Figure 4.

Introduction of an E-P motif into malate importer, ABCB14, increases its transport activity. A and B, simultaneous malate (A) and IAA (B) export of protoplasts prepared from tobacco leaves transfected with WT and P990E-mutated ABCB14 (B14). Significant differences (unpaired t test with Welch's correction, p < 0.05) to vector control are indicated by an “a.” Mean ± S.E.; n ≥ 4 transport experiments generated from independent tobacco transfections. C, P990E mutation does not alter significantly expression or plasma membrane location of ABCB14 revealed by confocal imaging of ABCB1-GFP of tobacco leaves transfected with ABCB1-GFP and stained with PM marker, FM4-64. Bars, 10 μm. D, BRET analyses of microsomal fractions prepared from tobacco leaves co-transfected with TWD1-Rluc and WT and P990E mutation of ABCB14-GFP. AUX1-YFP/TWD1-Rluc and YFP/TWD1-Rluc were used as negative and ABCB1YFP/TWD1-Rluc as positive controls. Significant differences (unpaired t test with Welch's correction, p < 0.05) to control (TWD1-Rluc alone) are indicated by an “a.” Mean ± S.E.; n = 3 BRET measurements from 3 independent tobacco transfections. E, co-immunoprecipitation of TWD1-Rluc using WT and P990E mutation of ABCB14-YFP as a bait. Co-IP was performed from microsomal fractions prepared from tobacco leaves co-transfected with indicated combinations. Total input (T) and elution (E) were probed against anti-GFP and anti-Rluc detecting ABCB14-YFP and TWD1-Rluc, respectively.