Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination

Abstract

Brassinosteroids (BRs) are plant steroid hormones that control many aspects of plant growth and development. BRs are perceived at the cell-surface by the plasma membrane-localized receptor complex composed of the receptor kinase BRI1 and its co-receptor BAK1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Artificially ubiquitinated BRI1 is recognized at the trans-Golgi Network/Early Endosomes (TGN/EE) and rapidly routed for vacuolar degradation. Mass spectrometry analyses identified residue K866 as an in vivo ubiquitination target in BRI1 involved in the negative regulation of BRI1. Model prediction revealed several redundant ubiquitination sites required for the endosomal sorting and vacuolar targeting of BRI1. Using total internal reflection fluorescence microscopy (TIRF), we also uncovered a role for BRI1 ubiquitination in promoting internalization from the cell-surface. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is a fundamental control mechanism for BR responses in plants. Altogether, our results identify K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development.

Unraveling K63 Polyubiquitination Networks by Sensor-Based Proteomics

“…The fact that transporters are usually of low abundance in the cell and are overrepresented in the crude extract-based isolation of the K63 ubiquitinome suggests that K63 polyubiquitination does play an important role in their function. This is consistent with the recently characterized role of K63 polyubiquitination in plant plasma membrane protein endocytosis (Kasai et al, 2011;Leitner et al, 2012;Martins et al, 2015) and with the preferential localization of Vx3K0 to membranes in vivo (Fig. 1B).…”

“…Since we, and others, recently reported the role of both monoubiquitination and K63 polyubiquitination in the endocytosis of cell surface proteins (Barberon et al, 2011;Kasai et al, 2011;Leitner et al, 2012;Martins et al, 2015), we explored further the possible link between Vx3K0 and endocytosis. We showed that the vast majority of intracellular vesicles positive for Vx3K0 were also positive for the FM4-64 endocytic tracer, indicating that they are of endocytic nature (Fig.…”

“…These include the PIN2 auxin efflux carrier, the BOR1 boron transporter, and the BRI1 steroid hormone receptor (Kasai et al, 2011;Leitner et al, 2012;Martins et al, 2015). In all three cases, K63 polyubiquitination of these membrane proteins has been associated with their endocytosis and degradation in the vacuole.…”

ESCRT-I Component VPS23A Affects ABA Signaling by Recognizing ABA Receptors for Endosomal Degradation

“…We found that (Leitner et al, 2012;Martins et al, 2015). Collectively, in some cases, the recognition of PYL4 by the endosomal trafficking pathway might depend on a direct interaction with the sorting complexes; in other cases, this recognition might rely on post-translational modifications, such as ubiquitination or phosphorylation (Reyes et al, 2011).…”

Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases

“…To gain insight into BRI1 trafficking in N. benthamiana leaves, we pairwise-coexpressed BRI1-GFP with MEMB12-mCherry, Vha-A1-RFP, mCherry-ARA7/RabF2b, and ARA6/ RabF1-RFP BRI1-positive vesicles colocalized with the endosomal markers ARA7/RabF2b and ARA6/RabF1-RFP (Fig. S6A), in agreement with BRI1 vacuolar trafficking (22). Heterologous coexpression of BRI1-GFP and FLS2-mCherry in N. benthamiana revealed colocalization at vesicles after flg22 elicitation (Fig.…”