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  • To function as Ub ligases

    2021-01-19

    To function as Ub-ligases, E3 proteins must interact with E2s. As compared to the extensive studies on E3s, functional studies on E2s are relatively rudimentary in higher plants. We previously reported that there are 48 genes encoding Ub-conjugating (UBC) fold-containing putative E2 proteins in the rice genome that are divided into three 1625 [23]. In another study, Kraft et al. [24] sorted 37 Arabidopsis E2s into 14 groups (groups III–XVI) based on detailed sequence homology analyses. In addition, Arabidopsis has a single SUMO-conjugating enzyme (AtSCE1a) and two Related to Ub-conjugating (RUB) enzymes (RCE1 and RCE2) that were classified into groups I and II, respectively [24]. In this report, we further classified the 48 rice E2s into 15 different groups. Yeast two-hybrid (Y2H) assays were performed to examine the interaction profiles of 40 E2s with 17 ARM-U-box E3s in rice. Of the 40 E2s, 11 E2s belonging to groups VI, VII, and VIII accounted for 70% of the E2–E3 interactions. These E2–E3 interactions were further validated by in vitro self-ubiquitination assays with rice SPL11 ARM-U-box E3 and various E2 partners. SPL11 E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, and no ubiquitination, depending on the various E2s. Overall, these results suggest that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.
    Materials and methods
    Results
    Discussion Our results based on the Y2H analysis between the U-box-motif region of E3s and the UBC domain of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3 Ub-ligases (Fig. 2, Fig. 3). These 11 E2s belong to groups VI, VII, and VIII (Fig. 1 and Supplementary Table S1 and Fig. S2). Thus, a single E2 could interact with multiple ARM-U-box E3s and a relatively small number of E2s interact with a large number of functional ARM-U-box E3 Ub-ligases. This supports the hypothesis that the rice system contains E2 hubs for E2–E3 interactions. There are significant sequence identities between rice group VI and VII E2s and human E2s that function as hubs for interactions with E3s [30]. These results suggest that the human E2 hub system may be conserved in rice. However, Y2H results obtained in our current study were based on the interactions between the U-box-motif region of E3s and the UBC domain of E2s. Therefore, we could not exclude the possibility that interactions between full-length E2s and E3s would give rise to different interaction patterns. Unexpectedly, two group VIII E2s (OsUBC29 and OsUBC30), which lack the catalytic Cys residues in their UBC domain and have no Ub-conjugating activity, interacted with numerous ARM-U-box E3s (Fig. 2, Fig. 3). The human UEV protein MMS2 (UBE2V2) forms a functional E2 complex through hetero-dimerization with Ubc13 E2 [31], [32]. Thus, future studies will examine if rice UEVs are involved in the regulation of E3 Ub-ligase activity by dimerization with other E2s. The rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns based on its different E2 partners (Fig. 4). SPL11 exhibited poly-ubiquitination activity with group VI E2s (OsUBC14, OsUBC15, OsUBC16, and OsUBC18), mono-ubiquitination activity with group VII OsUBC25, and no activity with group VI OsUBC19. The interaction between SPL11 and OsUBC25 was relatively weak compared to other SPL11–E2 interactions (Fig. 2, Fig. 3). Thus, the mono-ubiquitination pattern of SPL11 with OsUBC25 may be due to the weak interaction. An alternative possibility is that SPL11 E3 and OsUBC25 E2 specifically mono-ubiquitinates substrate proteins. This possibility is supported by UBE2W, a human homolog of OsUBC25, that mono-ubiquitinates substrate proteins with its E3 partner [35], [36]. A more detailed functional relationship between SPL11 and OsUBC25 remains to be elucidated. Arabidopsis COP10, which is homologous to rice VI E2s, lacks the active site Cys residue and is an inactive E2 [37]. Instead of functioning as an active E2, COP10 interacts with other active E2s and enhances their Ub-conjugating activities. This suggests that seemingly inactive E2s may still participate in ubiquitination regulation. Similarly, rice group VI UBC19 E2 that also lacks the catalytic Cys residue interacts with several ARM-U-box E3s (Fig. 2, Fig. 3) without detectable E2 activity (Fig. 4). Thus, a possible function of OsUBC19 is interacting with other E2s during ubiquitination activity in rice.