MOLECULAR ANALYSIS OF AUXIN-SPECIFIC SIGNAL-TRANSDUCTION

The auxin-binding protein (ABP1) of maize has been purified, cloned an d 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 funct ion of ABP. ABP1 is a glycoprotein with two identical subunits of appa rent M(r) = 22 kDa. The regions recognised by our five monoclonal anti bodies (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 2 56, 259) are directed at the endoplasmic reticulum (ER) retention sequ ence KDEL at the C-terminus. Early biochemical data pointed to six ami no acids likely to be involved in the auxin binding site. Inspection o f the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containin g five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, su ggesting that the region is highly conserved. This is confirmed by mor e recent information showing that the selected polypeptide contains th e longest stretch of wholly conserved sequence in ABP1. Most strikingl y, the antibodies show auxin agonist activity against protoplasts in t hree different electrophysiological systems - hyperpolarization of tob acco transmembrane potential; stimulation of outward ATP-dependent HS current in maize; modulation of anion channels in tobacco. The biologi cal activity of these antibodies indicates that the selected peptide d oes form a functionally important part of the auxin binding site and s trongly supports a role for ABP1 as an auxin receptor. Although ABP co ntains a KDEL sequence and is located mainly in the ER lumen, the elec trophysiological evidence shows clearly that some ABP must reach the o uter 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 KDE L 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) cultur es reported by Jones and Herman [7]. However, recent silver enhanced i mmunogold studies on maize protoplasts have succeeded in visualizing A BP at the cell surface, as well as auxin-specific clustering of the si gnal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membran e and gene levels remain to be elucidated.