Extended Data Fig. 1: Analysis of auxin binding to Arabidopsis thaliana TMK1 and ABP1.

a, DARTS assay on TMK1::TMK1-GFP plants. Protein extracts were incubated with IAA (in blue) or benzoic acid (in grey). Afterwards, different pronase quantities were added. Blots and quantifications (normalized to actin levels) show comparable pronase-induced degradation in the presence of both IAA and benzoic acid, suggesting no specific IAA binding to TMK1. Representative of 3 independent experiments with similar outcome. b, MST analysis of auxin binding to TMK1. Normalized binding curves of IAA, NAA and benzoic acid in the presence of 150 nM TMK1 heterologously expressed and purified extracellular domain (ECD). Plots are mean ± SD from 10 measurements (3 or 4 independent experiments) but no curve fitting to determine binding kinetics was possible. c, GCI-assisted analysis of binding properties of TMK1 ECD to IAA or benzoic acid as a control ligand, using the Creoptix® WAVEsystem. Heterologously expressed and purified TMK1 ECD was immobilized to the surface at the indicated levels, following which the response to different concentrations of IAA or benzoic acid in running buffer at different pH (resp. 5.5 and 7.6) was monitored for analysis of binding kinetics. No binding of IAA to TMK1 ECD was detected. d, DARTS assay on heterologously expressed ABP1. Purified protein was mixed with different quantities of pronase enzyme mixture. Blots and quantifications show that pronase-induced proteolysis of tagged ABP1 occurred less in the presence of 10 μM IAA, which was consistent for multiple pronase dilutions, indicating IAA association with ABP1. This was verified both by the anti-HIS-HRP antibody to ensure specificity of the visualized band. The intensity profiles are plotted in the graph below the blots. Representative of 3 independent experiments with similar outcome shown. e, Control no-pronase samples for DARTS results represented in Fig. 1a. Protein extracts from 35S::ABP1-GFP expressing plants were incubated with benzoic acid (in grey) or IAA (in blue). Blot intensities were quantified and normalized to the average actin intensity for the no-pronase samples. In these no-pronase control samples, we verified that the presence of the respective potential ligand at their concentration did not affect target protein stability as such. Representative of 3 independent experiments with similar outcome. f, Overview table and graphs of all GCI-based binding analyses for ABP1. The potential ligands IAA and benzoic acid were evaluated in serial dilution ranging from 91.5 nM to 200 μM. IAA binding kinetics was detectable, giving a Kd estimate of 13.7 μM at pH 5.5 and 1943 μM at pH 7.6. g, MST analysis of IAA binding to ABP1 at varying pH. Normalized binding curve of IAA in presence of 75 nM ABP1 at pH 5.5 (blue), 100 nM ABP1 at pH 7.0 (grey) or 100 nM ABP1 pH 7.5 (black). The IAA concentration varied from 61 nM to 2 mM. Plots are mean ± SD from 10 measurements; 2 (pH 7.5) or 4 (pH 5.5, pH 7.0) independent experiments. The estimated Kd values confirm efficient IAA binding at the apoplastic pH of 5.5, whereas the estimated Kd values with large SD indicate no binding at the pH 7.0 and pH 7.5. h, MST analysis of ligand binding to ABP1 at pH 7.0. Normalized binding curve of IAA (blue; identical to grey in S1g) and L-Trp (black) to 100 nM ABP1. The ligand concentration varied from 61 nM to 2 mM. Plots are mean ± SD from 10 measurements; 4 independent experiments. The estimated Kd values with large SD indicate no binding of these ligands at pH 7.0. i, MST analysis of ligand binding to ABP1 at pH 7.5. Normalized binding curve of IAA (blue, same as black in Extended Data Fig. 1g) and control ligand benzoic acid (grey) to 75 nM ABP1. The ligand concentration varied from 61 nM to 2 mM. Plots are mean ± SD from 10 measurements; 2 independent experiments. The estimated Kd values and SDs are much higher than those obtained for pH 5.5 and indicate no binding of these ligands at pH 7.5.

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