The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

Receptor-homology-transmembrane-RING-H2 (RMR) sorting receptors are essential for directing soluble cargo proteins to protein storage vacuoles in plants. These type I integral membrane proteins comprise a single transmembrane domain, an N-terminal lumenal region containing a protease-associated domain for cargo recognition, and a C-terminal cytoplasmic region with an RING-H2 domain. Here, we determined the crystal structure of the RING-H2 domain of Arabidopsis RMR isoform-1, where the conserved C3H2C3 motif coordinates two Zn ions, a feature typical of RING-type E3 ligases. RING-H2 domain of Arabidopsis RMR isoform-1 was shown to interact with Arabidopsis E2 ubiquitin-conjugating enzyme and exhibits E3 ligase activity in an in vitro ubiquitination assay. Biochemical analysis reveals that I234Y substitution disrupted the E2-E3 interaction and greatly reduced E3 ligase activity. Furthermore, we showed that the conserved RING-H2 domains of AtRMR isoform 2, 3, and 4 are also E3 ligases. Inactivation of E3 ligase activity by the I234Y mutation resulted in Golgi retention of AtRMR1-C-terminal cytoplasmic region and AtRMR2. These findings suggest that the E3 ligase activity is essential for post-Golgi trafficking of RMR receptors, providing new insights into receptor-mediated protein sorting in plants.