The N-terminal tetra-peptide (IPDE) short extension of the U-box motif in rice SPL11 E3 is essential for the interaction with E2 and ubiquitin-ligase activity
Introduction
The post-translational modification of proteins with ubiquitin (Ub) or poly-Ub chains regulates many cellular processes, including protein degradation, DNA repair, and cellular trafficking [1], [2], [3], [4]. Ubiquitination involves a cascade of enzymatic reactions in which Ub is transferred from an E1 Ub-activating enzyme to an E2 Ub-conjugating enzyme, forming a thioester bond with a cysteine residue of E2 [5]. E2 subsequently associates with an E3 Ub-ligase, which recognizes a target protein and facilitates the transfer of Ub from E2 to a lysine residue of the substrate [5].
There are three classes of E3 Ub-ligases, defined by the presence of a HECT, RING, or U-box domain. HECT E3s form a catalytic Ub thioester intermediate before transferring Ub to the protein substrates, whereas RING and U-box E3s catalyze the direct transfer of Ub from E2 to target proteins [6]. Despite the very similar structures of the RING and U-box domains, the U-box does not coordinate Zn2+ ion, whereas the RING domain does [7], [8]. Higher plants contain a larger number of U-box proteins compared to that of yeasts and mammals. For example, Arabidopsis and rice, dicot and monocot model plants, respectively, have at least 64 and 77 U-box motif-containing E3 proteins, with the majority containing the protein–protein interacting ARM repeat motif [9], [10], [11], [12]. By contrast, yeasts and humans have 3 and 7 U-box E3s, respectively [13], [14]. Plant U-box E3s participate in many diverse plant-specific events, such as responses to phytohormones [15], [16], biotic stress [17], [18], and abiotic stress [19], [20], self-incompatibility [21], and control of flowering time [22]. These results are consistent with the proliferation of U-box proteins in plants relative to yeasts and mammals.
There are at least 37 and 48 E2 Ub-conjugating enzymes in Arabidopsis and rice, respectively [23]. Although the U-box proteins have to interact with E2s for their E3 ligase activities and there are large numbers of U-box E3s in plant genomes, studies of the U-box structure or E2-binding requirements are still limited in higher plants [24], [25]. We want to understand how the specific interactions between E3s and E2s are controlled in plants. The specific aim of this study was to explore the minimal E2-binding site of the U-box domain in E3. For this purpose, we used SPL11, an ARM repeat-containing U-box E3 Ub-ligase, which plays a role as a negative regulator of programmed cell death in rice (Oryza sativa L.) [17]. The yeast two-hybrid assay and studies of in vitro self-ubiquitination in conjunction with site-directed mutagenesis indicate that, in addition to an intact U-box motif, an N-terminal tetra-peptide (IPDE) short extension of the U-box is required for the interaction of rice SPL11 E3 with E2s. These results suggest that the short extension of the U-box motif critically affects the interacting interface between the U-box and E2 for the E3 Ub-ligase activity in rice.
Section snippets
Yeast two-hybrid assay
The cDNAs for full-length rice SPL11 and seven deletion mutants of SPL11were inserted into the pGAD T7 vector (Clontech, Mountain View, CA, USA). The gene constructs were then transformed into yeast strain Y187. Full-length rice E2 and human UBCH5b E2 cDNAs were inserted into the pGBK T7 vector, and subsequently transformed into yeast strain AH109. For mating experiments, each pair of individual Y187 and AH109 transformants were inoculated and grown for 4 days at 30 °C in –Leu/–Trp media for
Identification of the minimal E2-binding site of the U-box domain in rice SPL11 E3 Ub-ligase
An interacting network analysis of human E2-RING E3 Ub-ligase revealed that only 10 of 35 active E2s were able to account for more than 80% of the total E2–E3 interactions. This indicates that a relatively small number of E2s are closely connected with a larger number of E3s [27], [28]. These studies suggest that E2–E3 interactions are not uniformly distributed, but there are E2 hubs for the interaction with their E3 partners in humans. To identify which E2 proteins interact with SPL11 U-box E3
Discussion
Plant U-box E3 Ub ligases regulate diverse plant-specific cellular processes [1], [2], [3], [4]. However, structural and functional relationships between U-box E3s and their E2 partners are largely unknown in higher plants [24], [25]. 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
Acknowledgments
This work was supported by Grants from the National Research Foundation (2010-0000782) and 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.
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