Distinct activation of an E2 ubiquitin-conjugatingenzyme by its cognate E3 ligasesItamar Cohena, Reuven Wienerb, Yuval Reissa, and Tommer Ravida,1

aDepartment of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, The Edmond J. Safra Campus, Jerusalem 91904, Israel;and bDepartment of Biochemistry and Molecular Biology, Institute for Medical Research Israel–Canada (IMRIC), Hebrew University School of Medicine,Jerusalem 91120, Israel

Edited by Aaron Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, and approved January 12, 2015 (received for review August13, 2014)

A significant portion of ubiquitin (Ub)-dependent cellular proteinquality control takes place at the endoplasmic reticulum (ER) ina process termed “ER-associated degradation” (ERAD). Yeast ERADemploys two integral ER membrane E3 Ub ligases: Hrd1 (alsotermed “Der3”) and Doa10, which recognize a distinct set of sub-strates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature ofthe ERAD system that entails differential activation of Ubc7 by itscognate E3s. We found that residues within helix α2 of Ubc7 thatinteract with donor Ub were essential for polyUb conjugation.Mutagenesis of these residues inhibited the in vitro activity ofUbc7 by preventing the conjugation of donor Ub to the acceptor.Unexpectedly, Ub chain formation by mutant Ubc7 was restoredselectively by the Hrd1 RING domain but not by the Doa10 RINGdomain. In agreement with the in vitro data, Ubc7 α2 helix muta-tions selectively impaired the in vivo degradation of Doa10 sub-strates but had no apparent effect on the degradation of Hrd1substrates. To our knowledge, this is the first example of distinctactivation requirements of a single E2 by two E3s. We proposea model in which the RING domain activates Ub transfer by stabi-lizing a transition state determined by noncovalent interactionsbetween the α2 helix of Ubc7 and Ub and that this transition statemay be stabilized further by some E3 ligases, such as Hrd1,through additional interactions outside the RING domain.

ubiquitin-proteasome degradation system | ER-associated degradation |protein quality control | ubiquitin E3 ligase | ubiquitin conjugating enzyme

The ubiquitin (Ub) conjugation machinery employs three basicenzymatic activities, E1, E2, and E3, that work in concert to

transfer Ub to client substrates and to form polyUb chains (1).Initially, an E1 Ub-activating enzyme forms a high-energy thio-ester bond with the C terminus of Ub, after which the Ub mol-ecule is transferred to the active-site Cys of an E2 Ub-conjugating(Ubc) enzyme. The Ub-charged E2 binds to an E3 ligase andcatalyzes the transfer of Ub to the e-amino group of a Lys sidechain within the substrate. Additional Ubs then can be ligated tothe initial Ub molecule through sequential ubiquitylation cycles,ultimately forming a polyUb chain. Ub can be conjugated to itselfvia specific Lys residues, resulting in diverse types of chain linkages.Linkage through Lys48 is linked primarily to substrate degrada-tion. Consequently, protein substrates carrying Lys48-linkedpolyUb chains bind to and are degraded by 26S proteasome.Although it is well established that E3 ligases activate Ub li-

gation by E2s via their RING domains, very little is actually knownabout the underlying regulatory mechanism. Several recent studiesdetermined the structure of RING domain complexes with Ub-charged UbcH5 (2–4). In one of these studies, the structure insolution of Ub-charged UbcH5c together with the mouse E3 ligaseE4B U-box domain revealed that Ub can adopt an array of “open”and “closed” conformations (2). The productive closed confor-mation promotes a nucleophilic attack on the Ub∼E2 thioester byan incoming Lys (acceptor) residue (2). A similar closed confor-mation was identified in the structures of UbcH5a and UbcH5b,together with their cognate RING domains (3, 4). Taken together,

these structural studies suggest that RING domains can catalyzeUb transfer by stabilizing a transition state of a closed confor-mation of the E2-bound (donor) Ub (5).Among the fundamental intracellular functions of the Ub–

proteasome system is maintenance of cellular protein qualitycontrol (PQC) by targeting a diverse array of transiently or per-manently misfolded substrates for proteolysis. A central branchof PQC degradation takes place in the endoplasmic reticulum(ER) in a process termed “ER-associated degradation” (ERAD)(6). Despite the multitude of misfolded substrates, ERADemploys only a few E3–ligase complexes (7). In fact, the bakers’yeast S. cerevisiae ERAD system employs only two Ub-ligationcomplexes, specified by their E3 ligase components, Hrd1 andDoa10 (8–12). Importantly, each of the two E3 ligase complexesrecognizes a distinct set of substrates, with minor overlaps (13).Degradation by the yeast ERAD Ub-ligation system entails

the combined activity of two E2 enzymes: Ubc6 and Ubc7 forthe Doa10 pathway and Ubc1 and Ubc7 for the Hrd1 pathway(14, 15). The shared E2 enzyme, Ubc7, is a soluble cytosolicprotein whose binding to either of the E3–ligase complexes atthe ER membrane is mediated by the auxiliary ER membraneprotein Cue1. Binding to Cue1 not only mediates the interactionwith the E3–ligase complex but also protects Ubc7 from deg-radation and stimulates its Ub-transfer activity (16–20). Ubc7 ishighly conserved in evolution, as evident from substantial se-quence and structure similarities with its orthologs from otherspecies (21). The human Ubc7 ortholog, Ube2g2 (21), functionstogether with several ER membrane-embedded E3 ligases, thebest characterized of which is the tumor autocrine motility

Significance

Ubiquitin (Ub) conjugation triggers protein degradation by theproteasome. Here we describe an unexplored feature of the Ubconjugation system that entails differential activation of an E2-conjugating enzyme by its cognate E3 Ub ligases. In vitro and invivo analyses of activity-reducing mutants of the yeast Ub-conjugating (Ubc) enzyme, Ubc7, demonstrated selective acti-vation by one of its two cognate E3 ligases, Hrd1, but not bythe other, Doa10. Supported by structural modeling of theRING:Ubc7∼Ub complex, these findings are consistent witha model whereby the E2∼Ub transition state depends onnoncovalent interactions between helix α2 of Ubc7 and Ub thatare differentially stabilized by the two E3 RING domains. Thisdifferential activation represents a previously unidentifiedmechanism for regulating protein ubiquitylation.

Author contributions: I.C., R.W., Y.R., and T.R. designed research; I.C., Y.R., and T.R. per-formed research; R.W. contributed new reagents/analytic tools; I.C., Y.R., and T.R. ana-lyzed data; and I.C., Y.R., and T.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: travid@cc.huji.ac.il.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1415621112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1415621112 PNAS | Published online February 2, 2015 | E625–E632

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factor receptor, gp78 (22). Ubc7 and Ube2g2 are subjected tosimilar regulatory mechanisms: They bind to and are activatedby the RING domains of their cognate E3s as well as by the E2-binding regions and CUE domains within Cue1 and gp78 (19,20, 23–26). The evolutionarily conserved sequence, structure,and regulatory mechanisms of the Ubc7 E2s imply an essentialphysiological function.In this study we explored the role of helix α2 of Ubc7 in en-

zyme activation. Based on our in vivo and in vitro observationsand on the available structural information, we propose a mech-anism whereby activation of Ubc7, mediated by noncovalent in-teraction with Ub at helix α2, is differentially affected by theRING domains of its cognate E3 ligases Hrd1 and Doa10.

ResultsLys118 Is Required for Ubc7 Activity.We have shown previously thatwhen the Ubc7 level exceeds that of its binding partner Cue1,Ubc7 undergoes auto-polyubiquitylation at the active-site Cysresidue and subsequent degradation (18). However, a smallfraction of Cue1-bound, monoubiquitylated Ubc7 was dithio-threitol (DTT) resistant, suggesting an isopeptide bond with aninternal Lys residue(s) (Fig. S1A). Restoring individual Lys residuesin a Lys-less Ubc7 (Ubc7KO) and testing for the reappearance ofmonoUb-Ubc7 detected a preferred single-ubiquitylation site atLys118 (Fig. S1B). To investigate whether Lys118 was importantfor Ubc7’s Ub-conjugating activity, we measured the stability ofthe Doa10 substrate Ura3-CL1, composed of a fusion between theURA3 gene product orotidine-5′-phosphate decarboxylase (Ura3)and the CL1 degradation signal (27, 28), in cells expressing eitherwild-type or mutant Ubc7 (Fig. 1A). Ura3-CL1 was barely detectedin cells with intact Ubc7. Because Ura3-CL1 was expressed from astrong inducible promoter, its low steady-state level likely reflectedits rapid degradation rate. Indeed, upon longer exposure of theimmunoblot reaction, Ura3-CL1 was visible in all tested strains(Fig. S2). Cycloheximide (CHX)-chase degradation analysis re-vealed a substantial increase in the Ura3-CL1 steady-state levelat time 0 that remained almost constant throughout the CHX-chase period in cells expressing either Ubc7KO or a Lys118-to-Arg Ubc7 (Ubc7R118) mutant. Restoration of Lys118 alone toUbc7KO was sufficient to reinstate the low steady-state level attime 0 and complete disappearance thereafter. These findingsimply that Lys118 of Ubc7 plays an essential role in Ub-mediateddegradation.To test the effect of Lys118 mutations on the intrinsic activity

of Ubc7 directly, wild-type and mutant Ubc7 were coexpressed inbacteria together with Cue1 lacking the transmembrane domain(Cue1ΔTM) (23) and was purified at an approximately 1:1 ratio(Fig. 1B). The Ubc7:Cue1 heterodimers subsequently were in-cubated in an in vitro ubiquitylation reaction that revealedDTT-resistant Ubc7–monoUb conjugates that, in contrast to theselective monoubiquitylation in vivo, were equally present bothin wild-type Ubc7 and Ubc7R118 (Fig. 1C). However, comparedwith wild-type Ubc7, Ubc7R118 assembled polyUb chains verypoorly after a 45-min ubiquitylation reaction (Fig. 1D). There-fore, we set out to explore the possibility that Ubc7R118 inhibitionis independent of Lys118 monoubiquitylation and instead is de-pendent on an alternative mechanism.

Determination of the Role of Lys118 of Ubc7 in Catalysis. Previousanalyses of E2 enzymes activity considered Lys residues spatiallyadjacent to the active site as potential effectors of the enzymaticreaction (29, 30). The Ubc7 crystal structure indicates thatLys118 is located in helix α2 that is remote from the active site(31). However, Lys118 was both essential and sufficient forDoa10-mediated degradation (Fig. 1A). Apparently, the UbcH5∼Ubstructure also reveals a noncovalent interaction between helixα2 and the donor Ub (2–4) that possibly exposes the Ub thioester

to nucleophilic attack by an incoming amine group and thuspromotes Ub transfer (2).To understand further the role of Lys118 in E2 catalysis, we

generated an in silico tertiary model of Ubc7, Doa10, RING, andUb (Fig. 1E). The model was based on a superimposition of theUbc7 crystal structure [Protein Data Bank (PDB) ID code4JQU] and a solution structure of the Doa10 RING domain(PDB ID code 2M6M) with the structure of the RING fingerprotein 4 (RNF4) RING:UbcH5B∼Ub complex (PDB ID code4AP4) (3, 19). The resulting in silico model predicted a hydrogenbond between the side chain of Lys118 and the backbone car-bonyl of Leu8 of Ub (Fig. 1E). The model also predicted a hy-drophobic interaction between the side chains of Leu121 ofUbc7 and Val70 of Ub. Parallel mutations in respective residuesof UbcH5, Lys101 and Leu104, to Ala inhibited the E2 activity,likely by abolishing a critical Ub interaction with helix α2 (2–4).

Fig. 1. Mutations in helix α2 inhibit Ubc7 activity. (A) Ura3-CL1 degradationin cells expressing Ubc7 mutants. Cells were collected at the indicated timeperiods after the addition of the translation inhibitor CHX. Ura3-CL1 wasvisualized by immunoblotting with anti-HA Abs. Probing the blot with anti-G6PD Abs provided a loading control. (B) Coomassie staining of purifiedUbc7 or Ubc7R118 shows equal amounts of copurified Cue1. Ubc7-2HA andCue1 were expressed in bacteria from a bicistronic plasmid and subsequentlywere purified using a chitin-intein tag (Materials and Methods). (C) Self-monoubiquitylation of Ubc7. Purified Ubc7:Cue1 dimers were incubatedwith E1, ATP, and FlagUbR48 for 45 min at 30 °C. Proteins were resolved bySDS/PAGE under reducing conditions and were detected by immunoblottingwith anti-Flag (Ub) or anti-HA (Ubc7) Abs. (D) PolyUb chain formation bywild-type Ubc7 or Ubc7R118. The ubiquitylation reaction was performed asdescribed in C using FlagUb. (E) A structural model for the Doa10 RING:Ubc7∼Ub complex. The model is based on a superimposition of Ubc7 (PDB IDcode 4JQU), Doa10 RING (PDB ID code 2M6M), and the RNF4 RING:UbcH5B∼Ubcomplex (PDB ID code 4AP4). The area within the rectangle is enlarged be-low. (F and G) Formation of polyUb chains by the indicated Ubc7 mutants.

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We thus hypothesized that, as with UbcH5, the reduced activityof Ubc7R118 was caused by aberrant helix α2∼Ub interaction andthat additional mutations in this structure also might be delete-rious. Indeed, replacing Leu121 with Ala, (equivalent to theUbcH5A104 mutation) substantially inhibited Ubc7A121 activity ascompared with wild-type Ubc7 (Fig. 1F). Furthermore, accordingto the structure prediction (Fig. 1E), a critical hydrogen bondthat stabilizes the closed conformation exists between thee amine of Lys118 and Leu8 in the Ub backbone. To test therequirement for this putative interaction, we examined the ac-tivity of additional single-amino acid substitutions, representingdistinct side-chain properties, at position 118. As predicted, alltested amino acid substitutions substantially reduced the activityof Ubc7 relative to the wild type (Fig. 1G), underscoring thestrict requirement for a Lys residue at position 118.

Ubc7R118 Is an Intrinsic Activity-Reducing Mutant. The E2-mediatedUb transfer comprises two partial activities: initial formation of ahigh-energy Ub thioester at the active site (charging) and sub-sequent formation of an isopeptide bond with an acceptor Lys(transfer). Consequently, to understand the role of Lys118 incatalysis, we compared the partial activities of wild-type andmutant Ubc7. To measure Ubc7 charging, we used a Ub de-rivative labeled with fluorescein (UbFL) on Cys48 that replacesthe original Lys residue at this position. Thus, while serving as atag, the fluorescein attached to Lys48 prevents the subsequentformation of a Lys48-linked polyUb chain, ensuring a single chargecycle. Consequently, the E2 was incubated with E1 and UbFL forvarious time periods; then a Ubc7∼UbFL adduct was monitored.The kinetics of Ubc7∼UbFL formation (Fig. 2A) detected acharged E2 after 15 s, reaching a maximum after 2 min. Impor-tantly, no differences in UbFL charging between wild-type Ubc7and Ubc7R118 were observed, indicating that the Arg118 mutationdid not affect Ubc7 charging.Next, we tested whether the Ub transfer activity of Ubc7 was

affected. To this end, we set up a single-round Ub-turnover ex-periment, a modification of a similar assay previously used todetermine the activity of the mammalian Ubc7 ortholog,Ube2g2, that facilitates Ub conjugation by engaging donor andacceptor Ub molecules (Fig. 2B) (32, 33). Consequently, to en-sure a single-round Ub transfer, we used preformed thiol estersof Ubc7:Cue1 with FlagUbR48 as donor Ub species, and Ubc7(without Cue1), charged with wild-type Ub, as acceptor Ubspecies. Because FlagUbR48 can be conjugated to an acceptor Ubbut then blocks further Lys48‐linked conjugation, it serves onlyas a donor, whereas in the absence of Cue1, Ubc7 serves solely asan acceptor (19), because it cannot transfer the active-site boundUb (23, 26). Accordingly, purified Ubc7 or Ubc7R118 in complexwith Cue1 initially was charged with FlagUbR48 and, in parallelwild-type Ubc7 purified in the absence of Cue1, was charged withwild-type Ub (Fig. 2B). Both Ub-charging reactions were ter-minated by ATP depletion and then were coincubated furtherfor various time periods to allow Ub transfer. Measurement ofthe diUb, Ub–FlagUbR48, dimer formation (Fig. 2C) revealedthat when Ubc7∼FlagUbR48 was used as a donor, the diUb wasdetected after 10 min (marked by an arrow), whereas only a faintUb–FlagUbR48 signal was observed after 30 min in the presenceof donor Ubc7R118∼FlagUbR48. The diminished Ub transferactivity of Ubc7R118 thus explains the substantial difference be-tween the activity of the wild-type and the mutant enzyme in theformation of the polyUb chain.

Differential Requirements for Ubc7 Activation by the Doa10 and Hrd1RING Domains. Doa10 and Hrd1 activate Ubc7, but, unlike otherE2s, Ubc7 can form polyUb chains in vitro even in their absence(Fig. 1 D, F, and G) (19, 20, 23). We next tested whether theRING domains of either Doa10 or Hrd1 can still activate polyUbchain formation by Ubc7R118. To this end, we analyzed the E2

activity in the presence of purified Doa10 RING domain (Fig.2D and Fig. S3). The Doa10 RING domain activated Ubc7 in atime-dependent manner (Fig. 2D) but failed to enhance Ubc7R118activity. Unexpectedly, the Hrd1 RING domain (Hrd1 RING)enhanced wild-type as well as Ubc7R118 activities, although thelatter were enhanced to a lesser extent (Fig. 2E). Notably, in-cubating Ubc7 with Hrd1 RING produced high-molecular-weightUb adducts, considerably larger than those produced in the ab-sence of RING E3 or upon incubation with Doa10 RING. Thecopurification of these high-molecular-weight Ub species withGST-tagged Hrd1 RING (Fig. S4) confirmed that they were pri-marily polyUb–Hrd1 RING conjugates, as previously shown (5).

A Selective Effect of the Lys118-to-Arg Mutation in Ubc7 on ERAD. Itwas possible that the apparent inability of Doa10 to activateUbc7R118 compared with Hrd1 was caused by the inherently lowerin vitro activity of Doa10 relative to that of Hrd1. To exclude thispossibility, we tested the ability of increasing concentrations

Fig. 2. The reduced catalytic activity of Ubc7R118 is partially rescued by theHrd1 RING domain. (A) Kinetics of Ubc7∼Ub thioester formation. The Ubc7:Cue1 dimer was incubated with E1 and Ub48FL for the indicated time periodsat 30 °C. Proteins were separated by SDS/PAGE with or without DTT, afterwhich fluorescence was detected using a Typhoon reader (GE Healthcare).(B, Left) Flowchart for single-round Ub-turnover experiments. Ubc7 and theUbc7:Cue1 dimer were charged separately with Ub and FlagUbR48, re-spectively, in the presence of E1 and ATP. Both reactions were terminated bythe addition of Apyrase to deplete ATP. Equal volumes of each reaction thenwere mixed for further incubation with either buffer or an E3 ligase; thendiUb (Ub–FlagUbR48) formation was determined by immunoblot analysiswith anti-Flag Abs. (Right) Graphic depiction of the single-round Ub-turn-over reaction. (C) Rates of Ub–FlagUbR48 formation by Ubc7 and Ubc7R118,determined in a single-round Ub-turnover assay in the absence of E3. TheE2∼Ub thioesters were combined and incubated at 30 °C for the indicatedtime periods. The reaction was terminated by the addition of SDS samplebuffer. Ubiquitylated proteins were resolved by SDS/PAGE and visualized byimmunoblot analysis with anti-Flag Abs. Arrow indicates the position ofdiUb. (D and E) Activation of polyUb chain formation by E3 RING domains.Either Ubc7 or Ubc7R118 was incubated in an ubiquitylation reaction mixture,as described in Materials and Methods in the presence of equal amounts ofrecombinant Doa10 or Hrd1 RING domains. Ub chains were resolved by SDS/PAGE and visualized by an immunoblot analysis with anti-Flag Abs.

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of Doa10 to activate ubiquitylation. The results presented inFig. S3 showed that, indeed, increasing the Doa10 concentra-tion enhanced wild-type Ubc7-mediated polyubiquitylation but didnot affect the Ubc7R118 activity whatsoever. We further in-vestigated the differential activation of Ubc7 by the two E3 ligasesby comparing the stability of a battery of Doa10 and Hrd1 sub-strates (Fig. 3A) (10, 12, 13, 34–37) in cells expressing either wild-type Ubc7 or Ubc7R118. To this end, we initially tested whether theR118 mutation affected Ubc7 stability. We found no significantdifferences in the steady-state levels of Ubc7, Ubc7R118, andUbc7A89 that were expressed chromosomally under control of theUbc7 promoter and no changes in Ubc7 levels during a CHX-chase degradation experiment (Fig. 3B). These results indicatedthat the R118 mutation did not destabilize Ubc7 and excluded thepossibility that different E2 levels might affect the stability of theE3 substrate in subsequent experiments. Consequently we next

tested whether Lys118 was essential for the in vivo degradation ofDoa10 and Hrd1 substrates (Fig. 3 C and D). Determining thesteady-state levels of the Doa10 substrate Deg1–Vma12–Ura3 (28)revealed that, among the various Lys-to-Arg mutants, onlyUbc7R118 had a stabilizing effect, although to a lesser extent thanthe active-site mutant Ubc7A89 (Fig. 3C). In contrast, none of theLys-to-Arg Ubc7 mutants had a stabilizing effect on the Hrd1substrate mutant carboxypeptidase Y (CPY*) (Fig. 3D and Fig.S5) (12). Thus, among the 11 Lys residues within Ubc7, onlyLys118 was essential and sufficient for substrate degradation bythe Doa10 pathway but dispensable for degradation by the Hrd1pathway. These in vivo degradation studies correlated well withthe in vitro activities of wild-type and mutant Ubc7.To ascertain further the selective inhibition of the Doa10

pathway by Ubc7R118, we measured the degradation kinetics ofadditional ERAD substrates by CHX-chase (Fig. 3 E and F).Both Doa10 substrates, Ubc6 (10) and Vma12–DegAB (36), weremarkedly stabilized in cells expressing Ubc7R118 (Fig. 3 E and F),whereas the degradation of the Hrd1 substrates KHN, contain-ing the yeast Kar2 signal sequence, fused to the simian virus 5HA-neuraminidase ectodomain (37), and KWW, composed ofthe KHN luminal domain, fused to the single transmembraneER protein Wsc1 (13), remained unaffected (Fig. 3 G and H).Noteworthy was the shift in the molecular weight of KHN andKWW caused by the O-linked glycosylation of the proteins that,having evaded ERAD, were modified in the Golgi apparatus (13,37). Evidently, the Lys-to-Arg substitution at position 118 wasan activity-reducing rather than an inactivating mutation, be-cause its effect varied between Doa10 substrates and the Lys-to-Arg mutant was consistently and substantially more active thana Cys-to-Ala catalytic site mutant.To confirm that the reduced degradation of Doa10 substrates

in cells expressing Ubc7R118 was indeed caused by a defect inubiquitylation, we used Vma12–DegABDD, a variant of theDoa10 substrate Vma12–DegAB that normally is ubiquitylated byDoa10 but is not degraded (38). Consistent with the previousdegradation results, an immunoblot analysis revealed a reductionof polyubiquitylated Vma12–DegABDD in cells expressing Ubc7R118to the levels observed in the Ubc7KO cells (Fig. S6). Importantly,an analysis of isolated Ubc7–Doa10 complexes showed no dif-ferences between wild-type and mutant Ubc7 in their associationwith Doa10 (Fig. S7), suggesting that the observed differences inUbc7 activity were conferred by the Ub-conjugation activity ofthe E2. Taken together, these results indicate that the activationof Ubc7R118 by its cognate E3s was selective: The activationprofoundly disrupts the degradation of Doa10 substrates but hadlittle effect on Hrd1 substrates.The ERAD pathway is a key mechanism for protein quality

control, as evidenced by the induction of the unfolded proteinresponse (UPR) when the ER is overloaded with misfoldedproteins (39) or when ERAD components are compromised (10,14, 39). Because the tested helix α2 mutants selectively inhibitedDoa10- but not Hrd1-mediated degradation, we predicted thattheir expression might induce a substantial UPR, albeit a milderone than that induced by the complete abolishment of Ubc7activity. Indeed, expression of a LacZ reporter under the controlof the UPR-activated Kar2 promoter (UPRE-LacZ) (40) in-dicated that the UPR was increased by ∼75% over basal levels incells expressing helix α2 mutants of Ubc7, compared with an∼200% increase in ubc7Δ cells or in cells expressing Ubc7A89(Fig. 3I). We attribute the induction of UPR by helix α2 mutantsto their inability to ubiquitylate the physiological ERAD sub-strates of Doa10.

Hrd1 Activation of Ubc7 Requires Alignment of Helix α2 with Ub. TheUbcH5 helix α2:Ub interactions determine the transition state ofthe Ub transfer reaction (2–4). The effect of the Lys118 mutationsuggests a similar transition state for Ubc7-mediated Ub transfer.

Fig. 3. Ubc7R118 differentially influences the in vivo degradation of Doa10and Hrd1 substrates. (A) Schematic presentation of the different ERADsubstrates used in this study. (B) Determination of Ubc7 stability in cellsexpressing wild-type Ubc7, Ubc7A89, or Ubc7R118. Cells at logarithmic phasewere collected at time 0 or after incubation with CHX for the indicated timeperiods and were lysed by incubation with 0.1 N NaOH for 5 min, followedby boiling in sample buffer containing 50 mg/mL DTT. Ubc7 was visualizedby immunoblot using anti-HA Abs. (C–H) Determination of the stability ofthe indicated Doa10 and Hrd1 substrates (shown in A) in cells expressingwild-type Ubc7, Ubc7A89, or Ubc7R118. Cells were treated and as described inB. Proteins were visualized by immunoblotting with anti-Ubc6 Abs, anti-FLAG Abs (for visualizing Flag-tagged Deg1-Vma12-Ura3 and Vma12-DegAB), or anti-HA Abs (for visualizing CPY*, KHN, or KWW). In all experi-ments G6PD staining was used for loading control. (I) Induction of UPR inUbc7 mutant cells. The indicated cells, expressing a LacZ reporter under thecontrol of the UPR-activated Kar2 promoter (UPRE-LacZ), were lysed, andβ-galactosidase steady-state levels were determined by an activity assay us-ing ortho-Nitrophenyl-β-galactosidase as a substrate. Data shown are themean ± SE of three or more independent experiments.

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Nevertheless, our findings that Hrd1 tolerated Arg at position 118suggested that it either compensated for suboptimal interactionsat the Ubc7∼Ub interface or that it employed a spatially distinctUbc7∼Ub transition state. If activation by Hrd1 requires the samehelix α2:Ub alignment, then a local repulsion force in this in-terface would be expected to inhibit Ub transfer. To test thisexpectation, we replaced Lys118 with Glu, introducing an oppo-site negative charge. This substitution would be expected to repelthe carbonyl of Leu8 at the Ub backbone, which is predicted toform an electrostatic bond with Lys118 (Fig. 1E). Indeed, Hrd1activated Ubc7R118- but not Ubc7E118-dependent Ub transfer andpolyubiquitylation (Fig. 4 A and B), although Ubc7E118 was ableto form a thioester with Ub efficiently (Fig. 4C). Notably, severalhigh-molecular-weight bands that were present in the Ub transferreaction corresponded to Hrd1 RING polyUb conjugates. Theirformation correlated well with the efficiency of the Ub transferreaction, as determined by the formation of diUb conjugates (Fig.4A). In agreement with these findings, in vivo expression ofUbc7E118 completely stabilized a Hrd1 substrate (Fig. 4D). To-gether, these results indicated that activation of Ubc7 by Hrd1was through the closed (helix α2:Ub) conformation of thedonor Ub∼Ubc7.

Next we examined the role of the critical helix α2 in vivo bymeasuring the stability of Doa10 and Hrd1 substrates in cellsexpressing a different helix α2 mutation, Ubc7A121. Consistentwith our previous results, this Ubc7 mutant stabilized the Doa10substrate Ubc6 (10) (Fig. 5A), but it had no affect on the degra-dation of the Hrd1 substrate KHN (Fig. 5B). Similar results wereobtained subsequently with additional E3-specific substrates,GFP-DegAB and CPY* (Fig. S8), confirming that Hrd1 did notrequire optimal helix α2:Ub interactions.To confirm this conclusion further, we investigated the ability

of Hrd1 to stimulate Ubc7-mediated Ub transfer and poly-ubiquitylation activities in vitro in the presence of Ala sub-stitutions of either Leu8 or Val70 Ub. According to the structuralmodel (Fig. 1E), Leu8 and Val70 interact with Lys118 andLeu121, respectively, and thus are expected to have an effect onHrd1 activity similar to that of the respective helix α2 mutations.Indeed, the Hrd1 RING activated both Ala8 and Ala70 polyUbchain formation, the latter to a much greater extent (Fig. 5C). Todemonstrate specifically that Hrd1 RING enhances Ub transferby stabilizing the transition state, we employed the single-turn-over Ub-transfer assay using Ub mutants as donors, acceptors, orboth (Fig. 5D). In agreement with the polyubiquitylation results,in which most of the observed polyUb chains were conjugated toHrd1 RING (Fig. S4), the Hrd1 RING stimulated transfer ofboth Ub mutants as either donors or acceptors. However, Hrd1self-ubiquitylation was substantially reduced in the presence ofAla8 as a donor.

DiscussionThe present study employs Ubc7 mutants to explore a possiblydifferential activation of Ubc7 by its two cognate E3 ligases,Doa10 and Hrd1. Initially Lys118 was identified as an essentialresidue for the degradation of a Doa10 substrate (Fig. 1A). Thein vitro studies further revealed that the Lys residue at position118 of Ubc7, rather than ubiquitylation at this site, was critical

Fig. 4. Hrd1 RING is incapable of activating Ubc7E118. (A) Hrd1-mediatedactivation of diUb formation. A single-round Ub-turnover assay was per-formed as described in Fig. 2C using Ubc7, Ubc7R118, or Ubc7E118 as Ubdonors in the presence of Hrd1 RING. The reaction was incubated for 30 minat 30 °C; then diUb formation was determined. Arrow indicates the migra-tion distance of diUb. (B) Hrd1 activation of polyUb chain formation by theindicated Ubc7 variants. The ubiquitylation reaction was carried out as de-scribed in Fig. 2D and was terminated after 30 min. (C) Kinetics of Ub∼Ubc7thiol ester formation. The Ubc7:Cue1 dimer was incubated at 30 °C with E1and FlagUbR48 for the indicated time periods. Proteins were resolved on SDS/PAGE and were subjected to immunoblot with anti-Flag Abs. (D) Degrada-tion of KHN in cells expressing the indicated Ubc7 variants was determinedby CHX-chase as described in Fig. 1A.

Fig. 5. Hrd1 overcomes mutations disrupting helix α2:Ub interaction.(A and B) Degradation of Ubc6 (Doa10 substrate) (A) and KHN (Hrd1 sub-strate) (B) in cells expressing wild-type Ubc7, Ubc7R118, or Ubc7A121. Proteindegradation was determined by CHX-chase, followed by immunoblottingusing anti-Ubc6 or anti-HA (for KHN) Abs. The G6PD staining providesa loading control for Ubc6. The asterisk indicates a cross-reacting proteinthat serves as a loading control for KHN. (C) PolyUb chain formation by Ubc7in the presence of Hrd1. The ubiquitylation assay was performed as describedin Fig. 2D using Ub, UbA8, or UbA70, as indicated. PolyUb conjugates werevisualized by immunoblot analysis using anti-Ub Abs. (D) Hrd1-mediatedactivation of diUb formation. A single-round Ub-turnover assay was per-formed as described in Materials and Methods and in the legend of Fig. 2C,using a mixture of wild-type and mutant Ub as donors and acceptors, asindicated. Arrow indicates the migration distance of diUb.

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(Fig. 1 C and D). The in silico structure prediction (Fig. 1E)implicated helix α2 of the enzyme, where Lys118 is located, tothe Ub transfer reaction. This hypothesis was confirmed by ex-amination of additional amino acid substitutions at this site, allof which impaired Ubc7 activity. To identify the functional sig-nificance of Lys118, a single-round Ub-transfer reaction wasdesigned using wild-type or mutant Ubc7 as the donor E2. Theseexperiments revealed that Lys118 was essential for the donor E2activity (Fig. 2C), consistent with previous observations thatpolyUb chain formation by Ube2g2, the mammalian ortholog ofUbc7, requires distinct activities of donor and acceptor E2∼Ubspecies (32, 33). Incubation of wild-type and mutant Ubc7 witheither Doa10 or Hrd1 RING produced an unexpected result:Although Hrd1 RING activated polyubiquitylation by both Ubc7variants, Doa10 activated only the wild-type E2 (Fig. 2 D and E).The physiological relevance of these in vitro activity studies wasestablished by showing a selective requirement for helix α2 forthe Doa10 and Hrd1 degradation pathways in intact yeast cells.In accordance with the in vitro Ubc7 activity results, the degra-dation of Doa10 substrates was markedly inhibited in cellsexpressing selected helix α2 mutants, whereas the Hrd1 sub-strates were essentially unaffected (Fig. 3). Furthermore, Ubc7helix α2 mutations induced an intermediate UPR response be-cause misfolded substrates are expected to accumulate frompartial inhibition of ERAD (Fig. 3I). It is noteworthy that thetested helix α2 mutations are not inactivating but rather areactivity reducing, as evidenced by the intermediate levels ofsubstrate stabilization they confer, compared with the full sta-bilization conferred by active-site C89A mutation. Inhibition ofHrd1 activation of Ubc7E118 (Fig. 4) indicated that Hrd1 usesa similar helix α2:Ub transition state. However, the ability ofHrd1 to tolerate mutations that weakened the helix α2:Ub in-teraction suggested that it stabilized the transition state by ad-ditional interactions, most likely outside the RING domain.Based on the data regarding the in vitro and in vivo Ubc7 ac-

tivity presented in this study and on the established structure ofRING domain complexes with Ub-charged UbcH5 (2–4), wepropose a model for the underlying RING:Ubc7∼Ub interac-tions (Fig. 6). According to the model, Ub binding at the activesite is in a loose (open) conformation. Because Ub wobbles, itonly infrequently attains the transition state that ensures opti-mal positioning of the donor Ub for a nucleophilic attack byan acceptor Ub. Hence, Ub transfer in the absence of RING isrelatively inefficient. Binding of a cognate RING domain toUbc7∼Ub stabilizes the transition state (closed conformation)through noncovalent interaction of helix α2 with the active site-bound Ub. These Helix α2/Ub interactions are critical for Doa10

and are less critical for Hrd1, which can compensate for sub-optimal Ubc7:Ub interactions by stabilizing the transition statefor effective Ub transfer by additional interactions with regionsflanking the RING domain.The physiological significance of selective Ubc7 activation in

the ERAD pathway is still unclear. Although both Doa10 andHrd1 are situated at the ER membrane, each recognizes a dis-crete set of substrates with a distinct membrane topology (13).The faster conjugation rate observed for the Cue1:Ubc7:Hrd1complex relative to its Doa10 counterpart may reflect a re-quirement for the extraction of luminal Hrd1substrates into thecytosol. For example, ubiquitylation of luminal ERAD substratesrecruits the Cdc48 ATPase complex, the binding of which pre-vents reverse translocation of the substrate into the ER lumen(41, 42). Accordingly, rapid ubiquitylation of the substrate maybe an important factor to ensure efficient extraction of thedegradation substrate. This stringent requirement for ubiq-uitylation may be redundant for Doa10 substrates, the majorityof which are cytosolic and nuclear proteins.The activation of polyUb chain elongation by Hrd1 in the

presence of Ala8 and Ala70 Ub variants was surprising in thelight of studies showing that the mutant Ub is largely inactive (2,3, 43). Notably, unlike most RING E3s, Hrd1 RING requirescytosolic flanking regions for its in vitro ubiquitylation activity(23). These regions may be those that are required for stabili-zation of the α2:Ub transition state and thus also compensate forthe omission of otherwise missing critical interactions, such asthe one absent between Lys118 of Ubc7 and Leu8 of Ub. Weattempted to test the role of these regions by creating a hybridenzyme containing the Doa10 RING domain flanked by theHrd1 cytosolic regions, assuming that they may elicit RINGDoa10 activity in the presence mutant Ubc7, similar to that ofHrd1. However, this hybrid protein was completely inactive. Theobserved differential activation of polyUbA8 and polyUbA70chain formation (Fig. 5C) is puzzling, because most studies in-dicate no activation of these mutants (2, 3, 43). The more severeeffect of the Ala8 mutation may suggest that this residue createsadditional essential interactions with RING E3s (2–4) that, whendisrupted by the mutation, cannot be compensated for, evenby Hrd1.More than three decades after the discovery of the Ub ligation

system, it now appears more complex than initially described(44). Although the system is still perceived as a linear, sequentialprocess, apparently there are multiple levels of regulation. Thedistinct requirements for Ubc7 activation by its cognate E3ligases Doa10 and Hrd1 provide evidence of a potential retro-grade regulatory mechanism of ERAD and possibly of other Ub-conjugation systems.

Materials and MethodsPlasmids, Yeast Strains, and Antibodies. Yeast strains and plasmids used in thisstudy are listed in Tables S1 and S2. Details of yeast and plasmid preparationare provided in SI Materials and Methods. The following Abs were used:anti-GFP (Roche), anti–glucose-6-phosphate dehydrogenase (G6PD) (Sigma-Aldrich), anti-Flag (Sigma-Aldrich), anti -HA (Roche), anti-myc (9E10) (Cova-nce), anti-GST (Santa Cruz Biotechnology), anti-Doa10 (45), anti-Cue1 (raisedagainst Cue1ΔTM), and anti-Ubc6 antiserum (raised against the N-terminal225-residue fragment of Ubc6).

Site-Directed Mutagenesis and Transfer PCR. Site-directed mutagenesis wasperformed using PfuUltra polymerase (Stratagene) according to the manu-facturer’s instructions. All mutations were confirmed by sequencing. TransferPCR was performed using Phusion polymerase (New England Biolabs),according to Erijman et al. (46). All mutations were confirmed by sequencing.

CHEX-Chase and Immunoblot. CHEX-chase was performed as previously de-scribed (35).

Fig. 6. A proposed model for the activation of Ubc7 by the RING domains ofDoa10 and Hrd1. Ub bound to Ubc7 at the active site is largely in a loose(open) conformation. Both Doa10 and Hrd1 RING promote the closed tran-sition state conformation by stabilizing noncovalent interaction of Ub withhelix α2. The Hrd1 RING domain can form additional interactions that sta-bilize the closed conformation and thus can overcome compromised helixα2:Ub interactions. Arrows indicate the direction of nucleophilic attack by anacceptor Lys. Joined arrowheads indicate the degree to which the donor Ubis free to wobble.

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Immunoprecipitation.Determination of Ubc7–Ub conjugates. ubc7Δ cells coexpressing chromosomallyintegrated Ubc7-3HA and CUP1p-induced UbA76 were incubated with100 uM CuSO4 until late logarithmic phase. Cells lysis, protein separation,and immunoblotting were done essentially as described (18).Determination of Doa10:Ubc7 complexes. Doa10 immune complexes were iso-lated from a preparation of crude microsomal fraction treated with1% digitonin, as previously described (47). Myc-Doa10:Ubc7-3HA complexeswere isolated from microsomal extracts by immunoprecipitation with anti-myc Abs, followed by binding to protein A beads. The immunocomplexeswere subjected to immunoblotting after separation on SDS/PAGE using anti-myc and anti-HA Abs.

In Vivo Ubiquitylation. In vivo ubiquitylation analysis was performed as pre-viously described (35).

β-Galactosidase Activity. The UPR was measured in yeast cells expressinga LacZ reporter under the control of the UPR-activated Kar2 promoter UPRE-LacZ. Cells were harvested, and β-galactosidase activity was measured aspreviously described (48).

Protein Purification. Ubc7 and Cue1, coexpressed in a bicistronic plasmid, werepurified from bacteria using a chitin-intein tag as previously described (23). Thetag was removed by incubating the purified protein with 50mMDTT, followedby dialysis (23). RING domains of Hrd1 (amino acids 287–531) or Doa10 (aminoacids 1–128) coupled to GST were purified from bacteria using glutathioneaffinity resin beads (Novagene) and were eluted with 50 mM glutathione. Ubvariants were purified by precipitation in perchloric acid according to Beal et al.(49). Briefly, 500 mL of isopropyl β-D-1-thiogalactopyranoside–induced BL21cells were lysed using a microfluidizer, and Ub was precipitated on ice by theslow addition of 1% (vol/vol) of 70% perchloric acid (Sigma-Aldrich) to thestirring extract. The soluble fraction containing Ub was dialyzed and con-centrated using Amicon Ultra centrifugal filters (3 K molecular weight cut-off). Synthesis of UbFL was done according to Berndsen et al. (50). The purityof the isolated proteins was checked by separating on 15% SDS/PAGE afterCoomassie brilliant blue staining.

In Vitro Ubiquitylation. The in vitro ubiquitylation assay was done as describedby Bazirgan et al. (23). Briefly, 1 μM wild-type or mutant Ubc7, 1 μM Cue1,1 μM Doa10 or Hrd1 RING, 50 nM E1, 4 μM FlagUb, and reaction buffercontaining 5 mM MgCl2, 4 mM ATP, and 50 mM Tris (pH 7.6) were incubatedat 30 °C for the indicated times, resolved by 5–15% SDS/PAGE, and immu-noblotted with anti-Flag or anti-Ub Abs, as indicated.

Charging of Ubc7 at the Active Site. Ubc7 charging with Ub on the active sitewas done using UbFL or a Flag-tagged UbR48 mutant. For charging with UbFL,Ub Lys48 first was replaced with Cys by site-directed mutagenesis. Next,fluorescein was attached to Cys48, as described by Berndsen et al. (50), toobtain UbFL. Ubc7 in the Ubc7:Cue1 complex was charged with UbFL at theactive site by incubation at 30 °C for the indicated time periods. The reactionmixture contained 1 μM Ubc7:Cue1, 80 μM UbFL, 1 μM E1, 10 mM MgCl2,100 mM NaCl, 200 μM ATP, and 30 mM Hepes (pH 7.6). Reactions wereterminated by adding SDS/PAGE loading buffer with or without DTT. Pro-teins were separated on 15% SDS/PAGE, and images were taken using aTyphoon laser scanner (GE Healthcare). For charging with FlagUbR48, theUbc7:Cue1 complex was charged with FlagUbR48 in a reaction mixture con-taining 1 μM Ubc7:Cue1, 4 μM FlagUbR48, 1 μM E1, 5 mM MgCl2, 4 mM ATP,and 50 mM Tris (pH 7.6). Reactions were resolved as above, and immuno-blotting was done using anti-Flag Abs.

Single-Round Ub Turnover. The single-round Ub-turnover assay is based on themethod described by the Liu et al. (33) for studying the activity of the Ubc7human homolog Ube2g2 (33). Purified Ubc7 (2 μM), without Cue1, wascharged with untagged Ub for 15min, as described above. In parallel, 2 μMof the Ubc7:Cue1 complex was charged with FlagUbR48 for 15 min. Then5,000 mU/mL Apyrase (New England Biolabs) was added to deplete the ATP.Equal volumes of both mixtures (with or without Cue1) were mixed togetherwith 2.5 μM GST‐Doa10 or Hrd1 RING and were incubated at 30 °C for theindicated times. Reactions were stopped by SDS/PAGE loading buffer with50 mg/mL DTT and immunoblotted with anti-Flag, anti-GST, or anti-Cue1 Abs.

ACKNOWLEDGMENTS.We thank A. Ciechanover, R. Hampton,M. Hochstrasser,R. Kulka, and D. Ng for plasmids and strains, Dr. A. Shiber for technicalassistance, and Dr. W. Breuer for critically reviewing the manuscript. Thiswork was supported by Israel Science Foundation Grant 786/08.

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