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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

The Binding of Auxin to the Arabidopsis Auxin Influx Transporter AUX1^1,[OA]

School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom (D.J.C., I.D.K.); School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, United Kingdom (N.T.A.B., R.S., M.J.B.); Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom (R.C.); and Warwick HRI, University of Warwick, Wellesbourne, Warwick CV5 9EF, United Kingdom (R.M.N.)

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K_d of 2.6 µM, comparable with estimates for the K_m for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ian D. Kerr (ian.kerr{at}nottingham.ac.uk).

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.122044

^* Corresponding author; e-mail ian.kerr{at}nottingham.ac.uk.

Received April 29, 2008; accepted June 24, 2008; published July 9, 2008.