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

Abstract

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and 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.

Figure 1. BRI1 carries K63 polyubiquitin chains in vivo, independently of ligand binding

(a) In vivo ubiquitination analyses of BRI1. Immunoprecipitation was performed using an anti-GFP antibody on solubilized protein extracts from wild-type and BRI1-mCitrine plants and subjected to immunoblotting with anti-GFP (left), anti-Ub P4D1 (middle) and anti-K63 polyUb Apu3 (right) antibodies. IB, immunoblotting; IP, immunoprecipitation. (b) Ligand-dependency of BRI1 ubiquitination. Ubiquitination assays were performed on wild-type and BRI1-mCitrine plants treated with mock (−BL) or 1μM BL (+BL) for 1 hour. (C) BRI1 ubiquitination in mutants affected in receptor complex activation. Ubiquitination assays were performed on BRI1-mCitrine, kinase-dead BRI1_K911R-mCitrine, and bak1-3/BRI1-mCitrine plants. The asterisk indicates aspecific signals used as loading control. See also Figure S1.

Figure 2. Artificial ubiquitination of BRI1 triggers vacuolar targeting from TGN/EE

(a) Phenotypic analysis of transgenic plants expressing BRI1-mCitrine, BRI1-mCitrine-Ub and BRI1-mCitrine-Ub_I44A in the bri1 null background. (b) Western blot analyses of BRI1 protein accumulation in bri1/BRI1-mCitrine, bri1/BRI1-mCitrine-Ub and bri1/BRI1-mCitrine-Ub_I44A plants using an anti-GFP antibody. The asterisk indicates aspecific signals used as loading control. (c) Confocal microscopy analyses of bri1/BRI1-mCitrine, bri1/BRI1-mCitrine-Ub and bri1/BRI1-mCitrine-Ub_I44A roots. Similar detection settings were used to compare the different lines. Inset, higher laser power and gain. (d) Drug sensitivity of bri1/BRI1-mCitrine and bri1/BRI1-mCitrine-Ub plants. Plants were exposed to BFA (50μM) and ConcA (2μM) for 30 minutes and 1 hour, respectively. Similar detection settings were used to compare the different lines. See also Figure S2.

Figure 3. Ubiquitination of residue K866 negatively regulates BRI1

(a) Identification of in vivo ubiquitination sites in BRI1. ED, extracellular domain; PM, plasma membrane, ID, intracellular domain; JM, juxtamembrane domain; KD, kinase domain; CT, C-terminal domain. The ubiquitinated peptide carrying the GG signature on K866 is shown. (b) Western blot analyses on bri1/BRI1-mCitrine and bri1/BRI1_K866R-mCitrine plants. Protein levels were detected with anti-GFP and anti-BES1 antibodies, respectively. Quantification of dephosphorylated BES1 protein, normalized to BRI1 levels is shown. (c) Phenotypic analysis of 4-week-old bri1/BRI1-mCitrine and bri1/BRI1_K866R-mCitrine plants. (d) Average hypocotyl lengths of 3-day-old etiolated bri1/BRI1-mCitrine and bri1/BRI1_K866R-mCitrine seedlings (n=15). Error bars indicate standard deviation. (e) Confocal microscopy analyses of bri1/BRI1-mCitrine and bri1/BRI1_K866R-mCitrine roots. See also Figure S3.

Figure 4. Loss of BRI1 ubiquitination impairs internalization and vacuolar targeting

(a) Phenotypic analysis of 4-week-old bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine plants. (b) Average petiole lengths of the fourth true leaf from of 4-week-old bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine plants. (c) Western blot analyses on bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine plants using an anti-GFP antibody. The asterisk indicates aspecific signals used as loading control. (d) Confocal microscopy analyses of bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine roots. (e) Sensitivity of bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine plants to BFA. Plants were pretreated with 100μM CHX for 1 hour and exposed to 50μM BFA for 30 minutes. (f) Representative kymograph obtained from TIRF movies of bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine. The time scale is shown. (g) Time of residency at the plasma membrane of bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine. The distribution was obtained from kymograph-based track length analyses coming from 3 independent experiments (n=350). (h) Sensitivity of bri1/BRI1-mCitrine and bri1/BRI1_K25R-mCitrine plants to dark growth conditions. Light-grown seedlings were kept in the dark for 2 hours before confocal imaging. See also Figure S4.