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PUB22-E2 pairing is determined by both the U-box domain and ARM repeats and can be induced by flg22. (A) PUB22-E2 pairing as determined by BiFC in Arabidopsis protoplasts. Detected interactions with cYFPUBCs are denoted with a yellow dot for nYFP-PUB22 U-box, blue for nYFP-PUB22 full-length, and green

PUB22-E2 pairing is determined by both the U-box domain and ARM repeats and can be induced by flg22. (A) PUB22-E2 pairing as determined by BiFC in Arabidopsis protoplasts. Detected interactions with cYFPUBCs are denoted with a yellow dot for nYFP-PUB22 U-box, blue for nYFP-PUB22 full-length, and green

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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 catalyse the attachment of ubiquitin and have emerged as ke...

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... PUB22-E2 pairs displayed distinct subcellular localizations. Most pairs, including UBC5, UBC8 and UBC36, showed a dual nuclear and cytoplasmic localization ( Figure 1B and Table S1). By contrast, the interaction with UBC26 was localized exclusively in the nucleus and was more pronounced in the nucleolus ( Figure 1B). ...
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... pairs, including UBC5, UBC8 and UBC36, showed a dual nuclear and cytoplasmic localization ( Figure 1B and Table S1). By contrast, the interaction with UBC26 was localized exclusively in the nucleus and was more pronounced in the nucleolus ( Figure 1B). UBC32 was shown to localize in the ER where it participates in ERAD (21). ...
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... was shown to localize in the ER where it participates in ERAD (21). Accordingly, the interaction with UBC32 displayed reticulate and perinuclear localization, reminiscent of the ER ( Figure 1B). ...
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... 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 ten E2s: UBCs 8, 10, 16, 17, 18, 28, 29, 30, 35 and 36 (Table S1, Figure 1A, S1 and S2). In most cases the ARM repeats shifted the subcellular localization of PUB22-E2 pairs from the nucleus and the cytoplasm to punctae in the cytoplasm ( Figure 1C and Table S1). ...
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... PUB22 interaction was restricted to ten E2s: UBCs 8, 10, 16, 17, 18, 28, 29, 30, 35 and 36 (Table S1, Figure 1A, S1 and S2). In most cases the ARM repeats shifted the subcellular localization of PUB22-E2 pairs from the nucleus and the cytoplasm to punctae in the cytoplasm ( Figure 1C and Table S1). This localization is reminiscent of that observed for PUB22 and its cognate substrate Exo70B2 in BiFC (9). ...
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... localization is reminiscent of that observed for PUB22 and its cognate substrate Exo70B2 in BiFC (9). By contrast, interaction with UBC35 or UBC36 was also cytoplasmic ( Figure 1D), similar to the subcellular localization detected for PUB22-MPK3 interaction in BiFC (8). On the other hand, full-length PUB22 and UBCs 1, 2, 3, 5, 11, 26, 32, 33, which interacted with the U-box only, were not detected ( Figure 1A and Table S1). ...
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... contrast, interaction with UBC35 or UBC36 was also cytoplasmic ( Figure 1D), similar to the subcellular localization detected for PUB22-MPK3 interaction in BiFC (8). On the other hand, full-length PUB22 and UBCs 1, 2, 3, 5, 11, 26, 32, 33, which interacted with the U-box only, were not detected ( Figure 1A and Table S1). ...
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... 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 ( Figure 1C). However, in all other cases no major effects could be observed. ...
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... we tested the specificity of PUB22- E2 pairing by including the Trp40Ala PUB22 point mutant variant, which is impaired in its autoubiquitination activity (4,10). Without exception, interaction of all PUB22-interacting E2s with the Trp40Ala mutant variant was inhibited ( Figure 1D and Table S1). This additionally confirms that the PUB22-E2 pairing occurs via the canonical U-box and UBC surfaces (22). ...
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... interacted with the majority of the eight E2s belonging to group VI ( Figure 1A). Group VI are prototypical E2s, which are almost exclusively composed of the UBC domain (Figure 2A and S3). ...
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... remains unknown whether E2-E3 pairing is regulated in response to specific cellular cues. Our BiFC analysis already suggested that interaction between PUB22 and UBC26 is induced by treatment with the immunogenic peptide flg22 ( Figure 1C and Table S1). Indeed, SLCA confirmed that activation of the immune response by flg22 resulted in enhanced interaction ( Figure 5A and S8B). ...
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... domains may restrict E2 interaction by sequestering PUB22 to its determined subcellular localisation. Indeed, localization of the BiFC signal was distinct between the U-box only and the full-length PUB22 ( Figure 1B and 1C). However, many of the E2s that interacted only with the truncated form were cytoplasmic and should in principle have access to the U-box domain of the full- length protein. ...
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... E2s that were unable to interact with PUB22 (e.g. UBC9 and UBC12; Figure 1A) also contained the motif (Figure S3), suggesting that this feature is not critical for specificity, but generally required for interaction. ...
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... note, our observations reveal that some PUB22-E2 pairings are dynamic and change in response to cellular status. Interaction with UBC30 was reduced, while PUB22 association to UBC26 and UBC35 was induced after activation of the immune response by flg22 ( Figure 1C, 5A to 5C). These results therefore open the possibility that in addition to a "division of labour", the catalytic properties of PUB22, as conveyed by the paired E2, change in response to altered cellular status, such as the activation of the immune response. ...
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... carrying genes encoding selected E2s (UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35) or COP10 and E3s (PUB20, PUB22, PUB24, PUB4, PUB13, MIEL1, and AvrPtoB) were generated with the use of Golden Gate cloning (71). Briefly, the expression vector encoding E2 protein with N-terminal cLuc or nLuc portion of Firefly Luc and HA-or Myc-tag also carried a gene encoded Renilla Luc, and the expression of the E2 fusion protein and Renilla Luc protein was driven by separate 35S promoters. ...
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... PUB22-E2 pairs displayed distinct subcellular localizations. Most pairs, including UBC5, UBC8 and UBC36, showed a dual nuclear and cytoplasmic localization ( Figure 1B and Table S1). By contrast, the interaction with UBC26 was localized exclusively in the nucleus and was more pronounced in the nucleolus ( Figure 1B). ...
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... pairs, including UBC5, UBC8 and UBC36, showed a dual nuclear and cytoplasmic localization ( Figure 1B and Table S1). By contrast, the interaction with UBC26 was localized exclusively in the nucleus and was more pronounced in the nucleolus ( Figure 1B). UBC32 was shown to localize in the ER where it participates in ERAD (21). ...
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... was shown to localize in the ER where it participates in ERAD (21). Accordingly, the interaction with UBC32 displayed reticulate and perinuclear localization, reminiscent of the ER ( Figure 1B). ...
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... 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 ten E2s: UBCs 8, 10, 16, 17, 18, 28, 29, 30, 35 and 36 (Table S1, Figure 1A, S1 and S2). In most cases the ARM repeats shifted the subcellular localization of PUB22-E2 pairs from the nucleus and the cytoplasm to punctae in the cytoplasm ( Figure 1C and Table S1). ...
Context 20
... PUB22 interaction was restricted to ten E2s: UBCs 8, 10, 16, 17, 18, 28, 29, 30, 35 and 36 (Table S1, Figure 1A, S1 and S2). In most cases the ARM repeats shifted the subcellular localization of PUB22-E2 pairs from the nucleus and the cytoplasm to punctae in the cytoplasm ( Figure 1C and Table S1). This localization is reminiscent of that observed for PUB22 and its cognate substrate Exo70B2 in BiFC (9). ...
Context 21
... localization is reminiscent of that observed for PUB22 and its cognate substrate Exo70B2 in BiFC (9). By contrast, interaction with UBC35 or UBC36 was also cytoplasmic ( Figure 1D), similar to the subcellular localization detected for PUB22-MPK3 interaction in BiFC (8). On the other hand, full-length PUB22 and UBCs 1, 2, 3, 5, 11, 26, 32, 33, which interacted with the U-box only, were not detected ( Figure 1A and Table S1). ...
Context 22
... contrast, interaction with UBC35 or UBC36 was also cytoplasmic ( Figure 1D), similar to the subcellular localization detected for PUB22-MPK3 interaction in BiFC (8). On the other hand, full-length PUB22 and UBCs 1, 2, 3, 5, 11, 26, 32, 33, which interacted with the U-box only, were not detected ( Figure 1A and Table S1). ...
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... 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 ( Figure 1C). However, in all other cases no major effects could be observed. ...
Context 24
... we tested the specificity of PUB22- E2 pairing by including the Trp40Ala PUB22 point mutant variant, which is impaired in its autoubiquitination activity (4,10). Without exception, interaction of all PUB22-interacting E2s with the Trp40Ala mutant variant was inhibited ( Figure 1D and Table S1). This additionally confirms that the PUB22-E2 pairing occurs via the canonical U-box and UBC surfaces (22). ...
Context 25
... interacted with the majority of the eight E2s belonging to group VI ( Figure 1A). Group VI are prototypical E2s, which are almost exclusively composed of the UBC domain (Figure 2A and S3). ...
Context 26
... remains unknown whether E2-E3 pairing is regulated in response to specific cellular cues. Our BiFC analysis already suggested that interaction between PUB22 and UBC26 is induced by treatment with the immunogenic peptide flg22 ( Figure 1C and Table S1). Indeed, SLCA confirmed that activation of the immune response by flg22 resulted in enhanced interaction ( Figure 5A and S8B). ...
Context 27
... domains may restrict E2 interaction by sequestering PUB22 to its determined subcellular localisation. Indeed, localization of the BiFC signal was distinct between the U-box only and the full-length PUB22 ( Figure 1B and 1C). However, many of the E2s that interacted only with the truncated form were cytoplasmic and should in principle have access to the U-box domain of the full- length protein. ...
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... E2s that were unable to interact with PUB22 (e.g. UBC9 and UBC12; Figure 1A) also contained the motif (Figure S3), suggesting that this feature is not critical for specificity, but generally required for interaction. ...
Context 29
... note, our observations reveal that some PUB22-E2 pairings are dynamic and change in response to cellular status. Interaction with UBC30 was reduced, while PUB22 association to UBC26 and UBC35 was induced after activation of the immune response by flg22 ( Figure 1C, 5A to 5C). These results therefore open the possibility that in addition to a "division of labour", the catalytic properties of PUB22, as conveyed by the paired E2, change in response to altered cellular status, such as the activation of the immune response. ...
Context 30
... carrying genes encoding selected E2s (UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35) or COP10 and E3s (PUB20, PUB22, PUB24, PUB4, PUB13, MIEL1, and AvrPtoB) were generated with the use of Golden Gate cloning (71). Briefly, the expression vector encoding E2 protein with N-terminal cLuc or nLuc portion of Firefly Luc and HA-or Myc-tag also carried a gene encoded Renilla Luc, and the expression of the E2 fusion protein and Renilla Luc protein was driven by separate 35S promoters. ...

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... Our assay could also be used with E3 and substrates from other plant species. Additionally, our assay can also be applied to single-subunit E3 ligases by replacing the SCF components with a monomeric E3 ligase, while considering the replacement of suitable E2 [41]. ...
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... Along with these genes, three other genes related to the immune system were also found to show footprints of recent selection. For example, UBP13 (Fig. 5e) encodes a ubiquitin-specific protease that is responsible for initial pathogen perception 60 , and UBC36 encodes an E2 ubiquitin-conjugating enzyme involved in dampening immune signaling 61 . The wall-associated receptor-like kinase gene, WAK2, also plays an important role in disease resistance 62 . ...
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... A0A173FEH2 is an E3 ubiquitin ligase SCF complex subunit SKP1/ASK1 family protein and an ortholog of ARABIDOPSIS SKP-LIKE 2 (ASK2); this protein forms a complex with TIR1 in the SCFTIR1 complex and is required for the auxin response in Arabidopsis thaliana [41,42]. A0A1D5STP8 is a ubiquitin-conjugating enzyme E2 whose Arabidopsis ortholog UBC35, together with UBC36, positively regulates plant auxin responses [43]. Therefore, our data imply that auxin synthesis and signaling were repressed in the wheat seedling roots under relatively high-B stress. ...
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... For example, RGLG1/2 ubiquitin E3 ligase can interact with StUBC13 to form a E2-E3 complex in potato using Y2H, SLC, and BiFC technology assays, and the complex is involved in the iron deficiency response of Arabidopsis roots [30]. In this regard, future research will focus on how StUBC13-StUEV1s conjugates different E3s to form Lys-63-linked poly-Ub chains and influences target proteins activities to regulate multiple cellular processes, such as [8,[31][32][33][34][35]. ...
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... Moreover, PAMP treatment leads to an increase in PUB4 accumulation, which promotes its role in the stabilization of activated BIK1. Both accumulation of PUB4 and phosphorylation of residues in the hinge region after immunostimulation are reminiscent of the stabilization mechanism first described for PUB22, in which phosphorylation inhibits autoubiquitination and degradation (Furlan et al, 2017), and may also contribute to regulate association to E2s (Kowarschik et al, 2018;Turek et al, 2018). While we reveal here that PUB4 positively regulates activated BIK1 accumulation, the E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B were recently shown to promote BIK1 activation , thus illustrating that distinct BIK1 ubiquitination events positively regulate both its accumulation and activation. ...
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... The dimeric E3s RNF4 and Birc7, contact the E2 via a single protomer, and Ub is folded back onto the E2 contacting both E3 RING domains to induce the 'closed state' [33,44]. Increased reactivity of E2s, in the presence of their cognate E3, is as expected conserved in plants [45]. The closed conformation has emerged as a hallmark of activation for ubiquitin, SUMO and Nedd8 E2s [25]. ...
... Combined with the conserved features in RING/U-box and HECT E3 ligases, it clarifies how E2s interact with several E3s. Identification of physiological E2-E3 pairs has remained one of the key challenges to elucidate ubiquitination mechanisms [9,40,[45][46][47]. ...
... However, both approaches miss important cellular determining factors, such as intracellular localization, interacting cofactors, post-translational regulation, as well as other competing E2s, which may be a defining feature. An in vivo screen using U-box E3s, confirmed that one E3 interacts with various E2s, and may therefore, modify substrates with different chain types [45]. Moreover, while the U-box itself confers pairing selectivity, the substrate-interaction domain adds an additional layer of selectivity by restricting the number of interacting E2s. ...
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... For instance, OsUBC47 showed a potential association with D3 in LC-MS analysis and formed DTT-sensitive thioester bonds with OsUb in vitro, but it could not facilitate formation of the polyubiquitination chain when incubated with the SCF D3-GFP complex purified from transgenic calli (supplemental Table 1 and supplemental Figure 4C and 4D). The Arabidopsis OsUBC47 orthologs AtUBC35/36 need to be combined with a UEV counterpart to work sufficiently in chain formation (Kowarschik et al., 2017;Turek et al., 2018), which provides new clues to further investigate the activity of OsUBC47 in coordinating with SCF D3 E3 ligase. ...
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Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes. Detecting E3 ligase activity in vitro is important for exploring the molecular mechanism of protein ubiquitination. However, in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve, especially in plants, mainly due to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs. In this study, we characterized components of the rice SCFDWARF3 (SCFD3) E3 ligase, screened the coordinated E2, and reconstituted active SCFD3 E3 ligase in vitro. We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1 (eSCR) protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53, which is the key transcriptional repressor in strigolactone signaling. Finally, we replaced D3 with other F-box proteins from rice and humans, and reconstituted active eSCF E3 ligases, including eSCFGID2, eSCFFBXL18 and eSCFCDC4 E3 ligases. Together, this work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins, providing a powerful platform to study the mechanisms of multisubunit SCF E3 ligases in eukaryotes.
... Hence, the physiological significance of physical interaction between AtUbc13 and AtCPR1 re-mains to be elucidated. AtUbc13 has been found to interact with two U-box E3 ligases, AtPUB22 and AtPUB24 (Turek et al., 2018), that are involved in plant immune responses (Trujillo et al., 2008). Whether these two Ub ligases serve as cognate E3s for AtUbc13 in plant immunity and whether this process requires K63-linked polyubiquitination need to be explored. ...
... tomato (Pst) DC3000 (Song et al., 2015). Interestingly, PARylation and K63-linked ubiquitination coordinately regulate pathogen-associated molecular pattern-triggered immunity (Turek et al., 2018;Yao et al., 2021). Both ubc13a,b and parp1,2 double mutants not only display enhanced disease susceptibility but also are compromised in flg22-or SA-induced resistance to Pst DC3000. ...
Article
Ubiquitination is one of the best known post-translational modifications in eukaryotes, in which different linkage types of polyubiquitination result in different outputs of the target proteins. Distinct from the well-characterized K48-linked polyubiquitination that usually serves as a signal for the target protein degradation, K63-linked polyubiquitination often requires a unique E2 heterodimer Ubc13-UEV and alters the target protein activity instead of degradation. This review focuses on recent advances on roles of Ubc13-UEV mediated K63-linked polyubiquitination in plant growth, development and response to environmental stresses.
... The priming mechanism can require the formation of a dimer, in which each protomer contacts ubiquitin to position it for catalysis (104). In line with this, an E2 mutant that irreversibly binds ubiquitin displays stronger interaction with PUB22, suggesting that ubiquitin contributes to E2-E3 pairing (127). However, yeast and human UFD2s are monomeric and utilize an allosteric mechanism for conformational restriction (105). ...
... Studies using in vitro autoubiquitination activity had previously shown a pairing specificity between E2 and E3s (21,65,66). A pairwise screen to identify E2s that interact with PUB22 in vivo detected 11 UBCs belonging to 4 different groups, out of 37 tested Arabidopsis E2s (127). Interaction specificity was dictated by both the U-box as well as the ARM repeats. ...
... Further analyses with a subset of E2s showed that PUB22 and the closely related PUB20 and PUB24, as well as the UND-containing PUB4, interact with UBC35 in vivo. Because UBC35 is dedicated to building Lys63-linked chains, these E3s most likely mediate the modification of substrates with this chain type (107,127). By contrast, PUB13, which was shown to control FLS2 levels and mediate BRI1 internalization, did not interact with UBC35 (127), suggesting that it may pair with other E2s to mediate endocytosis or may require activation. ...
Article
Posttranslational modifications add complexity and diversity to cellular proteomes. One of the most prevalent modifications across eukaryotes is ubiquitination, which is orchestrated by E3 ubiquitin ligases. U-box-containing E3 ligases have massively expanded in the plant kingdom and have diversified into plant U-box proteins (PUBs). PUBs likely originated from two or three ancestral forms, fusing with diverse functional subdomains that resulted in neofunctionalization. Their emergence and diversification may reflect adaptations to stress during plant evolution, reflecting changes in the needs of plant proteomes to maintain cellular homeostasis. Through their close association with protein kinases, they are physically linked to cell signaling hubs and activate feedback loops by dynamically pairing with E2-ubiquitin-conjugating enzymes to generate distinct ubiquitin polymers that themselves act as signals. Here, we complement current knowledge with comparative genomics to gain a deeper understanding of PUB function, focusing on their evolution and structural adaptations of key U-box residues, as well as their various roles in plant cells. Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.