Retention of maize auxin-binding protein in the endoplasmic reticulum: quantifying escape and the role of auxin

Abstract

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs 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 necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested the ER, namely that auxin binding induces a conformational change 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 distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretions of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP 1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected 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, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants.

Posttranslational Removal of the Carboxyl-terminal KDEL of the Cysteine Protease SH-EP Occurs Prior to Maturation of the Enzyme

“…The function of auxin-binding protein remains unknown, although it is widely assumed that the protein may be involved in signal transduction of plant hormone auxin. Auxinbinding protein is localized primarily in the ER as if it were a reticuloplasmin (64,65), although a small fraction of the auxinbinding protein is secreted to the cell surface (59,66,67) with its KDEL sequence intact (59). The binding of ligand, auxin, may alter the exposure of the KDEL sequence, and this could change the trafficking of the protein (68) so that its removal is not necessary to allow its exit from the ER.…”

ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis

“…For example, it is possible that ABP1 has some ligand-regulated chaperone role. ABP1 is found predominantly in the lumen of the endoplasmic reticulum, although it is certainly at detectable levels throughout the endomembrane system (Jones and Herman 1993;Henderson et al 1997) and at the plasma membrane/cell wall interface (Deikman et al 1995). Our working hypothesis is that ABP1 regulates the direction or flux of wall materials.…”

Auxin signaling and regulated protein degradation

“…The bulk of the ABP1 is localized to the endoplasmic reticulum, where the pH is too high for optimal auxin binding. However, a small proportion of ABP1 is on the cell surface and this might function as an extracellular receptor [44]. Although some evidence suggests that ABP1 is an auxin receptor, so far there are no reports linking ABP1 to auxin-induced gene expression or protein degradation.…”