Fig. 5: L704Y mutation in ABCG36 widens the transport selectivity to indolic compounds.
IBA (a) CLX (b), IAA (c), and indole (d) export from N. benthamiana protoplasts after transfection with indicated mutant versions ABCG36. Means of mutant ABCG36 that are significantly different from Wt ABCG36 (grey fill) are indicated in red, while non-significant ones are in blue. ABCG36A1357V (green) is included as a negative control. Significant differences (p < 0.05) of means ± SE (n = 14 independent protoplast preparations) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. Confocal imaging (e) and quantification (f) of GFP-tagged versions of mutated ABCG36 after transfection of N. benthamiana leaves. Short treatment of FM4-64 was used as PM markers; bar, 50 μm. Note that slightly enhanced PM signals of some mutant versions of ABCG36 in comparison to Wt are also found in respective FM4-64 controls and thus do not reflect higher expression levels; bar, 10 μm. Significant differences (p < 0.05) of means ± SE (n = 25 epidermal cells) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. g Free energy surfaces (FES) were calculated from metadynamics simulations with complexes of indole with Wt and L704Y ABCG36, respectively. h–j Pulling simulations of indole from the central binding pocket to the extracellular space were performed in simulations with Wt and L704Y ABCG36, respectively. Frequencies of contacts between the small molecule and the protein are plotted. j. A logistic regression model was employed to identify the key residues involved in defining transport. The model’s coefficients were color-coded from blue to red, indicating residues whose interactions negatively (blue) or positively (red) support transport, respectively. Data are presented as box-and-whisker plots, where median and 25th and 75th percentiles are represented by the box itself and the middle line, respectively; means are indicated by a “+”. Source data is provided as a Source Data file.
