Doa10 is a membrane protein retrotranslocase in ER-associated protein degradation
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 ) . 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 TFoF1 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 ( Ubc6C87A ) was not ubiquitinated , but an N-terminal fusion of ubiquitin with inactive Ubc6 ( Ub-Ubc6C87A ) 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 . 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 Cdc48E588A 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-Ubc6C87A 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 ) . 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 . 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 ( Ubc6SybTM and SybUbc6TM , 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-Ubc6C87A , 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 Ubc6SybTM . Doa10-dependent monoubiquitination of Ubc6SybTM 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 . 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 ( Ubc6SybTM ) 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 . 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 Cdc48E588A , 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 .
For protein expression in E . coli , BL21-CodonPlus ( DE3 ) -RIPL competent cells ( Agilent ) were used . Where indicated , BL21 ( DE3 ) competent cells ( NEB ) were used instead . To express Doa10 in S . cerevisiae , we used a doa10 deletion strain derived from BY4741 . All sequences were from S . cerevisiae , except for SNARE proteins which were from rattus norvegicus . For expression of Doa10 , yeast cells were grown in synthetic complete medium containing 2% ( w/v ) Glucose and amino acid drop-out supplements at 30°C ( Formedium ) . An overnight culture ( grown for 24 hr ) was diluted 1:50 into fresh medium . After 24 hr of growth , expression was induced by addition of yeast extract , peptone and galactose to final concentrations of 1% , 2% , and 2% ( w/v ) , respectively . After 17–19 hr , the cells were harvested at 3000 x g , washed once with ddH2O , resuspended in a minimal amount of H2O and stored at −80°C . To prepare cell lysates , 150 g of cells were resuspended in 900 mL of cold H2O and incubated with 2 mM DTT for 15 min on ice . All subsequent steps were done at 4°C . The cells were pelleted at 3000 x g and resuspended in lysis buffer ( 20 mM HEPES/KOH pH 7 . 5 , 5 mM potassium acetate , 600 mM mannitol , 0 . 5 mM EDTA ) . PMSF ( 1 mM ) and Pepstatin A ( 2 µM ) were added freshly . The cell suspension was then added to a bead beating chamber ( total volume 300 mL ) filled up to 1/3 with zirkonia beads . Cells were lysed in a Bead Beater ( Biospec Products ) with cycles of 20 s on and 2 min breaks in between for 50 min . Beads were filtered off , and the lysate was centrifuged at 1500 x g for 10 min . The supernatant was subsequently centrifuged at 40 , 000 x g for 45 min in a Ti45 rotor . The pelleted crude membrane fraction was resuspended in 200 mL lysis buffer by douncing and again pelleted at 180 , 000 x g for 30 min . The pellet was resuspended in 40 mL buffer , frozen in liquid nitrogen , and stored at −80°C . The total protein concentration of the membrane fraction was determined using the Pierce660 nm Protein Assay ( Thermo Scientific ) . To purify SBP-tagged Doa10 , the membrane fraction was solubilized with 1 . 3% ( w/v ) GDN ( Anatrace ) at a protein concentration of 3–4 mg/mL in 20 mM HEPES/KOH pH7 . 4 , 300 mM potassium chloride , 0 . 5 mM TCEP , 5 mM magnesium acetate , supplemented with 1 mM PMSF and 1 Pierce cOmplete EDTA-free protease inhibitor cocktail ( Roche ) per 100 mL solubilization volume . After 1 hr solubilization , insoluble material was pelleted at 40 , 000 rpm for 30 min in a Ti45 rotor . The supernatant was added to 4 mL Pierce High Capacity Streptavidin Agarose slurry ( Thermo Fisher Scientific ) and incubated for 3 hr . The beads were filtered off and washed with 4 × 25 mL of wash buffer ( 20 mM HEPES/KOH pH 7 . 4 , 150 mM potassium chloride , 5 mM magnesium acetate , 0 . 5 mM TCEP , 150 µM GDN ) . Doa10 was eluted with wash buffer supplemented with 2 mM biotin . Doa10 was further purified by sucrose density gradient ultracentrifugation . Gradients were prepared with two solutions where the less dense solution contained GDN ( solution A: 20 mM HEPES/KOH pH 7 . 4 , 150 mM potassium chloride , 2 mM magnesium acetate , 10% ( w/v ) sucrose , 0 . 5 mM TCEP , 100 µM GDN , solution B: 20 mM HEPES/KOH pH7 . 4 , 150 mM potassium chloride , 2 mM magnesium acetate , 25% ( w/v ) sucrose , 0 . 5 mM TCEP ) . Gradients were prepared using a gradient mixer ( Gradient Master , Biocomp Instruments ) at RT and kept at 4°C . 500 µL sample was loaded on top of the gradient . After centrifugation at 40 , 000 rpm for 19 hr in a SW41Ti rotor , the gradient was harvested in 500 µL fractions . Doa10-containing fractions were concentrated with Amicon Ultra Centrifugal Filters ( Merck ) using a 100 kDa cut-off . The same protocol was used for purification of SBP-tagged Doa10-N and Doa10-C . For bacterial expression , an overnight culture was diluted 1:50 into Terrific Broth and grown at 37°C . At an OD600 of 0 . 5 , the cells were shifted to 18°C and expression was induced with 0 . 5 mM IPTG . After approximately 20 hr of induction , cells were harvested at 4000 rpm , resuspended in buffer Im30 ( 50 mM Tris/HCl pH8 . 0 ( at 4°C ) , 500 mM NaCl , 30 mM Imidazole ) and stored at −20°C . Cells were lysed using a microfluidizer ( 17 , 000 psi , two passages ) . Immediately afterwards , 1 mM PMSF was added . Cell debris and unbroken cells were pelleted ( 1500 x g , 10 min ) . A membrane fraction was prepared by ultracentrifugation of the supernatant ( 40 , 000 rpm , 45 min , Ti45 rotor ) . The pellet was resuspended in buffer Im30 by douncing , frozen in liquid nitrogen , and stored at −80°C . Ubc6 and its variants were purified as described ( Vasic et al . , 2020 ) . To purify Ubc6-SBP , an additional purification step was included to ensure that only full-length Ubc6-SBP was purified . After size-exclusion chromatography , the protein was diluted to 0 . 5 mg/mL and bound to Pierce High Capacity Streptavidin Agarose ( Thermo Scientific ) . After washing the beads with buffer containing 20 mM HEPES/KOH pH 7 . 4 , 250 mM sodium chloride , 0 . 2 mM TCEP , 0 . 03% ( w/v ) n-dodecyl-β-D-maltopyranoside ( DDM , Carl Roth ) , Ubc6-SBP was eluted with buffer supplemented with 2 mM biotin . To purify SUMO-Ubc6 ( containing a C-terminal TEV cleavage site ) , protein was eluted from the Ni-NTA resin with buffer containing 50 mM Tris/HCl pH 8 . 0 ( at 4°C ) , 500 mM sodium chloride , 0 . 03% ( w/v ) DDM and 500 mM imidazole , and then further purified by size-exclusion chromatography ( Superdex 200 ) , as for Ubc6 . To purify Get3 , bacterial lysate was cleared by ultracentrifugation ( 40 , 000 rpm , 45 min , 4°C , Ti45 rotor ) and the supernatant incubated with Ni-NTA slurry ( 6 ml slurry for 6 L of culture ) for 2 hr . Beads were filtered off and washed with 4 × 50 mL buffer Im30 and 50 mL of buffer Im10 ( 20 mM Tris/HCl pH8 . 0 ( at 4°C ) , 200 mM NaCl , 10 mM Imidazole ) . Get3 was eluted from beads by cleavage with Ulp1 as described above . The elution fractions were supplemented with 1 mM DTT and further purified by size-exclusion chromatography using a Superdex 200 HiLoad16/60 column ( GE Healthcare ) equilibrated with 20 mM HEPES/KOH pH 7 . 4 , 200 mM NaCl , 1 mM DTT . Uba1 , Ubc7 , Cdc48 and Ufd1/Npl4 were purified as described ( Stein et al . , 2014 ) . Cue1 was purified as described ( Vasic et al . , 2020 ) . To express the t-SNARE complex , plasmids encoding syntaxin-1a ( aa 183–288 ) , synaptobrevin-2 ( aa 49–96 ) ( pETDuet-1 vector ) and SNAP-25A ( pET28a vector ) were co-transformed into BL21 ( DE3 ) E . coli cells ( NEB ) and expressed as described previously ( Stein et al . , 2007 ) . Briefly , at an OD600 of 0 . 5 , the cells were shifted to 18°C and induced with 0 . 5 mM IPTG . After approximately 20 hr of induction , the cells were harvested at 4000 rpm , resuspended in buffer Im8 ( 50 mM Tris/HCl pH8 . 0 at 4°C , 500 mM NaCl , 8 mM Imidazole ) and stored at −20°C . After cell lysis using a microfluidizer in the presence of 1 mM PMSF and Complete protease inhibitor , the lysate was cleared by ultracentrifugation ( 40 , 000 rpm , 30 min , Ti45 rotor ) . The pellet was solubilized in buffer Im8 supplemented with 5% ( w/v ) sodium cholate ( Sigma ) , 2 M urea , 200 mM sucrose and 1 mM PMSF ( 30 min , RT ) . Insoluble material was pelleted by ultracentrifugation ( 40 , 000 rpm , 30 min , 4°C , Ti45 rotor ) . HisPur Ni-NTA resin ( 6 mL for 6 L of culture ) was added to the supernatant and incubated for 3 hr at 4°C while rotating . Beads were filtered off and washed with 4 × 50 mL wash buffer ( 20 mM Tris/HCl pH 8 . 0 ( at 4°C ) , 500 mM NaCl , 8 mM imidazole , 200 mM sucrose , 2% ( w/v ) octyl glucoside ( OG , Glycon Biochemicals ) ) . Protein was eluted with wash buffer supplemented with 400 mM Imidazole . 1 mM DTT and 0 . 05 mg/mL of thrombin ( 100x stock prepared in 50% ( w/v ) glycerol ) were added to the elution fractions and incubated at 4°C overnight . The solution was then diluted to a conductivity of 15 mS/cm with buffer A ( 20 mM Tris/HCl pH7 . 4 ( RT ) , 1 mM DTT , 200 mM sucrose , 2% ( w/v ) OG ) . The protein was further purified by ion exchange chromatography on a MonoQ column ( GE healthcare ) equilibrated with 20 mM Tris/HCl pH7 . 4 ( RT ) , 150 mM NaCl , 1 mM DTT , 200 mM sucrose , 2% ( w/v ) OG and eluted in a gradient until 450 mM NaCl ( elution at ~25 mS/cm ) . ATP synthase ( from Bacillus PS3 ) was expressed and purified as described previously ( Schenck et al . , 2009; Suzuki et al . , 2002 ) . Shortly , ATP synthase was purified in the detergent DDM via a His10-tag attached to the β subunit . After Ni-NTA affinity chromatography , the dialysis step was omitted and the sample was directly further purified via ion exchange chromatography ( MonoQ ) . Detergent was exchanged to GDN subsequently by size exclusion chromatography ( Superose 6 16/60 , equilibrated with 10 mM HEPES , 100 mM KCl , 5 mM MgCl2 and 50 µM GDN , pH 7 . 4 ) . The protein was stored at 4°C for up to two weeks . To express Syb , a plasmid encoding His6-thrombin-Syb was transformed into BL21 ( DE3 ) E . coli cells ( NEB ) . Expression and preparation of a membrane fraction were done as described above , in buffer Im15 ( 50 mM Tris/HCl pH 8 . 0 , 500 mM NaCl , 15 mM imidazole ) . The membrane fraction was solubilized in buffer Im15 supplemented with 2 . 5% ( w/v ) sodium cholate for 30 min . After ultracentrifugation , solubilized material was incubated with Ni-NTA slurry ( 6 mL for 6 L culture ) for 3 hr . Beads were filtered off and washed with 2 × 50 mL wash buffer Im15 supplemented with 1 . 5% ( w/v ) sodium cholate and subsequently with 4 × 50 mL wash buffer Im15 supplemented with 5 mM decylmaltoside ( DM , Glycon Biochemicals ) . Protein was eluted with buffer Im15 containing 400 mM imidazole and 5 mM DM . The solution was dialyzed overnight against 10 mM MOPS , 50 mM NaCl , 1 mM DTT , 1 mM EDTA pH 7 . 0 ( 10 kDa MWCO ) , in the presence of 0 . 05 mg/mL thrombin . The protein was further purified by ion exchange chromatography on a MonoS column ( GE healthcare ) equilibrated with 10 mM MOPS , 50 mM NaCl , 1 mM EDTA and 1 mM DTT pH 7 . 0 and eluted in a salt gradient to 500 mM NaCl . Proteins were labeled at their C-terminal LPETGG tag with the previously described technique sortase-mediated transpeptidation ( Popp et al . , 2009 ) . A peptide with the sequence GGGC was labeled at its cysteine residue with a maleimide dye . Peptide dissolved in 100 mM HEPES/KOH pH 7 . 4 was added to dye ( DyLight680 maleimide or DyLight800 maleimide , Thermo Scientific ) in 1 . 5-fold molar excess . After labeling at RT for 2 hr , the reaction was stopped with 10 mM DTT . To label peptide with AlexaFluor 488 C5 Maleimide ( Thermo Scientific ) , peptide and dye were both dissolved in 100 mM HEPES/KOH pH7 . 4 and then mixed in a 1:1 molar ratio . A pentamutant P94R/D160N/D165A/K190E/K196T of SrtA from S . aureus was purified from E . coli ( Chen et al . , 2011 ) . To label proteins , 3-fold molar excess of labeled peptide , 10 mM CaCl2 and SortA were added to the protein . SortA was added to 1/7 of the total concentration of reactants ( peptide and protein ) . After labeling for 16–20 hr at 4°C , the reaction was separated by size-exclusion chromatography . All ubiquitination reactions were performed at 30°C in a thermocycler . The fusion reaction was diluted 1:2 ( f . c . of 0 . 1 µM Ubc6 , 0 . 01 µM Cue1 and 0 . 04 µM Doa10 ) . The following components were used at the indicated concentrations unless stated otherwise: 0 . 1 µM Uba1 ( E1 ) , 1 µM Ubc7 , 120 µM ubiquitin ( from S . cerevisiae , R and D Systems ) and 2 . 5 mM ATP . All reactions contained 0 . 1 mg/mL BSA . The ubiquitin mutant K0 ( Lifesensors ) is derived from human ubiquitin . Reactions were stopped by adding reducing SDS-sample buffer and samples were analyzed by SDS-PAGE . Proteoliposomes were prepared with protein-free liposomes containing 2 mol% biotinyl-PE and 0 . 5 mol% Rhd-PE . Fusion and ubiquitination was carried out as described above . To immobilize liposomes after ubiquitination , the ubiquitination reaction was diluted 1:2 to a final lipid concentration of 0 . 2 mM total lipid ( f . c . of 0 . 05 µM Ubc6 ) and BSA was added to a f . c . of 0 . 25 mg/ml . The diluted mix was then added to an equal volume of Pierce Streptavidin Magnetic Beads ( Thermo Scientific , prewashed with buffer B ) . After incubation for 1 hr at RT ( rotating ) , the unbound fraction was removed and the beads were subsequently washed 3 x with buffer B . The beads were then resuspended in the same volume of buffer B and 30 µL of the suspension aliquoted in a PCR-strip . The buffer was removed and the beads resuspended in 1 x extraction mixes or 1 x SDS sample buffer . 1 x extraction mixes contained 0 . 25 mg/mL BSA and where indicated 0 . 1 µM Cdc48 ( hexamer ) and 0 . 1 µM Ufd1/Npl4 . Beads were incubated for 30 min at 30°C . The supernatant was removed ( containing extracted and soluble proteins ) . After washing the beads 3 x with buffer B , the bound proteins were eluted by adding 30 µL of 1x SDS sample buffer . Samples of the supernatant and the elution fractions were analyzed by SDS-PAGE . To quantify the liposome immobilization efficiency , the Rhodamine fluorescence was measured in a Tecan Genios Pro microplate reader using 550/10 nm and 590/20 nm for excitation and emission , respectively . To quantify the protein immobilization efficiency , the DyLight680 ( Ubc6/Ub-Ubc6C87A ) fluorescence was measured using the Odyssey scanner ( 384-well plate , transparent bottom ) . To reconstitute Ubc6-SBP into proteoliposomes , Ubc6-SBP was preincubated with a 1 . 25-fold molar excess of tetrameric streptavidin ( NEB ) for 15 min at RT in the presence of 0 . 03% DDM to allow for complex formation . The reconstitution conditions were otherwise the same as for the co-reconstitution of Ubc6 and t-SNARE ( with 2 µM Ubc6-SBP and 2 . 5 µM Streptavidin ) . To assess the orientation of Ubc6-SBP in liposomes ( Figure 6—figure supplement 1B ) , a TEV protease protection assay was carried out as described above , except that liposomes were diluted 1:5 . A biotinylated nanobody was used for the biotinylated protein control . To test if the biotinylated nanobody ( anti-GFP , construct for expression kindly provided by Dirk Görlich; purified and biotinylated essentially as described in Pleiner et al . , 2018 is completely biotinylated ( Figure 6—figure supplement 1C ) , biotinylated nanobody was supplemented with 0 . 25 mg/mL BSA and incubated with magnetic streptavidin beads that were prewashed with buffer B or prewashed with buffer B supplemented with 10 mM biotin . After binding for 45 min at RT , samples of input and supernatant fractions were analyzed by SDS-PAGE and stain-free scanning using a GelDoc EZ Imager . For the antibody accessibility assay , the fusion reaction was first incubated with 1 . 5 µM biotin or biotinylated protein for 10 min at RT , before the fluorescence measurements were started as described above . A 6-fold molar excess of biotin/biotinylated protein over streptavidin was used . Fluorescence traces were processed and analyzed as described above . To check if biotinylated protein is capable of releasing Streptavidin from Ubc6-SBP , a flotation assay was used . Liposomes containing Ubc6-SBP and t-SNARE were incubated with Streptavidin for 5 min at RT ( f . c . 0 . 9 µM Streptavidin , 1 . 4 µM Ubc6-SBP ) . A 10-fold excess of biotin or biotinylated nanobody was then added and after incubation for another 5 min , a sucrose density gradient ( 40% ( w/v ) , 30% ( w/v ) , 15% ( w/v ) and buffer T layer ) was assembled . Flotation was carried out as described above . Samples were analyzed by SDS-PAGE and stain-free scanning using a GelDoc EZ Imager . Streptavidin and Ubc6-SBP levels were quantified using ImageJ . Intensity values for Streptavidin were normalized to Ubc6-SBP levels and subsequently to the buffer control . To measure extraction of Ubc6-SBP by the Cdc48-complex using the antibody accessibility assay , fusion and ubiquitination reactions were carried out as described above , except that liposomes were diluted 1:5 instead of 1:10 for the fusion reaction . After 30 min of ubiquitination ( total volume of 30 µl with f . c . 0 . 2 µM Ubc6-SBP , 0 . 08 µM Doa10 , 0 . 02 µM Cue1 , 0 . 1 µM Uba1 , 1 µM Ubc7 , 120 µM ubiquitin and 2 . 5 mM ATP ) , additional 2 . 5 mM ATP were added and the fluorescence measurement was started ( plate reader preheated to 30°C ) . After 10 min , anti-A488 antibody ( 1:15 diluted ) and 3 µL of 10 x Cdc48/UN mix were added . 10 x Cdc48/UN mix contained 2 µM Cdc48 ( hexamer ) , 2 µM Ufd1/Npl4 as well as 1 mM ATP . To quantify the fraction of extracted Ubc6-SBP , fluorescence traces were processed as described above and the difference between the normalized values of samples with and without Cdc48/UN 30 min after antibody addition was calculated . To test , if streptavidin stays in the liposome lumen during extraction ( Figure 6E , F ) , samples were floated after the extraction assay in a Nycodenz step gradient as described above . The samples were prepared with the following modifications: To ensure sufficient detection levels , the liposomes were diluted 1:4 for the fusion reaction . The ubiquitination reaction ( f . c . 0 . 3 µM Ubc6 , 0 . 12 µM Doa10 , 0 . 03 µM Cue1 , 0 . 1 µM Uba1 , 1 µM Ubc7 , 120 µM ubiquitin and 2 . 5 mM ATP ) was carried out as described above . After 30 min of ubiquitination , the extraction assay was performed in the plate reader as described above , in the presence of 0 . 3 µM Cdc48/UN complex ( f . c . Ubc6 = 0 . 25 µM ) . After 30 min , 2 mM biotin was added to 50 µL sample , a Nycodenz step gradient was assembled and the flotation carried out as described above . Samples were analyzed by SDS-PAGE and stain-free scanning using a GelDoc EZ Imager . Streptavidin levels were quantified using ImageJ . Intensity values were normalized to the sample without Cdc48/UN . Samples were mixed with SDS sample buffer ( stock used as 3 x contained 12% ( w/v ) SDS , 30% ( w/v ) glycerol , 0 . 05% Coomassie blue G-250 , 150 mM Tris/HCl pH 7 . 0% and 6% ( v/v ) ß-mercaptoethanol for reducing sample buffer ( Schägger , 2006 ) . Samples were heated at 70°C ( Streptavidin-containing samples were boiled ) and analyzed by SDS-PAGE using CRITERION TGX stain-free precast gels ( Bio-Rad ) . Fluorescent proteins were detected using an Odyssey scanner ( Li-Cor ) for DyLight680 and DyLight800-labeled proteins , and an FLA-700 fluorescence scanner ( Fujifilm ) for AlexaFluor488-labeled proteins . To detect streptavidin , samples were run on CRITERION TGX stain-free precast gels ( Bio-Rad ) and scanned with a GelDoc EZ Imager ( Bio-Rad ) . Colloidal Coomassie staining was used ( Dyballa and Metzger , 2009 ) . SBP-tagged proteins were analyzed by western blotting where indicated . After transfer on a nitrocellulose membrane using the Trans-Blot Turbo Transfer System ( Bio-Rad ) , the membrane was blocked with 5% skim milk powder ( dissolved in TBS-T ) for 1 hr at RT . A 1:2500 dilution of anti-SBP antibody ( clone 20 , mouse monoclonal , MAB10764 , Millipore ) , and a 1:15 , 000 dilution of secondary antibody ( goat anti-mouse , IRDye 800 CW or IRDye 680RD ) were used for detection . For the analysis of His-tagged ATP synthase , the nitrocellulose membrane was blocked with 2% ( w/v ) BSA ( dissolved in PBS containing 0 . 1% ( v/v ) Tween and 0 . 1% ( v/v ) Triton-X-100 ) . A 1:500 dilution of anti-His6 tag antibody ( Dia-900 , Clone13/45/31‐2 , mouse monoclonal , Dianova ) , and a 1:15 , 000 dilution of secondary antibody ( goat anti-mouse , IRDye 800 CW ) were used for detection . Antibodies were diluted in the respective blocking buffer . Gels were quantified using ImageStudio Lite ( Li-Cor ) . Fiji ( ImageJ ) was used for quantification of ubiquitin chain profiles ( plot profile function ) as well as streptavidin ( gel analyzer function ) ( Schindelin et al . , 2012 ) .
In endoplasmic reticulum-associated protein degradation ( ERAD ) , membrane proteins are ubiquitinated , extracted from the membrane , and degraded by the proteasome . The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates . How hydrophobic transmembrane ( TM ) segments are moved from the phospholipid bilayer into cytosol , often together with hydrophilic and folded ER luminal protein parts , is not known . Using a reconstituted system with purified proteins from Saccharomyces cerevisiae , we show that the ubiquitin ligase Doa10 ( Teb-4/MARCH6 in animals ) is a retrotranslocase that facilitates membrane protein extraction . A substrate’s TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase . Luminal substrate segments cross the membrane in an unfolded state . Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action . Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction .
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 .