue1, polyubiquitination was abolished, and we observed ubiquitin adducts of lower molecular weight (Figure 2A,B). These represent multiple monoubiquitinations, as the ubiquitination pattern was very similar when we used a ubiquitin mutant in which all lysines are mutated to arginine (Ubiquitin K0) and that thus cannot form ubiquitin chains (Figure 2—figure supplement 1A). Monoubiquitination also occurred in the absence of Doa10 (Figure 2—figure supplement 1B), but was enhanced in its presence (Figure 2—figure supplement 1C,D). Kinetics of transfer of the first ubiquitin onto Ubc6 were independent of the presence of Ubc7/Cue1 (Figure 2C), suggesting that monoubiquitination is a prerequisite for Ubc7-dependent polyubiquitination. This is indeed the case, as a catalytically inactive Ubc6 mutant (Ubc6_C87A) was not ubiquitinated, but an N-terminal fusion of ubiquitin with inactive Ubc6 (Ub-Ubc6_C87A) was a substrate for Ubc7-dependent polyubiquitination (Figure 2D,E and Figure 2—figure supplement 1E). Together, these results establish that after active site loading of Ubc6 with ubiquitin, Doa10 catalyzes Ubc6-monoubiquitination, followed by Ubc7/Cue1-dependent polyubiquitination (Figure 2F). These results agree with observations made in intact cells and with recombinant soluble fragments of Doa10 and Ubc6 (Walter et al., 2001; Weber et al., 2016). Furthermore, they indicate that our reconstituted system faithfully recapitulates the in vivo ubiquitination pathway for Ubc6.

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This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Figure 2A, as shown in in Figure 2C.

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This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Figure 2D, as shown in Figure 2E.

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This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Figure 2—figure supplement 1C, as shown in Figure 2—figure supplement 1D.

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Next, we tested for membrane extraction of polyubiquitinated Ubc6 by the Cdc48 ATPase. To this end, we immobilized Doa10/Ubc6 liposomes after the ubiquitination reaction, then incubated with Cdc48 and its co-factors Ufd1 and Npl4 (UN), and analyzed soluble and membrane-bound fractions (Figure 3A and Figure 3—figure supplement 1,A to D). We observed Cdc48- and ubiquitination-dependent extraction of Ubc6 (Figure 3A,B and Figure 3—figure supplement 1E,F). Extraction efficiency was dependent on the length of ubiquitin chains, with five ubiquitin moieties being minimally required (Figure 3—figure supplement 1G). In the presence of Cdc48/UN, 45 ± 17% of Ubc6 molecules with more than five attached ubiquitin moieties were extracted, compared to 15 ± 4% in the absence of the Cdc48 complex (Figure 3A,C). No extraction above this background was observed when either Cdc48 or Ufd1/Npl4 were omitted. Furthermore, ATP hydrolysis by the Cdc48 complex was necessary, as Cdc48_E588A was inactive. Polyubiquitin chains were required because we found no extraction above background when ubiquitination was performed in the absence of Ubc7 or less than five ubiquitins were attached (Figure 3A,C,D). Similar observations were made when we used Ub-Ubc6_C87A instead of WT Ubc6 to increase the efficiency of polyubiquitination (Figure 3B,C). Together, these observations show that the Cdc48 complex provides the driving force for the extraction of a polyubiquitinated membrane protein. We currently do not understand what limits the efficiency of Cdc48 mediated extraction in our assay. It is possible that a stabilizing chaperone or an accessory factor such as the Cdc48 co-factor Ubx2 would contribute to complete extraction (Neuber et al., 2005; Schuberth and Buchberger, 2005).

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This file contains the quantification of the fraction of extracted Ubc6 from three experiments as in Figure 3A,B, as shown Figure 3C,D.

It also contains the quantification of the immobilization efficiency of Ubc6 (Figure 3—figure supplement 1C) and of Rhodamine-labeled lipids (Figure 3—figure supplement 1D), as well as the quantification of the extraction efficiency of Ubc6 modified with different ubiquitin chain lengths (Figure 3—figure supplement 1G).

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To define structural elements in Doa10 important for its retrotranslocase activity, we generated two truncated versions of Doa10 that encompassed either only the N-terminal RING domain and the first two TM segments (Doa10-N), or the C-terminal part containing TM segments 3–14 (Doa10-C) (Figure 1—figure supplement 1,F to H). The sites of truncation were chosen based on the finding that in the yeast Kluyveromyces lactis, Doa10 is expressed as two separate polypeptides with similar boundaries (Stuerner et al., 2012). We then tested if those Doa10 variants retrotranslocate Ubc6 using the antibody accessibility assay. Doa10-C behaved similarly to full-length Doa10, whereas Doa10-N resulted in only minor quenching above background (Figure 4A,B). Corresponding observations were made when we tested for retrotranslocation using the protease protection assay (Figure 4C). These results show that TM segments 3–14 in Doa10 are sufficient to mediate retrotranslocation of Ubc6.

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This file contains the quantification of the quenched fraction of Ubc6 in samples containing Doa10 or its variants compared to samples lacking Doa10 from three experiments as in Figure 4A, as shown in Figure 4B.

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This file contains the quantification of the TEV-protected fraction of Ubc6 from three experiments, as shown in Figure 4C.

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This file contains the quantification of the quenched fraction of Ubc6 or its variants in samples containing Doa10 compared to samples lacking Doa10 from three experiments as in Figure 4D, as shown in Figure 4E.

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To test for structural elements in Ubc6 relevant for retrotranslocation, we generated mutants in which either its TM anchor or its cytoplasmic part were replaced with the corresponding segments of the TA protein synaptobrevin (Ubc6_SybTM and Syb_Ubc6TM, respectively). We then tested for retrotranslocation of these mutants using the antibody accessibility assay (Figure 4—figure supplement 1A,B). We only observed retrotranslocation of the Ubc6 TM, but not of the Syb TM (Figure 4D,E). A similar experimental setup also allowed us to exclude leakage or liposome rupture as the cause for antibody accessibility (Figure 4—figure supplement 1,C to E). Thus, the identity of the substrate’s TM segment is important for retrotranslocation.

Next, we tested how these mutations in the TM domains of Doa10 and Ubc6 affect ubiquitination of Ubc6. To specifically test for effects on polyubiquitination, we again used Ub-Ubc6_C87A, for which the initial ubiquitination steps (ubiquitin loading and monoubiquitination) are bypassed. Replacement of the Ubc6 TM with the TM of Syb mildly affected polyubiquitination as seen by the emergence of shorter ubiquitin chains in the case of the Syb TM (Figure 5A,B; Figure 5—figure supplement 1A,B). To test for effects of TM replacement on monoubiquitination of Ubc6, we compared Ubc6 and Ubc6_SybTM. Doa10-dependent monoubiquitination of Ubc6_SybTM was impaired (Figure 5C,D; Figure 5—figure supplement 1C,D), while E3-independent autoubiquitination of this mutant was unaffected (Figure 5—figure supplement 1E,F). Thus, the Ubc6 TM anchor contributes to the efficient Doa10-dependent ubiquitination of Ubc6, indicating a more efficient recruitment to Doa10.

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This file contains the quantification of the number of ubiquitins (n) transferred per Ubc6 or Ubc6_SybTM (Figure 5—figure supplement 1D) as well as the quantification of the number of total ubiquitin transferred from three experiments as in Figure 5C, as shown in Figure 5D.

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This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Figure 5E, as shown in Figure 5F.

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Moreover, efficient ubiquitination of Ubc6 requires the TM domain of Doa10. Ubc6 polyubiquitination by Doa10-N was less efficient compared to full-length Doa10 (Figure 5E,F). This was not due to impaired E3 activity, because co-reconstitution of Doa10-N and Doa10-C together restored ubiquitination to WT levels. Monoubiquitination in the absence of Ubc7 was similarly affected (Figure 5—figure supplement 2,A to C). We conclude that the Doa10 region that includes TMs 3–14 plays a role in ubiquitination of Ubc6. Both ubiquitination and retrotranslocation of Ubc6 are sensitive to changes in the membrane-embedded regions of Ubc6 and Doa10, indicating a specific interaction of Doa10 with the TM domain of Ubc6. Previous observations suggested that the identity of the TM segment played a role in substrate degradation (Habeck et al., 2015; Ruggiano et al., 2016; Walter et al., 2001). Our results show that the TM domain of Doa10 recognizes substrates and thereby contributes to the specificity of substrate selection. The observation that the mutant version of Ubc6 (Ubc6_SybTM) is still ubiquitinated to some extent suggests that other factors might contribute to substrate discrimination. Deubiquitinating enzymes have previously been shown to sharpen substrate selectivity in ERAD and might also play such a role in the context of Doa10-mediated ERAD (Zhang et al., 2013).

Substrates of Doa10 exhibit a wide range of topologies. They may contain multiple TM segments, such as the misfolded variants of the multi-spanning membrane proteins Pma1 and Ste6, called Pma* and Ste6*, respectively (Huyer et al., 2004; Wang and Chang, 2003), or luminal folded domains (Vashist and Ng, 2004). We next asked the question how the presence of an additional luminal polypeptide segment or an interaction with another luminal protein affects retrotranslocation. We appended a streptavidin binding peptide (SBP) to the C-terminus of Ubc6 (Ubc6-SBP), and formed a complex with streptavidin (Figure 6—figure supplement 1A,B). When we co-reconstituted this complex with Doa10, we observed no quenching over time upon antibody addition (Figure 6A,B). When we added biotin, which breaks the high affinity SBP-streptavidin interaction (Keefe et al., 2001), we observed Doa10-dependent quenching over time. This was only the case when biotin was used, but much reduced when we used a biotinylated protein, which is still capable of dissociating streptavidin from Ubc6-SBP on the outside of liposomes (Figure 6—figure supplement 1, C to E), but cannot pass the membrane. Together, this shows that a protein-protein interaction on the luminal side of the membrane, mimicking the presence of a folded domain, acts as an anchor and prevents retrotranslocation of Ubc6.

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This file contains the quantification of the quenched fraction of Ubc6 in samples containing Doa10 compared to samples lacking Doa10 from three experiments as in Figure 6A, as shown in Figure 6B.

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This file contains the quantification of the quenched fraction of Ubc6 in samples containing Cdc48/UN compared to samples lacking Cdc48/UN from three experiments as in Figure 6C, as shown in Figure 6D.

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This file contains the quantification of streptavidin in the top flotation fraction from three experiments as in Figure 6E, as shown in Figure 6F.

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Finally, we tested if this anchoring can be overcome by the Cdc48 complex. Liposomes containing Doa10 and Ubc6-SBP in complex with streptavidin were incubated with ubiquitination mix followed by the addition of Cdc48 complex and anti-A488 antibody. Retrotranslocation occurred depending on polyubiquitination and Cdc48 activity (Figure 6C,D and Figure 6—figure supplement 1F). In the absence of ubiquitin, when Ubc7 was omitted, or when we used the catalytically inactive Cdc48_E588A, no fluorescence quenching above background was observed. Importantly, streptavidin remained encapsulated in liposomes in reactions where Ubc6-SBP was extracted (Figure 6E,F). Thus, Cdc48 action on the cytosolic side of the membrane leads to dissociation of streptavidin from the SBP-tag in the liposome lumen. As this reaction entails the breaking of bonds that are comparable to the intramolecular interactions that keep a protein folded, we interpret the dissociation of the SBP tag from streptavidin as unfolding. Our results thus show that Doa10 retrotranslocates a luminal protein segment in an unfolded state. Cdc48, acting on cytoplasmic polyubiquitin chains, generates a mechanical force that results in luminal unfolding und drives retrotranslocation. The integrity of the membrane is maintained in this process.

During extraction of a protein from the membrane, an energetic barrier must be overcome that depends on the hydrophobicity of its TM domain (Botelho et al., 2013; Guerriero et al., 2017). Our results provide evidence that Doa10 contributes to overcoming the energetic barrier for membrane protein extraction. This is demonstrated by passive movement of a tail-anchored membrane protein into the aqueous phase in the presence of Doa10. In the absence of a folded luminal domain, factors such as Get3 or an antibody that trap the retrotranslocated state are sufficient to drive the reaction, bypassing the requirement for ubiquitination and Cdc48.

ERAD also entails retrotranslocation of less hydrophobic sequences, such as those in luminal loops or domains, across the hydrophobic core of the membrane. We show that, once dissociated from streptavidin, a 57 amino acid long luminal protein segment that includes the SBP-tag is retrotranslocated by Doa10 without markedly affecting retrotranslocation kinetics. Thus, Doa10 can also accommodate these less hydrophobic sequences in the retrotranslocation process.

A major unresolved question concerns the fate of luminal domains during retrotranslocation. Some studies suggested that luminal domains cross the ER membrane in a folded state, based either on the observation that substrates containing tightly folded domains were retrotranslocated in the first place or that they were detected in a folded state after retrotranslocation into the cytosol (Fiebiger et al., 2002; Petris et al., 2014; Shi et al., 2019; Tirosh et al., 2003). Others suggested that retrotranslocation requires unfolding of luminal protein segments, including reduction of disulfide bonds, prior to retrotranslocation (reviewed in Ellgaard et al., 2018). Some of these differences might be explained by the fact that different ubiquitin ligase complexes were involved that might have different requirements for retrotranslocation. Our data show that Doa10 does not accommodate a folded domain during retrotranslocation. Instead, unfolding of polypeptide segments on the luminal side of the membrane is a direct consequence of Cdc48 acting on cytoplasmically attached polyubiquitin chains.

It is unclear if chaperone-driven retrotranslocation of Ubc6 in the absence of ubiquitination or Cdc48 action also occurs in the cell. Genetic and biochemical experiments in yeast showed that degradation of TM domain containing Doa10 substrates, including Ubc6, strictly depends on Cdc48, Ufd1 and Npl4 (Foresti et al., 2013; Habeck et al., 2015; Huyer et al., 2004; Neuber et al., 2005; Ravid et al., 2006; Ruggiano et al., 2016; Wang and Chang, 2003). In the case of the strongly hydrophobic, multi-spanning Doa10 substrate Ste6*, fractionation experiments also showed that the Cdc48 complex is required for the retrotranslocation step (Nakatsukasa et al., 2008; Nakatsukasa and Kamura, 2016; Neal et al., 2018). However, these experiments do not exclude the possibility that a relatively mildly hydrophobic protein such as Ubc6 retrotranslocates into the cytosol in a Doa10-dependent, but ubiquitination- and Cdc48-independent manner. Speculatively, such a chaperone-stabilized cytoplasmic pool would not be a substrate for proteasomal degradation but rather for chaperone-assisted reinsertion into the ER membrane, and might therefore be difficult to detect. Furthermore, as chaperones would only be able to capture a substrate that has emerged from the membrane, we expect such chaperone-driven retrotranslocation to be sensitive to the overall hydrophobicity of the TM segment. Interestingly, several studies showed that chaperones play an important role in ERAD of membrane proteins at different stages of the ERAD process. In yeast, Hsp70 and Hsp40 chaperones promote ubiquitination of Ste6* and a misfolded variant of Pma1 (Han et al., 2007; Nakatsukasa et al., 2008). In mammals, the Bag6-Ubl4a-Trc35 chaperone complex facilitates ERAD of membrane proteins by stabilizing a soluble cytoplasmic state (Claessen and Ploegh, 2011; Claessen et al., 2014; Ernst et al., 2011; Wang et al., 2011; Xu et al., 2012). If such chaperones also act as a sink in a putative Cdc48-independent retrotranslocation process, remains to be determined.

Using a reconstituted system, we disentangled different ERAD subreactions, that is substrate recruitment, ubiquitination, retrotranslocation, and membrane extraction. This allowed us to identify two activities of Doa10: retrotranslocase and ubiquitin ligase. The interplay of Doa10 and the Cdc48 complex during the extraction process remains to be further explored. Studies showed that Cdc48 is recruited to the Doa10-complex via Ubx2 (Neuber et al., 2005; Schuberth and Buchberger, 2005). This recruitment is dependent on the ubiquitination activity of Doa10, suggesting that Cdc48-recruitment requires either autoubiquitination of Doa10 or substrate ubiquitination. Moreover, the Derlin Dfm1, which also interacts with Cdc48 through its carboxy-terminal SHP-box, has been shown to be required for degradation of Ste6* (Neal et al., 2018). Our experimental system is expandable and can be used to explore how Dfm1, Ubx2 or other factors affect retrotranslocation by Doa10 and Cdc48.

How exactly Doa10 facilitates release of proteins from the membrane is unclear. Structural information on Doa10 is necessary to further elucidate its mechanism of action. We speculate that TM segments access a binding site in Doa10 through a lateral gate. This might promote delipidation of TM segments and breaking of helix-helix interactions in multi-spanning membrane proteins. Quality control pathways for membrane proteins that require their extraction from the membrane exist not only in the ER, but also in other organelles. In the Golgi, mitochondria and chloroplasts, membrane proteins are removed from the organelle for proteasomal degradation in the cytosol. These processes are dependent on ubiquitination and Cdc48 (Heo et al., 2010; Ling et al., 2019; Schmidt et al., 2019; Tanaka et al., 2010). Moreover, extraction of membrane proteins also occurs by membrane-bound AAA ATPases that often have not only unfolding, but also proteolytic activity. In mitochondria, Msp1 and the FtsH-related AAA metalloproteases m-AAA and i-AAA are examples for membrane-bound AAA ATPases (Glynn, 2017). For m-AAA mediated extraction into the mitochondrial matrix, a contribution of the TM domain was shown, suggesting a retrotranslocase activity (Korbel et al., 2004; Lee et al., 2017). We propose that membrane-bound retrotranslocases generally contribute to AAA protein-driven extraction of membrane proteins.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Claudia C Schmidt

   Research Group Membrane Protein Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7642-7081

2. Vedran Vasic

   Research Group Membrane Protein Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2575-1006

3. Alexander Stein

   Research Group Membrane Protein Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   alexander.stein@mpibpc.mpg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2696-0611

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