Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling 22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman 7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.