The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis

Abstract

Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, brassinosteroid insensitive1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.

Figure 1.

Colocalization and Interaction between BRI1 and Clathrin. (A) Colocalization of BRI1-GFP with CLC2-mCherry at the PM and in endosomes (arrows) in epidermal cells of the Arabidopsis root meristem. Bar = 5 µm. (B) Coimmunoprecipitation of CHC with BRI1. Total extracts of 7-d-old seedlings expressing BRI1-GFP, GFP, and the wild type (Col-0) were incubated with agarose-conjugated anti-GFP antibodies and probed with anti-CHC or anti-GFP antibodies. IN is equivalent to 20% of the protein incubated with the beads for immunoprecipitation. IP, immunoprecipitation; WB, protein gel blot; IN, input; B, bound fraction.

Figure 2.

Identification of the Arabidopsis AP-2 Complex. (A) and (C) BiFC assays in N. benthamiana depicting interactions (GFP signal) between AP1/2B1-nGFP and cGFP-AP2A1, AP1/2B1-cGFP and nGFP-AP2S, and AP1/2B1-cGFP and nGFP-AP2M (A) and between AP2A1-nGFP and cGFP-CLC2 and AP2A1-nGFP and CHC-cGFP (C). Bars = 20 µm. (B) Coimmunoprecipitation of AP2A with CLC2-GFP. Total protein extracts of 7-d-old seedlings expressing CLC2-GFP, GFP, and the wild type (Col-0) were incubated with agarose-conjugated anti-GFP antibodies and probed with anti-GFP or anti-AP2A antibodies. IN is equivalent to 20% of the protein incubated with the beads for immunoprecipitation. IP, immunoprecipitation; WB, protein gel blot; IN, input; B, bound fraction. (D) Schematic representation of the Arabidopsis AP-2 complex. Red, blue, and yellow lines represent interactions found via TAP, BiFC, and Co-IP, respectively. Co-IP, coimmunoprecipitation.

Figure 3.

Localization of AP2A1 in Arabidopsis Roots. (A) Discontinuous labeling of AP2A1-GFP (green) at the PM and colocalization with FM4-64 (magenta) at the PM (arrows). (B) Colocalization of AP2A1-GFP with CLC2-mCherry at the PM in epidermal cells of the Arabidopsis root meristem. Arrows indicate colocalization at the PM. (C) Dislodgement of AP2A1-GFP from the PM after treatment with Tyrphostin A23, Wortmannin, or BFA, but not Tyrphostin A51. 5-d-old seedlings were treated for 1 h with the drugs at the indicated concentrations. Bottom panels show recovery of the PM signal after a washout of 1 h with medium without drugs. TyrA23, Tyrphostin A23; TyrA51, Tyrphostin A51; WM, Wortmannin. Bars = 5 µm. (D) Quantification of the ratio between the PM and the intracellular AP2A1-GFP signal intensities after treatments in (C). Values were normalized against the DMSO control. Error bars indicate sd. P values (t -test), * < 0.05 relative to DMSO-treated control.

Figure 4.

Functional Characterization of AP-2. Impaired FM4-64 uptake in epidermal cells of the Arabidopsis root meristem constitutively expressing AP2A-RNAi (A) or after induction of AP2MΔC expression with 5 µM β-estradiol for 2 d (B). Five-day-old seedlings were stained with FM4-64 (2 µM, 10 min) before imaging. Graphs represent total number of endosomes labeled by FM4-64 divided by the number of cells. Error bars indicate sd. P values (t test): * < 0.05 relative to Col-0 (A) and AP2MΔC after treatment with β-estradiol (B). Bars = 5 µm.

Figure 5.

AP-2–Mediated BRI1 Endocytosis. (A) Colocalization of BRI1-GFP with AP2A1-mTagRFP at the PM (arrow) in epidermal cells of the Arabidopsis root meristem. (B) Coimmunoprecipitation of AP2A with BRI1-GFP. Total extracts of 7-d-old seedlings expressing BRI1-GFP, GFP, or the wild type (Col-0) were incubated with agarose-conjugated anti-GFP antibodies and probed with anti-AP2A or anti-GFP antibodies. IN is equivalent to 20% of the protein incubated with the beads for immunoprecipitation. IP, immunoprecipitation; WB, protein gel blot; IN, input; B, bound fraction. (C) and (D) Reduced vacuolar accumulation of AFCS in epidermal cells of the root meristem by induced AP2MΔC expression (5 μM β-estradiol, 2 d) or constitutive AP2A silencing, respectively. Five-day-old Arabidopsis seedlings were pulsed for 30 min in 20 μM AFCS and imaged after a chase of 20 min. Graphs represent quantification of the vacuolar AFCS normalized to the BRI1-GFP–expressing line (C) or Col-0 (D). (E) Reduced number of BRI1-GFP–positive endosomes after induction of AP2MΔC expression (5 μM β-estradiol for 2 d). Graphs represent the average number of FM4-64–positive endosomes per cell. (F) Reduced size of the BRI1-labeled BFA bodies after induction of AP2MΔC expression (5 μM β-estradiol for 2 d). Five-day-old seedlings were treated with BFA (50 μM, 1 h). Graphs represent the sizes of the BRI1-GFP–positive BFA bodies. Error bars indicate sd. P values (t test): * < 0.05 relative to the BRI1-GFP–expressing line after treatment with β-estradiol ([C], [E], and [F]) and Col-0 (D). Bars = 5 μm. (G) Protein gel blot analysis of 7-d-old Arabidopsis seedlings expressing BRI1-GFP in Col-0 or in an AP2MΔC background after DMSO or β-estradiol treatment (5 µM for 2 d), probed with anti-BES1 or antitubulin antibodies.