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. 2018 Oct 19;293(42):16324-16336.
doi: 10.1074/jbc.RA118.004226. Epub 2018 Sep 5.

Multi-tiered Pairing Selectivity Between E2 Ubiquitin-Conjugating Enzymes and E3 Ligases

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Free PMC article

Multi-tiered Pairing Selectivity Between E2 Ubiquitin-Conjugating Enzymes and E3 Ligases

Ilona Turek et al. J Biol Chem. .
Free PMC article

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.

Keywords: E3 ubiquitin ligase; cell signaling; innate immunity; ubiquitin; ubiquitin-conjugating enzyme (E2 enzyme).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
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 cYFP-UBCs are denoted with a yellow dot for nYFP-PUB22 U-box, blue for nYFP-PUB22 full-length, and green for interactions of full-length nYFP-PUB22 after treatment with 1 μm flg22 for 1 h. The experiment was repeated with similar results. Shown are the phylogenetic relationships between A. thaliana E2 UBCs, and roman numbers indicate subgroups. The well-characterized human HsUBE2D2 enzyme was included for comparison. The phylogenetic tree was generated using the neighbor-joining method. B, BiFC of the nYFP-PUB22 U-box with cYFP-UBC8, cYFP-UBC26, or cYFP-UBC32, as indicated. Arrowheads show the nucleus and arrows indicate perinuclear mesh-like structure. C, BiFC of full-length nYFP-PUB22 with cYFP-UBC8 or cYFP-UBC26, as indicated. Protoplasts were treated with 1 μm flg22 for 1 h. Arrowheads indicate the nucleus and arrows show punctate structures. D, BiFC of nYFP-PUB22 or the inactive nYFP-PUB22 W40A mutant variant with cYFP-UBC35. Protein expression was confirmed by immunoblot of total protein samples. Coomassie Brilliant Blue (CBB) shows equal loading. B–D, interactions were assayed using transient expression in A. thaliana Col-0 protoplasts. Free mCherry was coexpressed to label the cytoplasm and nucleus. Pictures are representative of three independent experiments with similar results. Y, YFP. Scale bar = 50 μm. IB, immunoblot.
Figure 2.
Figure 2.
PUB22 pairing selectivity with the highly homologous group VI E2s. A, structural model of UBC30 from group VI. U-box/RING interacting surfaces are highlighted in blue. B, SLCA of cLUC-PUB22 with nLUC-fused group VI E2s: UBC8, UBC9, UBC10, UBC11, UBC12, UBC28, UBC29, and UBC30, as indicated. Arabidopsis mesophyll protoplasts were transiently co-transformed with constructs containing the indicated genes. Transformation efficiencies were normalized by Renilla luciferase in the vector harboring the E2. Values indicate the average value of three independent biological experiments ± S.D. Statistically significant differences indicated by different letters were determined by one-way analysis of variance (ANOVA) and Tukey post hoc test (p < 0.05). C, in vitro autoubiquitination assay using MBP-tagged wildtype (WT) or W40A mutant variant of PUB22 (E3), His-UBA1 (E1), His-UBCs (E2s) from group VI, and fluorescein-tagged ubiquitin (FITC-Ub). D, in vitro time course autoubiquitination assay using MBP-PUB22, His-UBA1, His-UBC9, or His-UBC30, and FITC-Ub. Coomassie brilliant blue (CBB) shows equal loading. IB, immunoblot.
Figure 3.
Figure 3.
Characterization of PUB22-interacting E2s. A, SLCA of cLUC-PUB22 with nLUC-fused UBC1 (group III), UBC5 (group IV), UBC9 (group VI), UBC12 (group VI), UBC17 (group VII), UBC26 (group XI), UBC28 (group VI), UBC30 (group VI), COP10, and UBC35 (group XV) fused to cLuc with nLuc-PUB22 transiently co-transformed in Arabidopsis mesophyll protoplasts. Transformation efficiencies were normalized by Renilla luciferase harbored in the vector containing the E2. Values indicate the average value of three independent biological experiments ± S.D. Statistically significant differences indicated by different letters were determined by one-way ANOVA and Tukey post hoc test (p < 0.05). B, in vitro autoubiquitination assay with MBP-PUB22 in the presence of ubiquitin, Arabidopsis His-UBA1, and His-tagged E2s: UBC1 (group III), UBC5 (group IV), or UBC30 (group VI), as a positive control. Ubiquitination reactions were stopped after 2 h. C, in vitro autoubiquitination of MBP-PUB22 in the presence of ubiquitin, His-UBA1, and His-tagged UBC17 (group VII) or UBC30 (group VI), as a positive control. Ubiquitination reaction was incubated for 1 h or overnight (o/n). D, in vitro autoubiquitination of MBP-PUB22 in the presence of ubiquitin, His-UBA1 and His-tagged UBC35 (group XV) with His-tagged Uev1D, or UBC30 (group VI), as a positive control. Ubiquitination reaction was stopped after 2 h. Vertical line indicates high molecular weight species of poly-ubiquitinated PUB22. B–D, ubiquitination reactions were resolved in a 7 and 12% Tris glycine PAGE. Arrowhead indicates the expected size of the ubiquitin-E2 conjugate. CBB, Coomassie brilliant blue; Ub, ubiquitin; IB, immunoblot.
Figure 4.
Figure 4.
Pairing of selected E2s with different types of E3s. A–F, SLCA of PUB20 composed of U-box (yellow) and ARM repeats (A), PUB24 composed of U-box (yellow) and ARM repeats (B), PUB4 composed of UND domain, U-box (yellow) and ARM repeats (C), PUB13 composed of UND domain, U-box (yellow) and ARM repeats (D), MIEL1 composed of two zinc fingers and a RING domain (red) (E), and AvrPtoB composed of two kinase-interacting domains and a U-box-like domain (purple) (F). E3s were assayed against UBC1 (group III), UBC5 (group IV), UBC9 (group VI), UBC12 (group VI), UBC17 (group VII), UBC26 (group XI), UBC28 (group VI), UBC30 (group VI), UBC35 (group XV), and COP10 transiently co-transformed in Arabidopsis mesophyll protoplasts. Transformation efficiencies were normalized by Renilla Luc harbored in the E2-containing vector. Strongest signal intensities are shown in blue. Values indicate the average of three independent biological experiments ± S.D. Statistically significant differences indicated by different letters were determined by one-way ANOVA and Tukey post hoc test (p < 0.05).
Figure 5.
Figure 5.
Activation of the immune response by flg22 treatment modulates E2–PUB22 pairing. A, SLCA of cLuc-PUB22 with nLuc-UBC26 transiently co-transformed in Arabidopsis mesophyll protoplasts and treated with the indicated concentrations of flg22 for 1 h. B, SLCA of nLuc-PUB22 with cLuc-UBC35 transiently co-transformed in Arabidopsis mesophyll protoplasts and treated with the indicated concentrations of flg22 for 1 h. C, SLCA of nLuc-PUB22 with cLuc-UBC30 transiently co-transformed in Arabidopsis mesophyll protoplasts and treated with the indicated concentrations of flg22 for 1 h. A–C, transformation efficiencies were normalized by Renilla Luc harbored in the E2-containing vector. Values indicate the average of three independent biological experiments ± S.D. Statistically significant differences indicated by different letters were determined by one-way ANOVA and Tukey post hoc test (p < 0.05).
Figure 6.
Figure 6.
UBC35 and UBC36 participate in immunity. A, root growth inhibition in Col-0 WT, pub22 pub23 pub24, and ubc35 ubc36 seedlings 6 days after transplanting (dat) onto media containing 1 μm flg22. B, length of the main root of seedlings in A. Data shown as mean of three independent experiments ± S.D., n ≥ 38. Statistically significant differences indicated by different letters were determined by one-way ANOVA and Tukey post hoc test (p < 0.05). C, total production of ROS during 60 min after treatment with 100 nm flg22. Data are shown as median ± S.D. Statistical significance compared with Col-0 plants is indicated with asterisks (Student's t test, **, p < 0.01). ROS production was evaluated in three independent experiments with similar results. D, oxidative burst generated during 1 h post-treatment with 100 nm flg22. Data are shown as mean ± S.D., n = 9. ROS production was evaluated in three independent experiments with similar results. E, infection assays with the virulent bacterial pathogen Pst DC3000. Col-0, pub22 pub23 pub24, and ubc35 ubc36 plants were spray-inoculated with a bacterial suspension of 5 × 108 colony forming units/ml. Infection and bacterial growth were assessed at 3 days after inoculation (dai). Data shown as median ± S.D., n = 6. Similar results were obtained in four independent experiments. F, representative Col-0, pub22 pub23 pub24, and ubc35 ubc36 seedlings 3 days after inoculation with the virulent bacterial pathogen Pst DC3000.

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