Modelling of auxin-binding protein 1 suggests that its C-terminus and auxin could compete for a binding site that incorporates a metal ion and tryptophan residue 44

Sequence comparison indicates that auxin-binding protein 1 (ABP1) belongs to a family of proteins with the core β-barrel structure of the vicilins. Previous modelling within this family correctly predicted metal-ion binding and oligomeric properties of oxalate oxidase. ABP1 also contains a putative metal-ion-binding cluster of amino acids, adjacent to a tryptophan side chain, leading to a proposed auxin-binding site that incorporates metal-ion interaction with the auxin carboxylate. Modelling implicates W44 (Zea mays ABP1) in auxin binding, rather than W136 or W151. Reduced sequence similarity for the C-terminal region prevents model building. It is proposed that one of these C-terminal tryptophans, along with a neighbouring negatively charged side chain, occupies the binding pocket in the absence of auxin, thereby linking auxin binding to conformational change and C-terminal involvement in signalling. Received: 10 December 1999 / Accepted: 4 August 2000     

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.     

Mass spectrometric analysis reveals a cysteine bridge between residues 2 and 61 of the auxin-binding protein 1 from Zea mays L.

The major auxin-binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass-spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20,243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose-type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1.     

In vitro binding affinities of 4-chloro-, 2-methyl-, 4-methyl-, and 4-ethylindoleacetic acid to auxin-binding protein 1 (ABP1) correlate with their growth-stimulating activities

4-Chlorindole-3-acetic acid (4-CI-IAA), an endogenous auxin in certain plant species of Fabaceae, has a higher efficiency in stimulating cell elongation of grass coleoptiles compared with indole-3-acetic acid (IAA), particularly at low concentrations. However, some investigations reported a 1,000-fold discrepancy between growth stimulation and binding affinity of 4-CI-IAA to auxin-binding protein 1 (ABP1) from maize. Here we report binding data of 4-CI-IAA and three alkylated IAA derivatives using purified ABP1 in equilibrium dialysis. There is a clear correlation between the growth-promoting effects and the binding affinity to ABP1 of the different IAA analogues measured by competition of [³H]naphthalene-1-acetic acid binding. Our data are consistent with the hypothesis that ABP1 mediates auxin-induced cell elongation.Abbreviations ABP1 auxin-binding protein 1 - 4-CI-IAA 4-chloroindole-3-acetic acid - NAA naphthalene-1-acetic acid - ER endoplasmic reticulum - IAA indole-3-acetic acid - 2-Me-IAA 2-methylindole-3-acetic acid - 4-Me-IAA 4-methylindole-3-acetic acid - 4-Et-IAA 4-ethylindole-3-acetic acid - MES 4-morpholineethanesulfonic acid - PAA phenylacetic acid     

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.     

Comparison of Site I auxin binding and a 22-kilodalton auxin-binding protein in maize

Several properties of a 43-kilodalton (kDa) auxin-binding protein (ABP) having 22-kDa subunits are shared by a class of auxin binding designated Site I. The spatial distribution of the ABP in the maize (Zea mays L.) mesocotyl corresponds with the distribution of growth induced by naphthalene-1-acetic acid and with the distribution of Site I binding as previously shown by J.D. Walton and P.M. Ray (1981, Plant Physiol. 68, 1334–1338). The greatest abundance of both ABP and Site I activity is at the apical region of the mesocotyl. The ABP and Site I activity co-migrate in isopycnic centrifugation with the endoplasmic-reticulum marker, cytochrome-c reductase. Red light, at low and high fluence, far-red and white light were used to alter the elongation rate of apical 1-cm sections of etiolated maize mesocotyls, the amount of auxin binding, and the abundance of the ABP. Relative changes in auxin binding and the ABP were correlated, but the growth rate was not always correlated with the abundance of the ABP.Abbreviations ABP auxin-binding protein - ER endoplasmic reticulum - FR far-red light - kDa kilodalton - NAA naphthalene-1-acetic acid - PM plasma membrane - R red light - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Authentic processing and targeting of active maize auxin-binding protein in the baculovirus expression system.

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Monoclonal antibodies detect an auxin-induced conformational change in the maize auxin-binding protein

The monoclonal antibody MAC 256 precipitates specifically the auxin-binding protein (ABP) of maize membranes. Auxin-binding activity was recovered from the immunoprecipitate and MAC 256 can, therefore, bind undenatured, native ABP. A sandwich enzyme-linked immunosorbent assay was used to present native ABP to MAC 256 and under these conditions auxins inhibit antibody binding. Millimolar naphthalene-1-acetic acid completely blocks MAC 256 binding and the characteristics of monoclonal antibody MAC 259 are similar. The ability of a range of auxins and related compounds to displace MAC 256 correlates with the known structure-activity relationships of these compounds in vivo and in binding assays. The results are interpreted in terms of an auxin-induced conformational change in ABP, auxin binding leading to a change in, or concealment of, the epitope of the antibody. The epitope for MAC 256 and 259 lies close to the carboxy terminus of the protein, implying that the part of ABP containing the sequence of amino acids responsible for retention within the endoplasmic reticulum is conformationally active.Abbreviations ABP auxin-binding protein - ELISA enzyme-linked immunosorbent assay - IAA indole-3-acetic acid - Mab monoclonal antibody - NAA naphthalene-1-acetic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TIBA 2,3,5-triiodobenzoic acid - 2,4,5-T, 2,4,6-T 2,4,5-trichloro- and 2,4,6-trichlorophenoxyacetic acid, respectively We are grateful to Neville Huskisson and Pat Baker of the Microchemical Facility, AFRC IAPGR, Babraham, UK for the aminoacid sequencing and to the staff at the AFRC Monoclonal Antibody Centre, Babraham where the Mabs were produced. This work was partially funded by the Biotechnology Action Programme of the European Economic Community.To whom correspondence should be addressed.     

The Plant Oncogene rolB Alters Binding of Auxin to Plant Cell Membranes

We measured auxin-binding capacity of the membrane preparationsfrom tobacco cells transformed by rolB as compared to untransformedcontrols. In the transformed cells, the overall auxin-bindingactivity is severalfold enhanced through an increase in a bindingactivity removable from the membranes at 0.5 M salt, while thebinding activity still attached to the membranes after saltwashes remains unchanged. Antibodies against the 22 kDa maizeauxin binding protein (ABP) depress most of the membrane-attachedbinding activity in both normal and rolB-transformed cells,while they do not affect the salt-washable binding activity.In contrast, antibodies against the RolB protein prevent completelybinding of auxin to the latter activity in both normal and transformedcells, while substantially unaffecting the membrane-associatedbinding. These results point to the presence, in untransformedmembranes, of an auxin-binding activity associated with a proteinimmunologically related to RolB. This activity is much increasedin rolB cells. In contrast, the auxin-binding protein analogousto maize ABP present in tobacco membranes does not increasein the rolB-transformed cells. (Received October 1, 1993; Accepted April 22, 1993)     

Interaction of auxin-binding protein 1 with maize coleoptile plasma membranes in vitro

In a search for membrane “docking proteins” interacting with Zea mays auxin-binding protein (ABP1) the binding of purified ABP1 to maize coleoptile plasma-membrane vesicles was investigated. Concentration-dependent, saturable binding of ABP1 to the membrane vesicles was observed in binding assays using 10⁻⁸–10⁻⁶␣M ABP1. Biotinylated ABP1 was displaced from the membrane binding sites by competition with unlabeled ABP1, demonstrating specific binding. The association step proved to be pH-dependent with maximum binding at pH 5.0 or lower. Auxins did not influence the ABP1 binding to plasma-membrane vesicles, but ABP1 associated with plasma-membrane vesicles was still able to specifically bind [³H]naphthalene-1-acetic acid. The rather stable interaction of ABP1 with plasma-membrane vesicles was only affected by strong alkaline buffers or detergents. The binding capacity was calculated to be in the range of 0.2 pmol ABP1 per g coleoptile fresh weight. Received: 29 April 1996 / Accepted: 20 September 1996     

Purification and properties of an auxin-binding protein from maize shoot membranes

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.     

Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene. Received: 24 February 1999 / Accepted: 25 March 1999     

Organ distribution of auxin-binding protein 1 in the etiolated maize seedling

The auxin-binding protein designated ABP1 has been proposed to mediate auxin-induced cellular changes such as cell expansion. Its exact mode of action is unknown, but currently several approaches to elucidate its function are being pursued. One of these approaches, described here, is to determine the organ distribution of this putative auxin receptor in order to correlate spatially the abundance of the protein with some auxin-regulated activity such as cell elongation. The absolute and relative amounts of ABP1 were determined along the entire etiolated shoot, the root, and within the caryopsis of maize. ABP1 can be detected immunologically in all extracts of the etiolated maize seedling except the tip of the primary root and the endosperm. Within the shoot, but excluding the leaf roll, the highest levels compared on a fresh weight basis are in the apical mesocotyl and basal coleoptile regions, the areas of the most rapid cell elongation and the areas where there is the greatest capacity for auxin-induced growth. The relative abundance of ABP1 compared on a fresh weight basis changed more than fivefold in this organ. When compared on a total protein basis, the relative change in ABP1 abundance was approximately two-fold, which is less than the relative change in auxin-induced growth rate along the shoot. Differences in shoot growth rate among varieties of maize were compared with the relative amounts of ABP1 within the apical mesocotyl and basal coleoptile. A statistically significant but not perfect correlation was found between the auxin-induced growth rate of the apical mesocotyl and ABP1 abundance. These results demonstrate a general correlation between the amount of ABP1 and growth along the shoot and within maize hybrid varieties.Abbreviations ABP1 auxin-binding protein 1 - NAA naphthalene-1-acetic acid - SDS sodium dodecyl sulfate - PAGE poly-acrylamide gel electrophoresis.     

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.     

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization

The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.     

PROTEIN RETENTION IN THE ENDOPLASMIC RETICULUM OF INSECT CELLS IS NOT COMPROMISED BY BACULOVIRUS INFECTION

High level expression of the major auxin-binding protein (ABP1) from maize (Zea maysL.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immuno-localization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.