br Materials and methods br
Materials and methods
Discussion Plant U-box E3 Ub ligases regulate diverse plant-specific cellular processes , , , . However, structural and functional relationships between U-box E3s and their E2 partners are largely unknown in higher plants , . In this study, we investigated the minimal binding domain of rice SPL11 U-box E3–E2 Ub-conjugating enzyme by yeast two-hybrid assay and analysis of in vitro self-ubiquitination in combination with site-directed mutagenesis. Our results revealed that SPL11 U-box E3 interacted with sub-group I E2s, which were homologous with a hub-group of human E2s  (Fig. 1 and Supplementary Fig. S1). SPL11 failed to bind to E2s belonging to the sub-groups II and III E2s, both of which were more distantly related to the human hub. Consistent with these results, SPL11 exhibited Ub-ligase activity with sub-group I E2s, but not with sub-groups II and III E2s (Fig. 1). Recently published results showed that large numbers of Arabidopsis RING and U-box E3s displayed in vitro Ub-ligase activities with AtUBC8 and AtUBC10 E2s, which were similar to the human E3 hub group , , , . Thus, it could be possible to speculate that, similar to human systems, E2–E3 interactions in rice do not depend on a uniform distribution, but E3 ligases are active within specific E2 hubs. However, more detailed investigations must be performed to examine this possibility. The U-box motif was necessary but not sufficient for the interaction of SPL11 E3 with its E2 partners (Fig. 2). BIIB021 of the entire C-terminal region (amino-acid residues 222–575) of SPL11 did not significantly affect its binding activity with E2 (Fig. 2A and B). In contrast, serial deletions of the N-terminal region resulted in the gradual decrease in binding activity between SPL11 and its E2 partner (Fig. 2C). This suggests that the N-terminal extension of the U-box is involved, at least in part, in the binding of SPL11 E3 and E2s. When the short N-terminal extension that consists of the IPDE tetra-sequence from –4 to –1 position of the U-box was removed, the binding capacity of SPL11 to E2 completely disappeared (Fig. 2). Of these four amino-acid residues, Ile and Pro residues at the –4 and –3 positions were essential to SPL11 for both its interaction with E2 and its Ub-ligase activity (Fig. 3). The Pro residue is conserved in approximately 90% of total rice U-box proteins. The N-terminal extension, along with eleven amino-acid residues in the U-box motif, was predicted to form the loop-1 structure (Fig. 3A) . This raised the possibility that, although the N-terminal short extension of the U-box motif may not directly participate in the binding to the E2 partner, it critically affected the interacting interface between the U-box and E2 and the Ub-ligase activity of rice SPL11 E3. In contrast to the N-terminal extension, no C-terminal extension is necessary for the function of the U-box in SPL11 (Fig. 2). However, deletion of the C-terminal ten amino-acid residues of the U-box resulted in complete loss of binding activity to E2 (Fig. 2). This region is involved in the formation of the helix-2 structure (Fig. 4A). This helix-2 structure forms a three-dimensional hydrophobic core with the loop-1 in the N-terminal region of the U-box , . Thus, deletion of the C-terminal ten amino-acid residues of the U-box may cause disruption of the hydrophobic core surrounding the U-box. This result indicates its critical role in the interaction between the U-box and E2. Although the U-box E3 Ub-ligases from rice and Arabidopsis have different structures (e.g., locations of the U-box motif and presence of additional functional domains), all these plant U-box E3s contain well-conserved tetra-peptide N-terminal extension. On the other hand, the amino-acid sequences of the N-terminal extension of the human and yeast U-box E3s are relatively divergent. Of seven U-box E3s in human, three do not have a Pro residue at the –3 position of the U-box (Supplementary Fig. S2). Furthermore, the human and yeast Prp19 U-box protein homologs contain neither the N-terminal extension nor the C-terminal helix of the U-box domain with functional E3 Ub-ligase activity . Thus, it may be proposed that the N-terminal tetra-peptide extension, along with the greater number of U-box E3s, may be correlated not only with their association with E2 but also with the diverse cellular functions of U-box E3s in higher plants. Additional functional and structural studies will be necessary to further investigate the plant-specific roles of U-box E3 Ub-ligases in higher plants.