Fig. 1: TWD1 is a co-chaperone of HSP90 binding to helix 7 of TWD1.
From: HSP90 differentially stabilizes plant ABCB-type auxin transporters on the plasma membrane
a Mutation of F326 in helix 7 of TWD1 disrupts TWD1-HSP90.3 interaction analyzed by FRET-FLIM after co-expression of indicated Wt and mutated versions of 35S:TWD1-RFP and 35S:YFP-HSP90.3 in tobacco. Significant differences (p < 0.05) of means ± SE (n = 3 independent tobacco transfections, each with 6–8 cells) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. b Co-immunoprecipitation of HSP90.1 and HSP90.3 after co-transfection of 35S:TWD1-RFP or 35S:RFP with 35S:YFP-HSP90.3 in tobacco. IP was performed using anti-RFP columns, Western detection using anti-RFP and anti-GFP. n = 3 independent tobacco transfections and co-IPs; an exemplary result is shown. c TPR-HSP90 interaction is not essential for ABCB1 activation by TWD1 analyzed by protoplast transport assays after co-expression of indicated Wt and mutated versions of 35S:TWD1-YFP and 35S:RFP-HSP90.3 in tobacco. Significant differences (p < 0.05) of means ± SE (n = 12–18 independent tobacco transfections and protoplast preparations) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters; diffusion control in Supplementary Fig. 1h. d Mutation of F326 does not interfere with TWD1-ABCB1 interaction analyzed by FRET-FLIM after co-expression of indicated Wt and mutated versions of 35S:ABCB1-YFP and 35S:TWD1-mCherry in tobacco. Significant differences (p < 0.05) of means ± SE (n = 3 independent tobacco transfections with 6–8 cells) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. e–g Phenotypic complementation of ABCB:ABCB1-GFP (in twd1-3) with Wt and a helix 7 mutation (TWD1F326K) of 35S:TWD1-mCherry; phenotypes of soil-grown (e) and root lengths of plate-grown plants (f, g). Significant differences (p < 0.05) of means ± SE (n = 3 independent experiments with each 10 seedlings) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. h ABCB1-GFP and TWD1-mCherry imaging of ABCB:ABCB1-GFP (in twd1-3) lines complemented with Wt and a helix 7 mutation (TWD1F326K) of 35S:TWD1-mCherry. ABCB1-GFP in Wt and twd1-3 is shown as a reference. Arrowheads mark aberrant cell divisions in the root tip; bars, 100 μm. i, j Confocal imaging (i) and quantification (j) of ABCB1-GFP PM signals of ABCB:ABCB1-GFP (in twd1-3) lines complemented with Wt (TWD1), TPR (TWD1K265A) and helix 7 mutation (TWD1F326K) of 35S:TWD1-mCherry treated with 5 μM GDA (+) or solvent control (−). Note that imaging of ABCB1 was performed under equal excitation conditions allowing for direct comparison. Arrowheads mark aberrant cell divisions; bars, 50 μm. Significant differences (p < 0.05) of means ± SE (n = 3 independent GDA treatments with 18–20 cells) were determined using Ordinary One-way ANOVA (Tukey’s multiple comparison test) and are indicated by different lowercase letters. 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 are provided as a Source data file.
