Electrophysiological evidence of synergistic auxin transport by interacting Arabidopsis ABCB4 and PIN2 proteins

Abstract

To understand why both ATP-binding cassette B (ABCB) and PIN-FORMED (PIN) proteins are required for polar auxin transport through tissues, even though only the latter is polarly localized, we biophysically studied their transport characteristics separately and together by whole-cell patch clamping. ABCB4 and PIN2 from Arabidopsis thaliana expressed in human embryonic kidney cells displayed electrogenic activity when CsCl-based electrolytes were used. Current-voltage (I-V) analysis of the activities and modeling the effects of adding the auxin anion (IAA-) as a potential substrate with the Goldman-Hodgkin-Katz equation, demonstrated that ABCB4 and PIN2 were 9-fold and 10-fold more selective for IAA- than Cl-, respectively. Thus, these proteins directly transport IAA-, which was not unequivocally established by previous auxin retention assays. Co-expression of ABCB4 and PIN2 produced an especially significant result. Co-expression synergistically doubled the selectivity for IAA-. An area of two-fold higher selectivity for IAA- that this result indicates will occur in cells with asymmetric PIN2 and symmetric ABCB4 matches what early models found to be necessary to create observed levels of polar auxin transport through tissues. Thus, the requirement for two different proteins appears to be explained by a synergistic effect on selectivity. More substrate details and important pharmacological results are reported.