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

TL;DR

This study shows that the RING-H2 domain of the Arabidopsis RMR protein acts as an E3 ligase, which is crucial for transporting proteins after they leave the Golgi in plants.

Contribution

The study identifies the E3 ligase activity of the RING-H2 domain in RMR proteins and its role in post-Golgi trafficking.

Findings

The RING-H2 domain of Arabidopsis RMR isoform-1 interacts with E2 ubiquitin-conjugating enzyme and shows E3 ligase activity.

The I234Y mutation disrupts E2–E3 interaction and reduces E3 ligase activity.

E3 ligase activity is essential for post-Golgi trafficking of RMR receptors.

Abstract

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.

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