Unexpectedly two group VIII E s OsUBC

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 SB505124 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 Ubiquitin (Ub) has been considered as one of the most important regulatory proteins in eukaryotes since it was first discovered in 1978 (Rechsteiner, 1987; Severo et al., 2013; Collins and Brown, 2010; Jiang and Chen, 2011; Wenzel et al., 2011). Ubiquitination is an enzymatic process including three sequential steps performed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzymes (E2) and the ubiquitin-ligases (E3) (Dupre et al., 2004; Jiang and Chen, 2011; Wenzel et al., 2011), which involves in the bonding of the Ub to target proteins through the covalent attachment to regulate a multitude of cellular processes. The ubiquitination starts with the activation of Ub by E1 in an ATP-dependent reaction to generate an E1∼Ub conjugate, and then the activated Ub is handed over to an E2 via a transthiolation reaction. The E2∼Ub conjugate selectively interacts with E3s that recruits and binds to specific substrates (Hershko et al., 1982; Wijk and Timmers, 2010; Stewart et al., 2016).