Characterisation of auxin receptors

Many auxin-binding systems, both membrane-bound and soluble, have been studied, but most lack credibility as receptors. The exception and best candidate as a valid auxin receptor is the auxin-binding protein of maize coleoptile membranes. This protein can be readily solubilised from the membranes and has been purified to homogeneity. It is a glycosylated homodimer of 22 kDa subunits. Preparations of minimum 50% purity were used to immunise rats and five monoclonal antibodies have been derived. Two of these can be mapped to epitopes within a C-terminal 1 kDa region, while the epitope of a third is within 7 kDa of the N-terminus. Immunotitration of receptor abundance in different tissues of maize seedlings shows that roots contain 40-fold less receptor protein per gram of fresh weight than coleoptiles. Pure receptor was produced by native PAGE and used to generate a high titer polyclonal antiserum in rabbits, capable of detecting receptor protein from as little as 1 mg of coleoptile tissue. The polyclonal specifically precipitates the 22 kDa polypeptide from maize membrane extracts, concomitant with removal of auxinbinding activity. The antiserum also detects homologous polypeptides in maize supernatant and in several other species, both monocots and dicots. In some cases, differences in chromatographic behaviour or size have been found. An auxin-induced conformational change in the receptor has been detected with a sandwich ELISA. The receptor gene has been cloned in four laboratories and the sequence data with knowledge of antibody epitopes can be used to identify parts of the protein involved in conformational changes (and perhaps auxin action). We are currently raising antibodies to a polypeptide thought to be part of the auxin binding site. Most of the auxin-binding protein is found in the endoplasmic reticulum but electrophysiological evidence, using polyclonal antiserum and impermeant auxin conjugates on protoplasts, suggests that a small population of functional receptor is accessible at the exterior face of the plasma membrane. Evidence bearing on the localisation and structure of the receptors is discussed.