CLONING AND CHARACTERIZATION OF A GLUTATHIONE-S-TRANSFERASE THAT CAN BE PHOTOLABELED WITH 5-AZIDO-INDOLE-3-ACETIC ACID

Previously, we identified a soluble protein from Hyoscyamos moticus th at was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (CST; J. Bilang, H. Macdo nald, P.J. King, and A. Sturm [1993] Plant Physiol 102: 29-34). We hav e examined the effect of auxin on the activity of this H. muticus CST. Auxins reduced enzyme activity only at high concentrations, with 2,4- dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic ac id being more effective than indole-3-acetic acid (IAA) and naphthylac etic acid. IAA was a noncompetitive inhibitor, whereas inhibition by 2 ,4-D was competitive with respect to 1-chloro-2,4-dinitro-benzene. We also present the sequence of a full-length cDNA clone that codes for a CST and contains all partial amino acid sequences of the purified pro tein. The auxin-binding CST was found in high amounts in roots and ste ms and low amounts in leaves and flower buds. The steady-state mRNA le vel was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a mo del in which 2,4-D is a substrate for CST, whereas IAA binds at a seco nd site, known as a ligandin-binding site for the purpose of intracell ular transport.