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• W2890878422 abstract "Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin–conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyze the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, in vivo E2–E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 Arabidopsis E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signaling. We show that whereas the U-box domain, which mediates E2 docking, is able to interact with 18 of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with 11 E2s. In vitro activity assayed by autoubiquitination only partially recapitulated the in vivo selectivity. Moreover, in vivo pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, whereas interaction with the K63 chain-building UBC35 was increased. Functional analysis of ubc35 ubc36 mutants shows that they partially mimic pub22 pub23 pub24 enhanced activation of immune responses. Together, our work provides a framework to interrogate in vivo E2–E3 pairing and reveals a multi-tiered and dynamic E2–E3 network. Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin–conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyze the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, in vivo E2–E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 Arabidopsis E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signaling. We show that whereas the U-box domain, which mediates E2 docking, is able to interact with 18 of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with 11 E2s. In vitro activity assayed by autoubiquitination only partially recapitulated the in vivo selectivity. Moreover, in vivo pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, whereas interaction with the K63 chain-building UBC35 was increased. Functional analysis of ubc35 ubc36 mutants shows that they partially mimic pub22 pub23 pub24 enhanced activation of immune responses. Together, our work provides a framework to interrogate in vivo E2–E3 pairing and reveals a multi-tiered and dynamic E2–E3 network. To date no physiological E2 ubiquitin–conjugating enzyme–E3 ubiquitin ligase pair has been reported for plants. Most research has focused on E3 ligases because they are the specificity determinants of the ubiquitination process. However, E2s mediate the attachment of ubiquitin mainly onto lysine residues of a substrate, in most cases guided by one of the more than 1400 E3s encoded in the Arabidopsis genome. The surfacing complexity of the different functions carried out by E2s contradicts their early image as simple carriers of activated ubiquitin. They are first loaded with ubiquitin by the ubiquitin-activating enzyme E1 via transthiolation forming a thioester bond with their catalytic cysteine and subsequently are recognized by an E3 ligase. The E2 then discharges ubiquitin onto either the E3, a process commonly used to test E3 activity termed autoubiquitination, or onto a substrate, in which case the E3 acts as an adaptor. E2s have emerged as key mediators of ubiquitin chain assembly and have been demonstrated to be able to govern the switch from ubiquitin chain initiation to elongation, as well as regulate the processivity of chain formation (1.Stewart M.D. Ritterhoff T. Klevit R.E. Brzovic P.S. E2 enzymes: more than just middle men.Cell Res. 2016; 26 (27002219): 423-44010.1038/cr.2016.35Crossref PubMed Scopus (271) Google Scholar). Substrates can be modified by attachment of a ubiquitin polymer (polyubiquitination), in which single moieties are linked to one another through one of seven lysine residues present in ubiquitin, or its initial methionine (2.Kulathu Y. Komander D. Atypical ubiquitylation: the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.Nat. Rev. Mol. Cell Biol. 2012; 13 (22820888): 508-52310.1038/nrm3394Crossref PubMed Scopus (486) Google Scholar). Importantly, E2s to a large extent dictate the Lys residue within ubiquitin used to link the moieties in a chain (1.Stewart M.D. Ritterhoff T. Klevit R.E. Brzovic P.S. E2 enzymes: more than just middle men.Cell Res. 2016; 26 (27002219): 423-44010.1038/cr.2016.35Crossref PubMed Scopus (271) Google Scholar). The resulting different linkage types lead to alternative topologies of the polyubiquitin chains, which in turn are responsible for the different fates endowed onto the modified proteins (2.Kulathu Y. Komander D. Atypical ubiquitylation: the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.Nat. Rev. Mol. Cell Biol. 2012; 13 (22820888): 508-52310.1038/nrm3394Crossref PubMed Scopus (486) Google Scholar). These can include the degradation of proteins tagged with Lys48-linked ubiquitin chains by the 26S proteasome, or regulate the transit of integral membrane proteins through the endomembrane system. The Arabidopsis genome encodes 37 ubiquitin E2 enzymes, all of which display a highly conserved cysteine residue at the predicted catalytic site and are classified into 16 groups based on the similarity to each other (3.Kraft E. Stone S.L. Ma L. Su N. Gao Y. Lau O.S. Deng X.W. Callis J. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis.Plant Physiol. 2005; 139 (16339806): 1597-161110.1104/pp.105.067983Crossref PubMed Scopus (296) Google Scholar). Compared with ubiquitin ligases, relatively little is known about the biological processes in which specific E2s participate. However, we recently were able to show intrinsic biochemical activity of 31 E2s in an orthogonal system using synthetic biology (3.Kraft E. Stone S.L. Ma L. Su N. Gao Y. Lau O.S. Deng X.W. Callis J. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis.Plant Physiol. 2005; 139 (16339806): 1597-161110.1104/pp.105.067983Crossref PubMed Scopus (296) Google Scholar, 4.Kowarschik K. Hoehenwarter W. Marillonnet S. Trujillo M. UbiGate: a synthetic biology toolbox to analyse ubiquitination.New Phytol. 2018; 217 (29194629): 1749-176310.1111/nph.14900Crossref PubMed Scopus (19) Google Scholar), confirming that most of the predicted, but largely unexplored E2s, display activity. Previous studies spearheaded the analysis of E2–E3 pairing by assaying the ubiquitination activity of E2s in combination with a set of RING E3 ligases employing in vitro autoubiquitination assays (3.Kraft E. Stone S.L. Ma L. Su N. Gao Y. Lau O.S. Deng X.W. Callis J. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis.Plant Physiol. 2005; 139 (16339806): 1597-161110.1104/pp.105.067983Crossref PubMed Scopus (296) Google Scholar, 5.Wiborg J. O'Shea C. Skriver K. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.Biochem. J. 2008; 413 (18393940): 447-45710.1042/BJ20071568Crossref PubMed Scopus (97) Google Scholar). However, whether in vitro autoubiquitination activity faithfully recapitulates all aspects of E2–E3 pairing in vivo remains open. Therefore, data interpretation pertaining to its relevance in vivo must be made with caution for several reasons. (i) The E2s usually employed to perform in vitro ubiquitination assay display high processivity and promiscuity and are therefore active with a wide range of E3 ligases. These include the commonly used UBC8 and UBC11 from group VI, which are homologues of the human UBE2D group of E2s shown to prime ubiquitination (6.Jin L. Williamson A. Banerjee S. Philipp I. Rape M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex.Cell. 2008; 133 (18485873): 653-66510.1016/j.cell.2008.04.012Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar), but are unlikely to confer chain-linkage specificity (7.Brzovic P.S. Lissounov A. Christensen D.E. Hoyt D.W. Klevit R.E. A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination.Mol. Cell. 2006; 21 (16543155): 873-88010.1016/j.molcel.2006.02.008Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). (ii) Factors that may be required to determine the specificity of an E2–E3 pairing, such as additional interacting proteins or post-translational modifications, are absent. (iii) High concentrations of recombinant proteins employed in these assays can lead to unspecific E2–E3 interactions. (iv) Spatio-temporal resolution that restricts the encounter of E2–E3 pairs within the cell is absent. Therefore, to fully understand the potentially distinct cellular roles played by any E3, it is of central importance to identify its physiological E2 counterparts, as they define to a large extent its biochemical properties. Here we interrogated the in vivo E2–E3 pairing employing the well-characterized plant U-box protein 22 (PUB22), 3The abbreviations used are: PUB22plant U-box protein 22PRRpattern recognition receptorARMarmadilloBiFCbimolecular fluorescence complementationHAhemagglutininUBCubiquitin-conjugating enzymeCOP10constitutive photomorphogenic 10UNDU-box N-terminal domainROSreactive oxygen speciesANOVAanalysis of varianceMBPmaltose-binding proteinSLCAsplit luciferase complementation assayLucluciferaseUevubiquitin-conjugating enzyme variantYFPyellow fluorescent protein. which contributes to the regulation of immune signaling (8.Furlan G. Nakagami H. Eschen-Lippold L. Jiang X. Majovsky P. Kowarschik K. Hoehenwarter W. Lee J. Trujillo M. Changes in PUB22 ubiquitination modes triggered by mitogen-activated protein kinase3 dampen the immune response.Plant Cell. 2017; 29 (28280093): 726-74510.1105/tpc.16.00654Crossref PubMed Scopus (51) Google Scholar, 9.Stegmann M. Anderson R.G. Ichimura K. Pecenkova T. Reuter P. Žársky V. McDowell J.M. Shirasu K. Trujillo M. The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis.Plant Cell. 2012; 24 (23170036): 4703-471610.1105/tpc.112.104463Crossref PubMed Scopus (161) Google Scholar, 10.Trujillo M. Ichimura K. Casais C. Shirasu K. Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis.Curr. Biol. 2008; 18 (18771922): 1396-140110.1016/j.cub.2008.07.085Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 11.Chen Y.C. Wong C.L. Muzzi F. Vlaardingerbroek I. Kidd B.N. Schenk P.M. Root defense analysis against Fusarium oxysporum reveals new regulators to confer resistance.Sci. Rep. 2014; 4 (24998294): 5584Crossref PubMed Scopus (62) Google Scholar, 12.Jacobs S. Zechmann B. Molitor A. Trujillo M. Petutschnig E. Lipka V. Lipka V. Kogel K.H. Schäfer P. Broad-spectrum suppression of innate immunity is required for colonization of Arabidopsis roots by the fungus Piriformospora indica.Plant Physiol. 2011; 156 (21474434): 726-74010.1104/pp.111.176446Crossref PubMed Scopus (209) Google Scholar), as well as drought responses (13.Cho S.K. Ryu M.Y. Song C. Kwak J.M. Kim W.T. Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress.Plant Cell. 2008; 20 (18664614): 1899-191410.1105/tpc.108.060699Crossref PubMed Scopus (186) Google Scholar, 14.Seo D.H. Ahn M.Y. Park K.Y. Kim E.Y. Kim W.T. The N-terminal UND motif of the Arabidopsis U-Box E3 ligase PUB18 is critical for the negative regulation of ABA-mediated stomatal movement and determines its ubiquitination specificity for exocyst subunit Exo70B1.Plant Cell. 2016; 28 (27956469): 2952-297310.1105/tpc.16.00347Crossref PubMed Scopus (61) Google Scholar, 15.Seo D.H. Ryu M.Y. Jammes F. Hwang J.H. Turek M. Kang B.G. Kwak J.M. Kim W.T. Roles of four Arabidopsis U-box E3 ubiquitin ligases in negative regulation of abscisic acid-mediated drought stress responses.Plant Physiol. 2012; 160 (22829319): 556-56810.1104/pp.112.202143Crossref PubMed Scopus (107) Google Scholar, 16.Trujillo M. News from the PUB: plant U-box type E3 ubiquitin ligases.J. Exp. Bot. 2018; 69 (29237060): 371-38410.1093/jxb/erx411Crossref PubMed Scopus (71) Google Scholar). Mutants of PUB22 and its homologues PUB23 to PUB26, display additive hyper-activation of signaling mediated by plasma membrane-localized pattern recognition receptors (PRRs) that perceive pathogen-derived molecules (10.Trujillo M. Ichimura K. Casais C. Shirasu K. Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis.Curr. Biol. 2008; 18 (18771922): 1396-140110.1016/j.cub.2008.07.085Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 16.Trujillo M. News from the PUB: plant U-box type E3 ubiquitin ligases.J. Exp. Bot. 2018; 69 (29237060): 371-38410.1093/jxb/erx411Crossref PubMed Scopus (71) Google Scholar, 17.Wang J. Grubb L.E. Wang J. Liang X. Li L. Gao C. Ma M. Feng F. Li M. Li L. Zhang X. Yu F. Xie Q. Chen S. Zipfel C. Monaghan J. Zhou J.M. A regulatory module controlling homeostasis of a plant immune kinase.Mol. Cell. 2018; 69 (29358080): 493-504.e496Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). These include the bacterial protein flagellin, or its derived peptide flg22, which is recognized by the FLS2 receptor (18.Chinchilla D. Bauer Z. Regenass M. Boller T. Felix G. The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception.Plant Cell. 2006; 18 (16377758): 465-47610.1105/tpc.105.036574Crossref PubMed Scopus (568) Google Scholar). Similar to animals, plant PRRs activate innate immune responses to fend off pathogens (19.Couto D. Zipfel C. Regulation of pattern recognition receptor signalling in plants.Nat. Rev. Immunol. 2016; 16 (27477127): 537-55210.1038/nri.2016.77Crossref PubMed Scopus (631) Google Scholar). PUB22 mediates the degradation of components of the exocyst complex, which are required for full activation of PRR signaling, during the immune response (8.Furlan G. Nakagami H. Eschen-Lippold L. Jiang X. Majovsky P. Kowarschik K. Hoehenwarter W. Lee J. Trujillo M. Changes in PUB22 ubiquitination modes triggered by mitogen-activated protein kinase3 dampen the immune response.Plant Cell. 2017; 29 (28280093): 726-74510.1105/tpc.16.00654Crossref PubMed Scopus (51) Google Scholar). plant U-box protein 22 pattern recognition receptor armadillo bimolecular fluorescence complementation hemagglutinin ubiquitin-conjugating enzyme constitutive photomorphogenic 10 U-box N-terminal domain reactive oxygen species analysis of variance maltose-binding protein split luciferase complementation assay luciferase ubiquitin-conjugating enzyme variant yellow fluorescent protein. PUB22 is composed of an N-terminal U-box that mediates E2 pairing and four armadillo (ARM) repeats that interact with its substrates (Fig. 1A). The U-box is a domain of ∼70 amino acids present in proteins from yeast to human. It consists of a typical fold stabilized by a network of hydrogen bonds that is structurally similar to RING domains (20.Ohi M.D. Vander Kooi C.W. Rosenberg J.A. Ren L. Hirsch J.P. Chazin W.J. Walz T. Gould K.L. Structural and functional analysis of essential pre-mRNA splicing factor Prp19p.Mol. Cell. Biol. 2005; 25 (15601865): 451-46010.1128/MCB.25.1.451-460.2005Crossref PubMed Scopus (72) Google Scholar). Employing cell-based assays we determined in vivo E2–PUB22 pairing and uncover different layers of specificity. The U-box, which provides the docking surface, mediates interaction with a subset of 18 E2s. However, the ARM repeats impose higher specificity allowing interaction with 11 E2s. Of note, activation of the immune response-modulated interaction with E2s from different subgroups known to have distinct catalytic properties. This suggests that PUB22 mediates distinct catalytic activities. The observed pairing specificities in vivo were only partially recapitulated by in vitro autoubiquitination. Our work therefore reveals various factors that regulate E2–E3 pairing, whereas in addition providing a framework for their study. To reveal the cellular function of the E3 ligase PUB22 we screened for PUB22–E2 pairs using a cell-based assay employing bimolecular fluorescence complementation (BiFC) that allows the in vivo visualization of protein–protein interactions. We subcloned all 37 E2 genes encoded by the Arabidopsis genome (3.Kraft E. Stone S.L. Ma L. Su N. Gao Y. Lau O.S. Deng X.W. Callis J. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis.Plant Physiol. 2005; 139 (16339806): 1597-161110.1104/pp.105.067983Crossref PubMed Scopus (296) Google Scholar) to generate N-terminal HA-cYFP and Myc-nYFP E2 or E3 fusion proteins, respectively. We first assayed the interaction between the E2s and a truncated PUB22 carrying the U-box only to test the specificity conferred by this domain on its own. The UBC domain of E2s, which mediates pairing, is highly conserved. We therefore anticipated that the U-box of PUB22 would be able to interact with various E2s. Indeed, BiFC was detected for 18 out 37 tested E2s, including UBCs 1, 2, 3, 5, 8, 10, 11, 16, 17, 18, 26, 28, 29, 30, 32, 33, 35, and 36 (Fig. 1A, and Figs. S1 and S2). Therefore, the U-box domain on its own conferred only a low degree of specificity. U-box PUB22–E2 pairs displayed distinct subcellular localizations. Most pairs, including UBC5, UBC8, and UBC36, showed a dual nuclear and cytoplasmic localization (Fig. 1B and Table S1). By contrast, the interaction with UBC26 was localized exclusively in the nucleus and was more pronounced in the nucleolus (Fig. 1B). UBC32 was shown to localize in the endoplasmic reticulum where it participates in endoplasmic reticulum-associated degradation (21.Cui F. Liu L. Zhao Q. Zhang Z. Li Q. Lin B. Wu Y. Tang S. Xie Q. Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance.Plant Cell. 2012; 24 (22214659): 233-24410.1105/tpc.111.093062Crossref PubMed Scopus (186) Google Scholar). Accordingly, the interaction with UBC32 displayed reticulate and perinuclear localization, reminiscent of the endoplasmic reticulum (Fig. 1B). We next tested whether the ARM repeats, which mediate the interaction with substrates, influence the specificity of the PUB22–E2 pairing. The presence of the ARM domains had a dramatic impact on both the localization and the specificity of the PUB22–E2 pairing. Full-length PUB22 interaction was restricted to 10 E2s: UBCs 8, 10, 16, 17, 18, 28, 29, 30, 35, and 36 (Table S1, Fig. 1A, and Figs. S1 and S2). In most cases the ARM repeats shifted the subcellular localization of PUB22–E2 pairs from the nucleus and cytoplasm to punctae in the cytoplasm (Fig. 1C and Table S1). This localization is reminiscent of that observed for PUB22 and its cognate substrate Exo70B2 in BiFC (9.Stegmann M. Anderson R.G. Ichimura K. Pecenkova T. Reuter P. Žársky V. McDowell J.M. Shirasu K. Trujillo M. The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis.Plant Cell. 2012; 24 (23170036): 4703-471610.1105/tpc.112.104463Crossref PubMed Scopus (161) Google Scholar). By contrast, interaction with UBC35 or UBC36 was also cytoplasmic (Fig. 1D), similar to the subcellular localization detected for the PUB22-MPK3 interaction in BiFC (8.Furlan G. Nakagami H. Eschen-Lippold L. Jiang X. Majovsky P. Kowarschik K. Hoehenwarter W. Lee J. Trujillo M. Changes in PUB22 ubiquitination modes triggered by mitogen-activated protein kinase3 dampen the immune response.Plant Cell. 2017; 29 (28280093): 726-74510.1105/tpc.16.00654Crossref PubMed Scopus (51) Google Scholar). On the other hand, interactions between full-length PUB22 and UBCs 1, 2, 3, 5, 11, 26, 32, 33, which interacted with the U-box only, were not detected (Fig. 1A and Table S1). PUB22 is regulated by the activation of PRRs, including FLS2 (8.Furlan G. Nakagami H. Eschen-Lippold L. Jiang X. Majovsky P. Kowarschik K. Hoehenwarter W. Lee J. Trujillo M. Changes in PUB22 ubiquitination modes triggered by mitogen-activated protein kinase3 dampen the immune response.Plant Cell. 2017; 29 (28280093): 726-74510.1105/tpc.16.00654Crossref PubMed Scopus (51) Google Scholar, 9.Stegmann M. Anderson R.G. Ichimura K. Pecenkova T. Reuter P. Žársky V. McDowell J.M. Shirasu K. Trujillo M. The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis.Plant Cell. 2012; 24 (23170036): 4703-471610.1105/tpc.112.104463Crossref PubMed Scopus (161) Google Scholar). We therefore tested whether treatment with flg22 influenced PUB22–E2 pairing. Elicitation with flg22 induced the interaction of the full-length PUB22 with UBC26, which was detected in the nucleus and nucleolus (Fig. 1C). However, in all other cases no major effects could be observed. Finally, we tested the specificity of PUB22–E2 pairing by including the W40A PUB22 point mutant variant, which is impaired in its autoubiquitination activity (4.Kowarschik K. Hoehenwarter W. Marillonnet S. Trujillo M. UbiGate: a synthetic biology toolbox to analyse ubiquitination.New Phytol. 2018; 217 (29194629): 1749-176310.1111/nph.14900Crossref PubMed Scopus (19) Google Scholar, 10.Trujillo M. Ichimura K. Casais C. Shirasu K. Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis.Curr. Biol. 2008; 18 (18771922): 1396-140110.1016/j.cub.2008.07.085Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Without exception, interaction of all PUB22-interacting E2s with the W40A mutant variant was inhibited (Fig. 1D and Table S1). This additionally confirms that the PUB22–E2 pairing occurs via the canonical U-box and UBC surfaces (22.Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Chaperoned ubiquitylation: crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex.Mol. Cell. 2005; 20 (16307917): 525-53810.1016/j.molcel.2005.09.023Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Together, we identified 11 PUB22-interacting E2s and we show that both U-box, as well as the ARM repeats, contribute to the pairing specificity. Moreover, the data revealed that activation of the immune response can induce interaction between PUB22 and UBC26. PUB22 interacted with the majority of the eight E2s belonging to group VI (Fig. 1A). Group VI are prototypical E2s, which are almost exclusively composed of the UBC domain (Fig. 2A and Fig. S3). E2s dock onto U-box/RING domain of E3s via the L1 and L2 loops and the N-terminal α-helix (Fig. 2A, dark blue) with low affinity, which is thought to be a requirement for the rapid exchange between binding to the ubiquitin-E2 conjugate, the aminolysis reaction and the subsequent undocking of the unloaded E2 (1.Stewart M.D. Ritterhoff T. Klevit R.E. Brzovic P.S. E2 enzymes: more than just middle men.Cell Res. 2016; 26 (27002219): 423-44010.1038/cr.2016.35Crossref PubMed Scopus (271) Google Scholar, 23.Eletr Z.M. Kuhlman B. Sequence determinants of E2-E6AP binding affinity and specificity.J. Mol. Biol. 2007; 369 (17433363): 419-42810.1016/j.jmb.2007.03.026Crossref PubMed Scopus (53) Google Scholar). As a consequence, commonly used techniques such as co-immunoprecipitations are unsuitable to confirm the interaction. We therefore opted to use a split luciferase complementation assay (SLCA), which allows the detection of weak interactions, and in contrast to BiFC in which complementation is irreversible, maintains association-dissociation dynamics (24.Stefan E. Aquin S. Berger N. Landry C.R. Nyfeler B. Bouvier M. Michnick S.W. Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17942691): 16916-1692110.1073/pnas.0704257104Crossref PubMed Scopus (155) Google Scholar). Moreover, normalization of the transformation efficiency was performed via Renilla luciferase (Luc) harbored in the vector containing the UBCs by dual-luciferase detection. We validated the specificity of the interaction detected via SLCA by using two different variants of PUB22 carrying point mutations to alanine in the U-box known to interfere with autoubiquitination (10.Trujillo M. Ichimura K. Casais C. Shirasu K. Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis.Curr. Biol. 2008; 18 (18771922): 1396-140110.1016/j.cub.2008.07.085Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Trp40 is predicted to be located on an α-helix distant from the E2 docking surface and not to be required to maintain the core structure of the U-box (25.Ohi M.D. Vander Kooi C.W. Rosenberg J.A. Chazin W.J. Gould K.L. Structural insights into the U-box, a domain associated with multi-ubiquitination.Nat. Struct. Biol. 2003; 10 (12627222): 250-25510.1038/nsb906Crossref PubMed Scopus (223) Google Scholar). Cys13 is located on one of the two loops that make contact with the E2 (22.Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Chaperoned ubiquitylation: crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex.Mol. Cell. 2005; 20 (16307917): 525-53810.1016/j.molcel.2005.09.023Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). When compared with the WT PUB22, the W40A variant was still able to partially interact, whereas the C13A mutation abrogated the interactions with UBC30 as well as UBC10 and UBC11 (Fig. S4). These results show the high sensitivity and specificity of the SLCA to investigate E2–E3 interactions. Analysis of PUB22 and group VI E2 pairing by SLCA confirmed that PUB22 interacted with UBC8, UBC10, UBC11, UBC28, UBC29, and UBC30 (Fig. 2B and Fig. S5). Signal intensities for PUB22 interactions with UBC9 and UBC12, which were not detected by BiFC, were also significantly weaker in SLCA. Hence, we were able to confirm our initial results and show in vivo specificity in the interaction of PUB22 and group VI E2s. Previous reports show that the U-box domain binds with higher affinity to the loaded E2 (26.Soss S.E. Klevit R.E. Chazin W.J. Activation of UbcH5c∼Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B.Biochemistry. 2013; 52 (23550736): 2991-299910.1021/bi3015949Crossref PubMed Scopus (40) Google Scholar), which can be mediated by binding of the E2 and the loaded ubiquitin to the RING domain (27.Plechanovova A. Jaffray E.G. McMahon S.A. Johnson K.A. Navratilova I. Naismith J.H. Hay R.T. Mechanism of ubiquitylation by dimeric RING ligase RNF4.Nat. Struct. Mol. Biol. 2011; 18 (21857666): 1052-105910.1038/nsmb.2108Crossref PubMed Scopus (131) Google Scholar, 28.Dou H. Buetow L. Sibbet G.J. Cameron K. Huang D.T. BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer.Nat. Struct. Mol. Biol. 2012; 19 (22902369): 876-88310.1038/nsmb.2379Crossref PubMed Scopus (239) Google Scholar). To investigate a possible effect of ubiquitin in binding of the E2 with the U-box of PUB22 we replaced the catalytic cysteine residue at position 85 of UBC30 with alanine, which impairs ubiquitin loading, or a lysine, which is able to receive ubiquitin by forming an isopeptide bond (27.Plechanovova A. Jaffray E.G. McMahon S.A. Johnson K.A. Navratilova I. Naismith J.H. Hay R.T. Mechanism of ubiquitylation by dimeric RING ligase RNF4.Nat. Struct. Mol. Biol. 2011; 18 (21857666): 1052-105910.1038/nsmb.2108Crossref PubMed Scopus (131) Google Scholar). Compared with the WT UBC30, the inactive C85A variant displayed reduced, whereas the C85K variant displayed enhanced, Luc activity (Fig. S6). This suggests that ubiquitin loading may enhance UBC30 binding to PUB22 U-box. To test whether interaction specificity is maintained in vitro, we carried out autoubiquitination assays. In contrast to the differences observed between the interactions in vivo, ubiquitin chain formation activities of PUB22 in combination with group VI E2s did not show any major changes under the used conditions (Fig. 2C). Nevertheless, in all instances ubiquitination was inhibited in the case of the W40A mutant, indicating that interaction with the E3 was required for activity (Fig. 2C). However, detailed time course analyses of PUB22 autoubiquitination revealed differences in the processivity between the in vivo noninteracting UBC9 and the in vivo interacting UBC30 (Fig. 2D). We next selected representative E2s that were identified in the BiFC screen and compared the interaction using SLCA. Selected E2s included UBC17 (group VII), UBC26 (group XI), UBC28 (group VI), UBC30 (group VI), and UBC35 (group XV). In addition, we included as negative controls UBC9 and UBC12 (group VI), which did not interact with PUB22 in BiFC, as well as UBC1 (group III) and UBC5 (group IV), which interacted in BiFC only wi" @default.
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• W2890878422 date "2018-10-01" @default.
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• W2890878422 title "Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases" @default.
• W2890878422 cites W1612017191 @default.
• W2890878422 cites W1758089766 @default.
• W2890878422 cites W1963990261 @default.
• W2890878422 cites W1979019144 @default.
• W2890878422 cites W1980468156 @default.
• W2890878422 cites W1981975060 @default.
• W2890878422 cites W1988617344 @default.
• W2890878422 cites W1992219896 @default.
• W2890878422 cites W1996766766 @default.
• W2890878422 cites W2001701156 @default.
• W2890878422 cites W2005030601 @default.
• W2890878422 cites W2010517835 @default.
• W2890878422 cites W2017556424 @default.
• W2890878422 cites W2027792462 @default.
• W2890878422 cites W2029794768 @default.
• W2890878422 cites W2030264283 @default.
• W2890878422 cites W2034203606 @default.
• W2890878422 cites W2041517382 @default.
• W2890878422 cites W2043328334 @default.
• W2890878422 cites W2053384550 @default.
• W2890878422 cites W2055019434 @default.
• W2890878422 cites W2056998476 @default.
• W2890878422 cites W2059680729 @default.
• W2890878422 cites W2060295565 @default.
• W2890878422 cites W2060410804 @default.
• W2890878422 cites W2061671463 @default.
• W2890878422 cites W2066812847 @default.
• W2890878422 cites W2070272438 @default.
• W2890878422 cites W2072015681 @default.
• W2890878422 cites W2073376866 @default.
• W2890878422 cites W2089692504 @default.
• W2890878422 cites W2090412780 @default.
• W2890878422 cites W2092090989 @default.
• W2890878422 cites W2096993874 @default.
• W2890878422 cites W2098158539 @default.
• W2890878422 cites W2101066284 @default.
• W2890878422 cites W2103144304 @default.
• W2890878422 cites W2105595936 @default.
• W2890878422 cites W2107440564 @default.
• W2890878422 cites W2109954733 @default.
• W2890878422 cites W2110943831 @default.
• W2890878422 cites W2114097399 @default.
• W2890878422 cites W2115804728 @default.
• W2890878422 cites W2118683297 @default.
• W2890878422 cites W2122525664 @default.
• W2890878422 cites W2122672983 @default.
• W2890878422 cites W2124825049 @default.
• W2890878422 cites W2134320577 @default.
• W2890878422 cites W2135988226 @default.
• W2890878422 cites W2146149989 @default.
• W2890878422 cites W2149616074 @default.
• W2890878422 cites W2149863714 @default.
• W2890878422 cites W2151965686 @default.
• W2890878422 cites W2157873949 @default.
• W2890878422 cites W2161581369 @default.
• W2890878422 cites W2171984357 @default.
• W2890878422 cites W2254998690 @default.
• W2890878422 cites W2304335366 @default.
• W2890878422 cites W2479540212 @default.
• W2890878422 cites W2520799606 @default.
• W2890878422 cites W2562623982 @default.
• W2890878422 cites W2565884518 @default.
• W2890878422 cites W2588534968 @default.
• W2890878422 cites W2593432225 @default.
• W2890878422 cites W2774113045 @default.
• W2890878422 cites W2774984099 @default.
• W2890878422 cites W2775574987 @default.
• W2890878422 cites W2783088162 @default.
• W2890878422 cites W2790961781 @default.
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