Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination

Significance

The brassinosteroid (BR) receptor BRI1 provides a paradigm for understanding receptor-mediated signaling in plants. Different posttranslational modifications have been implicated in the regulation of BRI1 activity. Here, we show that BR perception promotes BRI1 association with plant U-box E3 ubiquitin ligases PUB12 and PUB13, which in turn directly ubiquitinate BRI1. Importantly, the BRI1 protein abundance and plasma membrane-residence time are increased while the endosomal pool of BRI1 is reduced in the pub12pub13 mutant, indicating that PUB12/PUB13-mediated ubiquitination regulates BRI1 endocytosis and degradation. BRI1 phosphorylates PUB13 on a specific residue to enhance its association with BRI1, suggesting a unique regulatory circuit of phosphorylation-regulated E3 ligase–substrate association. Our study elucidates a mechanism of BRI1 internalization through E3 ubiquitin ligase-mediated ubiquitination.

Abstract

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase–substrate association regulated by phosphorylation.