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We used 5-azido-[7-3Hjindole-3-acetic acid (5-azido-[7-3H]IAA), a photoaffinity analogue of the plant hormone indole-3-acetic acid (IAA), to search for auxinbinding proteins in Arabidopsis thaliana membranes. We identified an auxin-binding protein with a molecular mass of 24 kDa (Atpm24) in microsomes as well as in plasma membrane vesicles. Atpm24 was solubilized by 1% Triton X-100 and partially purified. A cDNA clone (Atpm24.1) corresponding to Atpm24 was isolated. The amino acid sequence predicted from the Atpm24.1 cDNA contains 212 amino acid residues with a relative molecular mass of 24,128 Da. Data base searches revealed that the predicted protein has homology to glutathione S-transferases (GSTs; EC 2.5.1.18). When Atpm24.1 was expressed in Escherichia coli, we found a high level of GST activity in the bacterial extracts. We have analyzed the substrate specificity of this protein and found that cumene hydroperoxide and trans-stilbene oxide but not trans-cinnamic acid or IAA-CoA were substrates. A role for this GST in physiological processes of plants is discussed.

Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous twophase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido- [7-3H]indole-3-acetic acid ([3H]N3IAA), we identified several IAAbinding proteins with molecular masses of 60 kDa (pm6O), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, we were able to selectively extract pm23 from the plasma membrane. We show that auxins and functional analogues compete with[3H]N3IAA for binding to pm23. We found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, we identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-1 14. We designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7000-fold purification.

ABSTRACT1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carriermediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, we have synthesized a tritiated, photolabile NPA The group of phytohormones known as auxins [indole-3-acetic acid (IAA) and related natural and synthetic analogues] influence fundamental processes in plant growth, differentiation, and development (for review see ref.

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.