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 that was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (CST; J. Bilang, H. Macdonald, P.J. King, and A. Sturm [1993] Plant Physiol 102: 29-34). We have 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 acid being more effective than indole-3-acetic acid (IAA) and naphthylacetic 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 protein. The auxin-binding CST was found in high amounts in roots and stems and low amounts in leaves and flower buds. The steady-state mRNA level was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a model in which 2,4-D is a substrate for CST, whereas IAA binds at a second site, known as a ligandin-binding site for the purpose of intracellular transport.