Dr. Lomax proposes a feasibility study of a simple but powerful approach to isolating the indole-3-acetic acid uptake symport. This involves photoaffinity labeling of the protein in purified plant plasma membranes with a tritiated azido-IAA analog, 3H-5N3IAA. The azido auxin after exposure to UV light in the presence of plasma vesicles will provide a covalent label which will allow identification of the protein during subsequent solubilization and purification by HPLC and SDS-PAGE. The pilot studies will establish: 1. the optimal conditions for the binding of 3H-5N3IAA to phase-purified plasma membrane vesicles, 2. the affinity of 3H-5N3IAA for the uptake carrier, 3. the ability of covalent binding of the azido auxin to block IAA transport by the uptake symport, and 4. the specificity of the azido IAA for the carrier in the presence of excess IAA, other auxin analogs, and auxin transport inhibitors. Taken together, the results of these studies should indicate if only the uptake symport or also other IAA-binding proteins (such as the efflux carrier) are labeled and whether or not the photoaffinity method will be appropriate for isolation of the auxin uptake carrier. %%% Very little is known about the mechanisms of action of plant hormones. The hormone auxin is essential to the control of plant development. This hormone is transported into the plant cells by a specific protein transport carrier. The aim of this proposal is to identify and isolate this transport protein in order that its structure and function can be studied.
