Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

Abstract

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of Exposure of N3IAA to UV light (300 nm) results in photolysis of the aryl azide (azido group). The products of this reaction are N2 gas and a highly reactive nitrene, which, via a nucleophilic reaction, would be expected to covalently label specific binding proteins. Using this compound, Jones et al. (16,17) were able to demonstrate quantitative differences in labeling of maize microsomal proteins in the presence of competing auxin or auxin analogues, although much background polypeptide labeling was evident (17). Ripp et al. (18) recently reported that a photoaffinity analogue of sucrose labeled a 62-kDa membrane protein associated with sucrose transport in soybean. They found that lowering the temperature of the reaction mixture to -1960C (liquid N2) resulted in reduced background labeling. Here we report that nonspecific [3H]N3IAA labeling of membrane proteins can also be largely eliminated by conducting the photolysis at -196C. Under these conditions, photolysis of zucchini (Cucurbita pepo L.) PM proteins with [3H]N3IAA results in the highspecific-activity labeling of a low-abundance PM polypeptide doublet that displays properties consistent with those expected for a PM auxin receptor. MATERIALS AND METHODS

Mechanisms of action of abscisic acid at the cellular level

“…Although there is recent evidence for a binding protein at the plasmalemma (Barbier-Brygoo et al, 1989;Hicks et al, 1989) and involvement of phosphatidylinositol metabolites (Ettlinger & Lehle, 1988) for auxin responses, and for Ca^^ in an ABA response (Napier et al, 1989), no complete response chain via a second messenger has been elucidated for any phytohormone. Approaches have been initiated with ABA in order to elucidate the pathway back towards tbe receptor.…”

Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase.

“…Initial biochemical characterization using equilibrium auxin-binding assays as a guide for purification ofthese proteins was met with limited success due to technical difficulties. Photoactivatable auxin analogues, such as 5-azido-[7-3H]indole-3-acetic acid (5-azido-[7-3H]IAA), turned out to be valuable tools to identify auxin-binding proteins (3)(4)(5)(6)(7)(8)(9). Here we report the photoaffinity labeling of a 24-kDa polypeptide in PM vesicles of Arabidopsis thaliana.…”

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases)

“…After the final centrifugation, PM vesicles were solubilized as described below or resuspended in a small volume of buffer 1 plus protease inhibitors, flash frozen in liquid Nz, and stored at -7OOC. Marker enzyme assays indicate that zucchini hypocotyl PMs prepared in this manner are >95% pure (Hicks et al, 1989). Protein concentrations were astimated using either the method of Schaffner and Weissmann (1973), which eliminates interference by detergents, or a kit from Bio-Rad based on the Bradford assay.…”

“…Microsomal pellets were resuspended in buffer 2 (5 m KPi [pH 7.81, 250 m SUC, 4 m KCl), flash frozen in liquid Nz, and stored at -7OOC until use. PM vesicles were prepared by aqueous two-phase (dextran/PEG) separation of microsomal membrane preparations from zucchini seedlings as described by Hicks et al (1989). Protease inhibitors were replenished after each centrifugation.…”