Fig. 1.

AtABCG40 mediates specific uptake of ABA in a heterologous system. (A) Time-dependent uptake of ³H-ABA by YMM12 yeast cells expressing AtABCG40 (ABCG40) or transformed with the empty vector (EV). Yeast was incubated in SG-URA medium containing 4.5 nM ³H-ABA (7.4 kBq, 1.63 Tba/mmol) at pH 7. Radioactivity taken up was normalized to the cell number. Data are mean ± SEM of n = 12 from three independent experiments. (B) AtABCG40 transcripts in independent BY2 cell lines expressing AtABCG40 (G1-G4) or EV. Actin was amplified as a loading control. (C) Time-dependent transport of 4.5 nM ³H-ABA (7.4 kBq, 1.63 Tba/mmol) by BY2 cells expressing AtABCG40 (G1-G4) or EV at pH 5.7. (D) Concentration-dependent uptake of ABA containing 1 nM ³H-ABA (from 1 nM to 5 μM ABA) or 2 nM ³H-ABA (from 10 to 15 μM ABA) in BY2 cells (line G2) for 18 min. Inset shows double reciprocal plot analysis of the data indicating a K _M of 1 μM. Mean is compiled from three experiments. K _Ms calculated for the single experiments vary between 0.7 and 1.2 μM. Values were corrected for the effective (S)-ABA concentrations. (E) Uptake of 1 μM (R, S)-ABA containing a trace amount (7.4 kBq) of ³H-ABA in BY2 cells (line G2) in the absence (C) or presence of an additional 3-fold (3 μM) unlabeled (R, S)-ABA, (R)-ABA, ABA-glucose ester (G), indole-3-acetic acid (I), or benzoic acid (B). (F) Inhibition of ABCG40-mediated 4.5 nM ³H-ABA (7.4 kBq, 1.63 Tba/mmol) uptake in BY2 cells (line G2) by 3 mM vanadate (VD), 10 μM verapamil (VP), and 25 μM glibenclamide (GC). C, control without inhibitor pretreatment. In D–F, ABA uptake mediated by AtABCG40 was obtained by subtracting the activity of the empty vector control incubated under the same conditions from the total radioactivity to exclude the portion of ABA transported by diffusion.