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  • br Materials and methods br


    Materials and methods
    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 Itraconazole synthesis 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.
    Acknowledgments This work was supported by a grant from the National Center for GM Crops (PJ008152) of the Next Generation BioGreen 21 Program funded by the Rural Development Administration, Republic of Korea, to W.T.K.
    Introduction The E2 enzyme is the central component in the transfer of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins to targets in diverse conjugation pathways. During the ubiquitination of proteins, an Ub/Ubl is first adenylated by the E1 enzyme, and then transferred to a conserved cysteine residue on the E1 protein through a thioester linkage formed with the terminal carboxyl group of the Ub/Ubl. A subsequent trans-thiolation reaction transfers the Ub/Ubl to a conserved cysteine residue on the E2 enzyme. In the final step of the cascade, the Ub/Ubl is transferred from the E2 to the ε-amino group of lysine residues on protein substrates or the amine group of phosphatidylethanolamine in the case of the autophagy pathway (Ciechanover et al., 2000, Ichimura et al., 2000). This final step is usually mediated by E3 ligases that may function in one of two distinct ways: The HECT-like E3 ligases transfer the Ub/Ubl from E2 to an internal cysteine through a further trans-thiolation step before transferring it to the target, whereas the RING (U-Box) and A20 finger-type E3 ligases appear to only bridge the E2 enzyme and the substrate with the target lysine residues (Aravind et al., 2003, Ardley and Robinson, 2005, Wertz et al., 2004). The role of the E2 enzymes in the Ub/Ubl conjugation cascade, and the importance of the conserved cysteine shared by all catalytically active E2 enzymes in the trans-thiolation reaction are well-established (Berleth and Pickart, 1996, Dye and Schulman, 2007, Hershko et al., 1983, Pickart, 2001). The transfer of Ub/Ubl from E1 to E2 involves a nucleophilic attack by the conserved E2 cysteine on the carbonyl group of the Ub/Ubl-E1 thioester linkage (Pickart, 2001). Experiments have suggested that this reaction is primarily catalyzed by residues on the E1 protein in addition to the E2 cysteine (Wu Itraconazole synthesis et al., 2003).