RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice

Rice EL5 is an ATL family gene characterized by a transmembrane domain at the N-terminal and a RING-H2 finger domain (RFD), which exhibits ubiquitin ligase (E3) activity. To elucidate the physiological roles of EL5, we analyzed transgenic rice plants overexpressing mutant EL5 proteins that are impaired in E3 activity to various degrees. Plants expressing EL5C153A and EL5W165A, which encode an inactive E3, showed a rootless phenotype accompanied by cell death in root primordia, and those expressing EL5V162A, with moderately impaired E3 activity, formed short crown roots with necrotic lateral roots. The dominant-negative phenotype was specifically observed in root meristems where EL5 is expressed, and not recovered by exogenous auxin. When wild-type EL5 was transcriptionally overexpressed, the EL5 protein was barely detected by Western blotting. Neither treatment with a proteasome inhibitor nor mutation of the sole lysine residue, a potential target of ubiquitination, resulted in increased EL5 accumulation, whereas mutations in the RFD led to increased EL5 accumulation. The stabilized EL5 without the RFD was localized in the plasma membrane. Deletion of the transmembrane domain prevented the EL5 from localizing in the membrane and from exerting an inhibitory effect on root formation. Deletion of the C-terminal region also neutralized the negative effect. We concluded that EL5 plays a major role as a membrane-anchored E3 for the maintenance of cell viability after the initiation of root primordial formation. In addition, we propose that EL5 is an unstable protein, of which degradation is regulated by the RFD in a proteasome-independent manner.