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Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly‐ubiquitylation

2020; Springer Nature; Volume: 39; Issue: 22 Linguagem: Inglês

10.15252/embj.2020104863

1460-2075

Christian Lips, Tobias Ritterhoff, Annika Weber, Maria K. Janowska, Mandy Mustroph, Thomas Sommer, Rachel E. Klevit,

Cancer-related Molecular Pathways

Article5 October 2020Open Access Source DataTransparent process Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation Christian Lips Christian Lips Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Tobias Ritterhoff Tobias Ritterhoff Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Annika Weber Annika Weber Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Maria K Janowska Maria K Janowska Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Mandy Mustroph Mandy Mustroph Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Thomas Sommer Thomas Sommer Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Lady Davies Guest Professor, Technion-Israel Institute of Technology, Haifa, Israel Search for more papers by this author Rachel E Klevit Corresponding Author Rachel E Klevit [email protected] orcid.org/0000-0002-3476-969X Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Christian Lips Christian Lips Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Tobias Ritterhoff Tobias Ritterhoff Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Annika Weber Annika Weber Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Maria K Janowska Maria K Janowska Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Mandy Mustroph Mandy Mustroph Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Search for more papers by this author Thomas Sommer Thomas Sommer Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany Lady Davies Guest Professor, Technion-Israel Institute of Technology, Haifa, Israel Search for more papers by this author Rachel E Klevit Corresponding Author Rachel E Klevit [email protected] orcid.org/0000-0002-3476-969X Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Author Information Christian Lips1,‡, Tobias Ritterhoff2,‡, Annika Weber1,4, Maria K Janowska2, Mandy Mustroph1, Thomas Sommer1,3 and Rachel E Klevit *,2 1Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany 2Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA 3Lady Davies Guest Professor, Technion-Israel Institute of Technology, Haifa, Israel 4Present address: MRC Laboratory of Molecular Biology, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +1 206 543 5891; E-mail: [email protected] The EMBO Journal (2020)39:e104863https://doi.org/10.15252/embj.2020104863 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s. Synopsis During polyubiquitination, RING E3 ligases cooperate with both ubiquitin chain priming and elongation E2 enzymes. Kinetic analyses reveals the functional coordination of yeast ERAD E3s Hrd1 and Doa10 with Ubc6 and Ubc7 E2s. Substrate modification with the first ubiquitin (priming), but not ubiquitin-chain building, is a rate-determining step in ERAD. Ubc7 activity depends on the action of an optimal RING E3 "linchpin" residue, while Ubc6 is largely linchpin-independent. An optimal linchpin residue and low affinity for Ubc6 promotes Hrd1 pairing with Ubc7 for priming. Doa10 has a sub-optimal linchpin residue, which still allows pairing with Ubc6 for priming. The Doa10/Ubc6 example provides a rationale to understand why about 50% of all RING E3s have a suboptimal linchpin. Introduction The post-translational modifier ubiquitin (Ub) controls virtually every process in eukaryotic cells. Protein modification with Ub is carried out by three sequentially acting enzymes. The E1 enzyme activates the Ub C-terminus for transfer to the active site of an E2 enzyme to form an E2~Ub thioester conjugate (in this text, "~" signifies the thioester linkage). E3 ligases are generally attributed with two functions: They bring an E2~Ub conjugate and a suitable substrate into proximity ("recruitment"), and they catalyze the transfer of Ub from the E2 to the substrate thus ensuring spatiotemporal control of the process ("stimulation"). All eukaryotic organisms from yeast to human have a hierarchy of E1/E2/E3 enzymes, with 1–2 human E1s, up to 36 E2s, and hundreds of E3s. The hierarchy dictates that a given E2 must work with numerous E3s, but it is also true that numerous E2s can work with a given E3. For the largest family of E3s, the RING E3s, the identity of the collaborating E2 defines the product of the reaction (i.e., mono- or poly-ubiquitylation) and, therefore, the biological outcome (Christensen et al, 2007). How an E3 pairs with a specific E2—from the set of enzymes it can physically interact with—to determine a distinct functional outcome remains an open question. The ability to stimulate E2 activity is rooted in the way RING E3 ligases function. They bind an E2~Ub conjugate and stimulate Ub transfer to a substrate without participating directly in the reaction. In solution, Ub conjugated to an E2 is highly flexible, creating a dynamic conformational equilibrium that is associated with low transfer activity (Fig 1A; Pruneda et al, 2011). RING E3s work allosterically by restricting the flexible Ub toward so-called closed conformations that involve non-covalent interactions between a hydrophobic surface patch on Ub and a surface proximal to the E2 active site (Reverter & Lima, 2005; Saha et al, 2011; Wickliffe et al, 2011; Dou et al, 2012; Plechanovová et al, 2012; Pruneda et al, 2012; Brown et al, 2014; Kelly et al, 2014; Branigan et al, 2020). In general, RING E3s achieve this shift in E2~Ub conformational equilibria through a conserved residue, the allosteric linchpin, that engages both the Ub and the E2 to restrict their relative orientations. A shift toward closed conformations is associated with a dramatic increase in Ub transfer activity from an E2 (Pruneda et al, 2012; Branigan et al, 2020). Structural data reveal that a RING linchpin engages Ub and the E2 through hydrogen bonding (Fig 1B; Dou et al, 2012; Plechanovová et al, 2012; Pruneda et al, 2012; Branigan et al, 2015). In a survey of yeast and human RING-type E3s, arginine, with its multiple hydrogen bond donor groups, is most commonly found at the linchpin position (Figs 1C and EV1 for yeast and human, respectively). Surprising, at least half of all human and yeast RINGs feature a residue other than arginine at the linchpin position. Some have been reported as functional, but less efficient linchpins than arginine (Yin et al, 2009; Pruneda et al, 2012; Scott et al, 2014; Stewart et al, 2017); some are residues that can potentially act as a hydrogen bond donor, but have not been experimentally verified as functional linchpins; some are residues that lack hydrogen bond potential altogether. Prominent examples include yeast RING E3s Rad16 (histidine) and Rad18 (leucine), both involved in DNA damage pathways, and Rbx1 (asparagine), the common RING module of the large family of cullin-RING ligases (CRLs). How these RINGs stimulate the ubiquitylation activity of their paired E2s is largely unknown. Figure 1. Ubiquitylating enzymes in yeast ERAD and mechanism of RING-mediated E2 stimulation A. Current model of RING E3-mediated E2 activation. B. RING linchpin-mediated interactions in an E2˜Ub/RING complex. Crystal structure of UbcH5a-Ub/RNF4 (PDB: 4AP4) highlighting the hydrogen bond interactions involving the RING linchpin arginine to side chain and backbone atoms on both the E2 and Ub. C. Histogram of amino acid frequencies at the linchpin position of all 42 yeast RING domains. The linchpin position was defined as the residue at the n + 1 position after the final Zn2+ ligand coordinating residues. "–" refers to the atypical RING of Pib1, which lacks the loop containing the linchpin. The SP-RING domains of the two yeast SUMO E3 ligases Siz1 and Siz2 as well as the RING1 domains of the two yeast RBR E3 ligases Hel1 and Itt1 were excluded from this analysis. D. Summary of the relevant properties of E2 enzymes and E3 ligases of the yeast ERAD system. E. Cartoon showing ERAD E2 pairings for both E3 ligases during priming and elongation (see text for details). F. Sequence alignment of Hrd1 and Doa10 for the final Zn2+-binding loops (C-X-X-C) showing the linchpin position. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Amino acid frequencies at the linchpin position of all human RING domainsHistogram of amino acids at the linchpin position of all 316 human RING domains. The linchpin position was defined as the residue at the n + 1 position after the final Zn2+ ligand coordinating residues. "–" refers to RING domains that feature a gap at the linchpin position in the multiple sequence alignment performed for all RING domains. SP-RING domains of the PIAS SUMO E3 ligases and RING1 domains of all human RBR E3 ligases were excluded from this analysis. Download figure Download PowerPoint The best-understood types of protein ubiquitylation involve the formation of poly-Ub chains of specific linkages (e.g., Ub K48- or K11-linked chains serve as signals for proteasomal degradation) (Komander & Rape, 2012; Metzger et al, 2013b). For RING-type E3s, substrate modification with a poly-Ub chain involves two types of Ub transfer reactions: (i) a "priming" step in which a single Ub is transferred to a protein substrate and (ii) "elongation" reactions in which Ub is attached to the substrate-modified Ub to generate a poly-Ub chain (reviewed in Stewart et al, 2016; Deol et al, 2019). The priming step is biochemically diverse due to the varied nature of potential substrates and Ub attachment sites, whereas chain elongation entails highly specific and repetitive reactions. The different biochemical requirements of priming and elongation have led to the recognition of dedicated E2s for each step. Dedicated chain-elongating E2s that specialize in the synthesis of specific Ub linkages such as K11-, K48-, and K63-linked chains are well known. However, the identities and mechanisms of specific mono-ubiquitylating priming E2s and of E2s that appear to carry out both functions are less well understood. For example, members of the UbcH5 (UBE2D) family are highly promiscuous in vitro and potentially function in both priming and elongation capacities. It is likely that their functional action, i.e., priming or elongation, is dictated by the particular E3 with which such E2s cooperate in a given case (summed up by Stewart et al, 2016), but how such a selection is achieved is not known. Examples of RING E3s that are confirmed to collaborate with separate priming and elongation E2s to catalyze attachment of poly-Ub chains to substrates continue to be reported (Rodrigo-Brenni & Morgan, 2007; Saha & Deshaies, 2008; Kleiger et al, 2009; Parker & Ulrich, 2009; Pierce et al, 2009; Williamson et al, 2009; Wu et al, 2010a; Kelly et al, 2014; Scott et al, 2014, 2016; Dove et al, 2016; Weber et al, 2016; Hill et al, 2019). Still, our understanding of how E3 ligases control the interplay of E2 enzymes during the two steps of poly-ubiquitylation is rather limited. To understand how RING E3s utilize E2s for priming and elongation reactions, we focused on a well-defined molecular poly-ubiquitylation system. ER-associated protein degradation (ERAD) is a highly conserved protein quality control pathway that targets misfolded ER-resident proteins and marks them for proteasomal degradation (Christianson & Ye, 2014). Yeast ERAD relies on two RING E3 ligases, Hrd1 and Doa10, and two E2 enzymes, Ubc6 and Ubc7, to modify substrates with K48-linked Ub chains (Fig 1D). Ubc6 harbors a C-terminal transmembrane domain and is thought to function primarily as a mono-ubiquitylating priming E2 (Weber et al, 2016). Ubc7 is recruited to the membrane by the accessory protein Cue1; binding to the Ubc7-binding region (U7BR) of Cue1 renders Ubc7 competent for Ub transfer, while the Cue1 domain of the protein serves to align a growing poly-Ub chain for K48-specific elongation by Ubc7 (Kostova et al, 2009; Bagola et al, 2013; Metzger et al, 2013a; von Delbrück et al, 2016). The E3 Doa10 employs Ubc6 and Ubc7 for priming and elongation, respectively (Weber et al, 2016), while Hrd1 relies predominantly on Ubc7 (Bays et al, 2001) (Fig 1E). Hrd1-targeted ubiquitylation is largely unaffected by the absence of Ubc6, implying that Ubc7 can carry out both priming and elongation with Hrd1. Nevertheless, no direct evidence for Ubc7's priming activity has been reported to date. Notably, Doa10 features a potentially suboptimal histidine as its linchpin, while Hrd1 harbors a canonical arginine (Fig 1F). This feature implies different requirements for E3-mediated E2 stimulation in the two RING E3s, but this notion has not been directly addressed experimentally. Our comparisons of the two non-redundant ERAD E3 ligases and their interacting E2 enzymes lead to several key observations and conclusions. First, we present direct in vitro evidence that chain-elongating Ubc7 can work as a priming E2. Second, E3-mediated E2 stimulation contributes to in vivo substrate degradation rates in the priming step, but is dispensable in the chain elongation step. Third, Hrd1 and Doa10 exploit different modes of E2 stimulation that are tailored to the properties of their preferred priming E2. Hrd1 has a high affinity for Ubc7 and relies on its canonical allosteric linchpin to stimulate the E2 in the priming reaction. Ubc6 has high basal activity and does not absolutely rely on stimulation by a functional linchpin for RING-mediated ubiquitylation. This enables Doa10 with its non-canonical linchpin residue to use Ubc6 and not Ubc7 for priming, while not affecting the E3's use of Ubc7 for chain elongation to a degree that impinges on protein degradation rates. Altogether, this study provides insight into how E3s functionally pair with their potential E2s to enable specific functional outcomes. Results Hrd1 selects Ubc7 through high binding affinity To understand the E2 preferences of Hrd1 and Doa10, we characterized the catalytically relevant combinations of each E3 ligase with Ub conjugates of either E2. Isothermal titration calorimetry (ITC) was performed using the E3 RING domains, a C-terminally truncated version of Ubc6, and Ubc7 in complex with the U7BR of Cue1, which renders the E2 competent for Ub transfer (Bagola et al, 2013). As the native Ubc6~Ub thioester is susceptible to hydrolysis (see also later Fig 5C), we engineered stable mimics of E2~Ub thioester conjugates by generating a covalent disulfide linkage between a Ub(G76C) mutant and the E2 active site cysteine [(Lorenz et al, 2016); "-SS-" signifies the disulfide linkage for these conjugates]. The Hrd1 RING binds the U7BR/Ubc7-SS-Ub conjugate more than one order of magnitude stronger than it binds the Ubc6-SS-Ub conjugate, with a KD in the low μM range (Fig 2A). This implies that Hrd1 selects between Ubc6 and Ubc7 on the basis of E2~Ub/E3 affinity, consistent with the reported Ubc6 independence of Hrd1 substrate degradation (Bays et al, 2001). In contrast, the Doa10 RING binds each E2 conjugate with similar weak affinity, with KDs in the high μM range, consistent with this E3 cooperating with both E2s. We note that the dissociation constants measured for the soluble versions of the RING domains and E2 conjugates reflect the intrinsic affinity for these functional protein–protein interactions and, therefore, the probability of an E3 engaging a particular E2~Ub. Tethering of the relevant components in the ER membrane (see Fig 1E) may overcome the relatively low intrinsic affinity by providing high local concentrations of an E3 and its E2~Ub conjugates. Figure 2. Hrd1 selects Ubc7 through high binding affinity A. Dissociation constants for ERAD E2-SS-Ub/E3 pairs. KD values were determined by ITC titration of Hrd1 or Doa10 to Ubc6-SS-Ub and U7BR/Ubc7-SS-Ub, respectively. Due to the weak binding, errors are high, so the constants are reported to a single significant figure. B. In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; "no E3" reactions do not contain S-Hrd1, "no ATP" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a non-specific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by one-way ANOVA test; *P < 0.05. For clarity, only significances related to the "no E3" control of a given E2 are shown. C. In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top—yellow) and Ubc6 (bottom—green) with ethanolamine as nucleophile. Representative Coomassie gels are shown. D. Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2˜Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by Student's t-test; *P < 0.05. E. Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by Hrd1. E2 stimulation is reported as the ratio of rates derived from RING-catalyzed reactions and "no E3" controls in D. Values are reported as means ± standard deviation (n = 3). Significance was determined by Student's t-test; *P < 0.05. Source data are available online for this figure. Source Data for Figure 2 [embj2020104863-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Hrd1 is presumed to collaborate with Ubc7 for both priming and chain elongation steps, but direct evidence of substrate priming by Ubc7 has not been reported. We implemented an in vitro substrate ubiquitylation assay in which a sequence derived from bovine RNase A ("S-peptide") is genetically fused to an E3 (e.g., "S-Hrd1") to serve as a high-affinity substrate recruiter for an RNase A variant that lacks its S-peptide sequence (referred to here as "RNase"; Bays et al, 2001). The Ub(K48R) mutant was used in reactions containing Ubc7 to limit reactions to the priming step by preventing K48-linked poly-Ub chain formation. Reactions that contain a functional E2/E3 pair will generate a product that corresponds to RNase modified with Ub. The pairing of Ubc7 and S-Hrd1 yields a robust RNase-Ub band and a fainter RNase-Ub2 band, which likely reflects double mono-ubiquitylated RNase (Fig 2B). In the absence of S-Hrd1, Ubc7-mediated ubiquitylation is negligible. Importantly, this result provides direct evidence that the efficient chain-elongating E2 Ubc7 is able to prime a substrate. The pairing of Ubc6 and S-Hrd1 also generated modified RNase, albeit to a lesser extent, and this reaction does not depend entirely on the presence of S-Hrd1. Importantly, the stimulatory effect of S-Hrd1, i.e., the difference of RNase modification in the presence of the E3 compared to its absence, is much greater in the case of Ubc7 than Ubc6. Altogether, the results indicate that Ubc6/S-Hrd1 can, in principle, form a functional E2/E3 pair for priming, but is much less efficient than the Ubc7/S-Hrd1 pair. Although the in vitro ubiquitylation assay provided insights into the capability of Ubc6 and Ubc7 to prime a substrate, RNase is an artificial substrate that interacts with the E3 ligase in a non-native way. To obtain a quantitative measure of E2 activity and the potential of E3s to stimulate it without confounding contributions from a substrate, we monitored the kinetics of Ub transfer from preformed E2~Ub conjugates to a small nucleophile (see Fig EV2A; Pickart & Rose, 1985; Wenzel et al, 2011). Nucleophile is present at huge molar excess thus circumventing the need for a specific substrate interaction. Ub discharge from Ubc6~Ub and U7BR/Ubc7~Ub was followed as a function of time (Fig 2C), and rates were extracted (Fig 2D), providing a metric for substrate-independent E2 discharge activity. While U7BR/Ubc7~Ub undergoes aminolysis like most E2s, Ubc6 has been reported to be hydroxy-reactive (Wang et al, 2009; Weber et al, 2016). We therefore used ethanolamine as the nucleophile, as it provides both an amino and a hydroxy group. While this experimental design allows for uniform reaction conditions, a direct comparison of the E2s' discharge rates is inadvisable due to the inherent reactivity profile differences. Also, the discharge rates reported here are not directly transferrable to ubiquitylation of substrate in vivo. Despite these caveats, the assays allow assessment of the stimulatory effect of an E3 for a given E2, which we report as the ratio of the discharge rates with and without E3 (Fig 2E). U7BR/Ubc7~Ub discharges very slowly in the absence of an E3 and Hrd1 greatly accelerates the process. In contrast, Ubc6~Ub discharges quite rapidly on its own and is only mildly stimulated by Hrd1. Quantitatively, Hrd1 is roughly an order of magnitude more potent at stimulating discharge from U7BR/Ubc7 than from Ubc6. These results are consistent with the observations made in the in vitro substrate ubiquitylation assays and in line with the difference in binding affinity. They suggest that the difference observed in the in vitro ubiquitylation assay (see Fig 2B) is due to Hrd1's more effective stimulation of Ubc7. Altogether, the binding and functional assays indicate that the known functional in vivo preference of Hrd1 for Ubc7 over Ubc6 for priming is dictated mainly by binding affinity. Click here to expand this figure. Figure EV2. In vitro Ub nucleophile discharge assay data A. Cartoon depiction of in vitro Ub nucleophile discharge assays. |Nu depicts the nucleophile with its electron lone pair, which in the reactions shown here is the bi-functional molecule ethanolamine. B. SDS–PAGE run under reducing conditions of samples at the beginning and end of indicated discharge reactions to show the degree of E2 auto-ubiquitylation ("-Ub") during each reaction. C, D. Quantification of Ub discharge assays with U7BR/Ubc7 and indicated Doa10 (C) and Hrd1 (D) variants. Plots of Ubc7˜Ub discharge as a function of time (dots) and first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show absolute reaction rates derived from these fits. For ease of comparison, the y-axis of these insets is the same in C–F. The "no E3" control is identical in both C and D. E, F. Quantification of Ub discharge assays with Ubc6 and indicated Doa10 (E) and Hrd1 (F) variants. Plots of Ubc6˜Ub discharge as a function of time (dots) and first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show absolute reaction rates derived from these fits. For ease of comparison, the y-axis of these insets is the same in C–F. The "no E3" control is identical in both E and F. G. Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by indicated RING variants. Direct comparison of all tested E2 stimulations, shown as the ratio of rates derived from RING-catalyzed reactions and respective "no E3" controls from C and E. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by one-way ANOVA test; *P < 0.05. For clarity, only significances related to the respective "no E3" control of a given E2 are shown. Gray background indicates data shown in main figures (Figs 2E, 3A, and 4C). Source data are available online for this figure. Download figure Download PowerPoint RING linchpin plays a key role in Ubc7 stimulation Having established that Ubc7 can carry out priming with Hrd1, we wondered why it reportedly does not do so with Doa10, especially as the competing E2 for priming, Ubc6, binds with comparably low affinity to Doa10 (see Fig 2A). Hrd1 and Doa10 differ in their linchpin residues (Arg and His, respectively; see Fig 1F), suggesting the answer might lie in how the two E3s stimulate Ubc7. To investigate the contribution of linchpins in the stimulation of Ubc7, we generated Hrd1 and Doa10 RING variants that harbor one of four amino acids at their linchpin positions (arginine—potent hydrogen bond donor and linchpin of Hrd1; histidine—potential hydrogen bond donor and linchpin of Doa10; alanine—no hydrogen bond potential; or glutamate—hydrogen bond acceptor and charge reversal of arginine). A strong dependence on linchpin identity was observed in Ub discharge assays for U7BR/Ubc7 acting with either E3 ligase (Fig 3A): For both, arginine is most effective, histidine and alanine are much less effective, and glutamate provides only minor stimulation of U7BR/Ubc7 activity over a "no E3" control. Wild-type Hrd1(400R) elicits the highest Ub discharge rates from U7BR/Ubc7 with rates achieved by wild-type Doa10(94H) being roughly three times lower. Notably, Doa10 with an arginine linchpin (94R) is as effective as wild-type Hrd1. These observations imply that Hrd1-mediated Ubc7 stimulation is driven by the identity of the linchpin. Figure 3. Ubc7-mediated Ub chain elongation is not rate-determining for protein degradation A. In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig EV2C and D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by one-way ANOVA test; *P < 0.05. For clarity, only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set. B. In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by one-way ANOVA test; *P < 0.05. For clarity, only significances related to the "no E3" control of given reaction set are shown. C. Steps of ERAD protein degradation with associated rate constants; S = ER protein; S* = misfolded protein, i.e., ERAD substrate; kobs = observed rate of protein degradation in assays; kUb1 = rate of the priming reaction; kUb2 = rate of the first elongation step, i.e., mono-Ub to di-Ub; kUb3 − kUbn = rates of subsequent elongation steps; k0 − kj = rates for other steps of ERAD; the black box identifies steps of substrate poly-ubiquitylation with priming highlighted by the green box and chain elongation by the yellow box. The light yellow background highlights the mono-Ub to di-Ub reactions. D. Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means