RETENTION OF MAIZE AUXIN-BINDING PROTEIN IN THE ENDOPLASMIC-RETICULUM- QUANTIFYING ESCAPE AND THE ROLE OF AUXIN

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) h as been studied using a variety of techniques. At the whole-tissue lev el, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. W ithin cells, the signals from immunofluorescence and immunogold labell ing of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi appara tus or cell wall. This distribution is consistent with targeting motif s in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessa ry on the cell surface for rapid, auxin-mediated protoplast hyperpolar isation. We have tested one proposed model to account for release of A BP1 from the ER, namely that auxin binding induces a conformational ch ange in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distributio n of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretion of ABP1 into the medium, but se cretion was below the limit of detection. This can be ascribed at leas t partly to the very active acidification of the medium by these cells and the instability of ABP1 in solution below pH 5.0. In the insect-b aculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detec ted under all conditions. Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, q uantitative glycan analysis was used to determine what proportion of A BP1 might reach the plasma membrane in maize coleoptile tissue. The re sults suggest that less than 15% of ABP1 ever escapes from the ER as f ar as the cis-Golgi and less than 2% passes further through the secret ory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are no t able to rule out the possibility that some ABP1 is carried through a ssociated with other proteins. The data are consistent with the presen ce of ABP1 both on the plasma membrane and in the ER. The relative siz es of the two pools explain the results obtained with immunofluorescen ce and immunogold labelling and illustrate the high efficiency of ER r etention in plants.