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• W2042009962 abstract "ABP57 is an auxin-binding protein that possesses receptor function. In this study, a protocol for ABP57 purification was developed on the basis of cross-reactivity shown between ABP57 and antisera raised against bovine serum albumin, which enabled us to purify ABP57 with a high yield and to further characterize it. ABP57 activates plant plasma membrane H+-ATPase (PM H+-ATPase) via direct interaction. The binding of indole-3-acetic acid (IAA) to the primary binding site on ABP57 caused a marked increase in the affinity of ABP57 for PM H+-ATPase, which was accompanied by a change in ABP57 conformation. Meanwhile, additional IAA binding to the secondary site on ABP57 nullified the initial effect without inducing further conformational change. When ABP57 with IAA occupying only the primary site interacted with PM H+-ATPase, no IAA could access the secondary site. These results suggest that IAA-induced biphasic alteration in the affinity of ABP57 for PM H+-ATPase correlates with a bell-shaped dose response of the enzyme to IAA. There is also a possibility that, whereas the stimulation phase of the response is associated with a conformational change of ABP57, the destimulation phase probably results from hindrance arising directly from the presence of IAA at the secondary site. ABP57 is an auxin-binding protein that possesses receptor function. In this study, a protocol for ABP57 purification was developed on the basis of cross-reactivity shown between ABP57 and antisera raised against bovine serum albumin, which enabled us to purify ABP57 with a high yield and to further characterize it. ABP57 activates plant plasma membrane H+-ATPase (PM H+-ATPase) via direct interaction. The binding of indole-3-acetic acid (IAA) to the primary binding site on ABP57 caused a marked increase in the affinity of ABP57 for PM H+-ATPase, which was accompanied by a change in ABP57 conformation. Meanwhile, additional IAA binding to the secondary site on ABP57 nullified the initial effect without inducing further conformational change. When ABP57 with IAA occupying only the primary site interacted with PM H+-ATPase, no IAA could access the secondary site. These results suggest that IAA-induced biphasic alteration in the affinity of ABP57 for PM H+-ATPase correlates with a bell-shaped dose response of the enzyme to IAA. There is also a possibility that, whereas the stimulation phase of the response is associated with a conformational change of ABP57, the destimulation phase probably results from hindrance arising directly from the presence of IAA at the secondary site. auxin-binding protein indole-3-acetic acid plasma membrane polyacrylamide gel electrophoresis bovine serum albumin 3-(cyclohexylamino)propanesulfonic acid 2-(N-morpholino)ethanesulfonic acid dimethyl suberimidate 4-morpholinepropanesulfonic acid Auxin-binding proteins (ABPs)1 are a class of low abundance proteins in plants that bind active auxins with high affinity and specificity. As a result of ABP-auxin binding, ABP might initiate the auxin signal pathways leading to various cellular responses and thus have a plant hormone receptor function. Extensive studies have led to the identification of a number of ABPs in both membrane and soluble fractions, of which the best characterized in terms of cellular location, biochemical nature, and putative receptor function is ABP1 (for reviews, see Refs. 1Jones A.M. Prasad P.V. Bioessays. 1992; 14: 43-48Crossref Scopus (27) Google Scholar, 2Jones A.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1994; 45: 393-420Crossref Google Scholar, 3Napier R.M. Venis M.A. New Phytol. 1995; 129: 167-201Crossref Scopus (83) Google Scholar, 4Venis M.A. Napier R.M. Crit. Rev. Plant Sci. 1995; 14: 27-47Crossref Scopus (66) Google Scholar, 5Macdonald H. Physiol. Plant. 1997; 100: 423-430Crossref Google Scholar). The main natural auxin in most plants is indole-3-acetic acid (IAA), which appears to exert many effects in plants, including, most importantly, cell elongation. In fact, the term “auxin” is used to describe chemical substances that stimulate elongation growth in coleoptiles and many stems. There is no clear explanation of the mechanism by which auxin regulates cell growth. The immediate effect of exposure of plant tissues to auxin is proton excretion, occurring within minutes. The resulting apoplastic acidification provides a favorable condition for cell wall loosening, which could be an early part of auxin-induced cell expansion (1Jones A.M. Prasad P.V. Bioessays. 1992; 14: 43-48Crossref Scopus (27) Google Scholar, 3Napier R.M. Venis M.A. New Phytol. 1995; 129: 167-201Crossref Scopus (83) Google Scholar, 6Rayle D.L. Cleland R.E. Plant Physiol. ( Bethesda ). 1992; 99: 1271-1274Crossref PubMed Scopus (627) Google Scholar). H+-ATPase activity of the plasma membrane (PM) in plants is responsible for proton extrusion from the cell. Therefore, the acidification of the cell wall space is thought to result from activation of the electrogenic H+-ATPase in some manner. There is evidence that auxin stimulates plant PM H+-ATPase, which is correlated with an increased affinity of the enzyme for ATP (7Gabathuler R. Cleland R.E. Plant Physiol. ( Bethesda ). 1985; 79: 1080-1085Crossref PubMed Google Scholar) but does not involve a direct effect of auxin on the enzyme itself (8Santoni V. Vansuyt G. Rossignol M. Planta. 1991; 185: 227-232Crossref PubMed Scopus (39) Google Scholar, 9Szpornarski W. Vansuyt G. Rossignol M. Phytochemistry. 1991; 30: 1391-1395Crossref Scopus (14) Google Scholar). We have previously isolated two isoforms of a soluble ABP in rice plants, one from shoots and the other from roots, and partially characterized them with respect to IAA binding and interaction with the H+-ATPase (10Kim Y.-S. Kim D. Jung J. FEBS Lett. 1998; 438: 241-244Crossref PubMed Scopus (21) Google Scholar, 11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). Both isoforms, found to be monomeric proteins with an estimated molecular mass of about 57 kDa on SDS-PAGE and thus designated ABP57, work together with IAA to elicit an auxin response of plant PM over a very wide range of IAA concentrations. That is, the root isoform elicits a response with IAA concentrations of 10−10–10−7m, and the shoot isoform elicits a response with IAA concentrations of 10−7–10−4m. ABP57 is believed to have an unambiguous IAA receptor function in many respects. The rates of ATP hydrolysis and proton translocation by the H+-ATPase activity of plant PM are regulated biphasically by IAA only in the presence of the ABP. There are two different types of IAA-binding sites on the ABP molecule, one showing a higher affinity for IAA and the other a lower affinity. On its own, the ABP functions as a simple activator of PM H+-ATPase, but the binding of IAA makes the efficiency of this activation biphasic. In the present investigation we found that ABP57 has a domain in its internal amino acid sequence that is identical to one known to exist in bovine serum albumin (BSA). This finding led us to conjecture that the domain might provide a motif that is recognized by anti-BSA antibody, because serum albumin has been known to have affinity for indoles, such as tryptophan and IAA (12Bertuzzi A. Mingrone G. Gandolfi A. Greco A.V. Ringoir S. Vanholder R. Clin. Chim. Acta. 1997; 265: 183-192Crossref PubMed Scopus (32) Google Scholar, 13GPeters, T., Jr. (ed) (1996) All about Albumin: Biochemistry, Genetics, and Medical Applications, pp. 76-132, Academic Press, San Diego, CAGoogle Scholar). Consistent with this hypothesis, significant cross-reactivity between ABP57 and antisera raised against BSA was observed. Based on this observation, immunoaffinity chromatography using IgGanti-BSA as a probe was employed for isolating the ABP from rice plants. This approach recovered ABP57 purified to homogeneity with a yield of about 60%. The high yield preparation allowed us to further characterize the ABP with respect to its role in IAA signal transduction in plant PM. We focused particularly on whether the binding of IAA causes a conformational change of the ABP and, if so, how it is related to regulating the proton-translocating activity of plant PM. An underlying mechanism for the IAA effect on the H+-ATPase via ABP57 is proposed, with the relevant evidence provided. An ABP57 preparation, purified from the shoots of dark-grown rice seedlings as described previously (10Kim Y.-S. Kim D. Jung J. FEBS Lett. 1998; 438: 241-244Crossref PubMed Scopus (21) Google Scholar), was freeze-dried, redissolved in 70% formic acid, admixed with molar excess cyanogen bromide, and incubated at room temperature for 24 h in the dark. The resulting CNBr-cleaved peptides were purified in a fast protein liquid chromatography system (Amersham Pharmacia Biotech) with a Superdex 75 HR column. The fractions of peptide with molecular mass of about 15 kDa were concentrated under vacuum, run on an SDS-PAGE gel, electroblotted onto a polyvinylidine difluoride membrane in CAPS buffer at 50 V for 1 h, and then subjected to analysis in an amino acid sequencer (Applied Biosystems). BSA and ABP57 were run on an SDS-PAGE gel and electroblotted onto a polyvinylidine difluoride membrane as above. The membrane was blocked with Tris-buffered saline containing 5% (w/v) casein, then incubated with anti-BSA (Sigma), washed with Tris-buffered saline-Tween 20 and Tris-buffered saline, and then reincubated with peroxidase-conjugated anti-IgG (Sigma), as described by Harlow and Lane (14Harlow E. Lane D. Antibodies. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The membrane was thoroughly washed in Tris-buffered saline-Tween 20; then sprayed with a peroxidase substrate (Supersignal West Pico chemiluminescent substrate, Pierce) consisting of luminol, chemiluminescence enhancer, and stable peroxide buffer; and immediately used to expose x-ray film for a few seconds. Soluble protein, prepared by ammonium sulfate (30–60%) fractionation of crude extracts from etiolated rice seedlings, was solubilized in 20 mm Tris-Mes buffer (pH 8.0) and loaded onto an immunoaffinity column (1.7 × 3.5 cm) filled with anti-BSA-agarose (Sigma). Elution was done with a linear gradient of ethylene glycol (0–20%) in the same buffer with a flow rate of 0.1 ml/min. The fractions showing ABP57 activity were pooled, concentrated under vacuum, and passed through a DEAE-Sepharose CL-6B column (2.5 × 5 cm). The flow-through fractions were then retrieved. PM vesicles were prepared from rice roots as in Larsson et al. (15Larsson C. Widell S. Kjellbom P. Methods Enzymol. 1987; 148: 558-568Crossref Scopus (366) Google Scholar). The H+-ATPase was isolated from PM vesicles essentially according to Serrano (16Serrano R. Methods Enzymol. 1988; 157: 533-544Crossref PubMed Scopus (233) Google Scholar), with a slight modification for the detergent-solubilizing step; that is, 0.1%n-dodecyl-d-maltoside was used instead of 0.6% lysophosphatidylcholine to solubilize the membrane. Vanadate-sensitive ATP hydrolysis catalyzed by PM preparations as well as by the isolated enzyme was measured as described by Dufour et al. (17Dufour J.P. Amory A. Goffeau A. Methods Enzymol. 1988; 157: 513-528Crossref PubMed Scopus (59) Google Scholar). H+ uptake by the inside-out PM vesicles was assayed by the procedure of Palmgren (18Palmgren M.G. Plant Physiol. ( Bethesda ). 1990; 94: 882-886Crossref PubMed Scopus (79) Google Scholar). Dimethyl suberimidate (DMS), which can chemically link closely associated amino groups (19Mattson G. Conklin E. Desai S. Nielander G. Savage D. Morgensen S. Mol. Biol. Rep. 1993; 17: 167-183Crossref PubMed Scopus (227) Google Scholar), was used for the cross-linking experiments. A mixture of isolated H+-ATPase (70 μg/ml) and ABP57 (40 μg/ml) in 1 m triethanolamine-HCl (pH 9.7) was incubated with 10 mm DMS for 2 h at room temperature. The reaction was terminated by boiling in Laemmli buffer, and cross-linking was identified by SDS-PAGE. ABP57 (86 μg/ml) was incubated with trypsin (0.75 benzoyl-l-arginine ethyl ester units/ml) and IAA (0–1 mm) in 50 mm potassium phosphate (pH 7.8) at 30 °C for 8 min. The digestion was stopped by the addition of type II-T trypsin inhibitor (0.1 μg/ml) immediately followed by boiling in Laemmli buffer. Samples were run on SDS-PAGE gel and stained, and the remaining intact ABP was measured with a Gel-Pro analyzer (Media Cybernetics). Rayleigh scattering by ABP57 in 0.2 m potassium phosphate buffer containing IAA (0–1 mm) was measured at 25 °C in a fluorescence spectrophotometer (Hitachi F-4500), with both excitation (incident light) and emission (scattered light) wavelengths set at 350 nm. The circular dichroism (CD) spectra of the ABP were measured under the same buffer and temperature conditions as above in a Jasco spectropolarimeter (Jasco J-715). Because a preliminary investigation revealed that the N terminus of the ABP is blocked, internal sequencing was performed with a peptide purified from CNBr-treated ABP57. Interestingly enough, the N-terminal sequence of the peptide with 18 amino acids identified, such as REKVLTSSARQRLRCASI, was found to show 100% sequence identity to a part of domain II of BSA. 2GenBankTM accession number M73993, submitted by E. W. Holowachuk (1991). Because this domain could be an antigenic determinant, we tested whether BSA-specific polyclonal antibody cross-reacts with ABP57and found a band emitting chemiluminescence in Western blots on a polyvinylidine difluoride membrane (Fig.1). The intensity of luminescence from the ABP band was estimated to be about one-half of that from a BSA band of the same amount, indicating that there is significant cross-reactivity between the ABP and anti-BSA antibody. Based on the immunoblotting result, we developed a simple procedure for purifying ABP57 with a high yield by the use of IgGanti-BSA as an immunoadsorbent. Because the new protocol often needed a final clean-up step, a DEAE-Sepharose CL-6B column was used to remove small amounts of several protein contaminants that might bind nonspecifically to the antibody, yielding electrophoretically pure preparations of ABP57 as shown (Fig.2). The overall scheme of purification is compiled in Table I. One unit of activity was defined as the amount of protein that caused a 2-fold increase in the rate of ATP hydrolysis by 1 mg of protein of plant PM in the presence of the optimal concentration of IAA at 37 °C and pH 7.0.Figure 2Isolation of ABP57 from shoots of dark-grown rice seedlings. The progress of purification by ammonium sulfate fractionation of crude extracts (lane I), immunoaffinity chromatography based on the ABP-IgGanti-BSAinteraction (lane II), and removal of protein impurities using a DEAE-Sepharose column (lane III) is demonstrated by SDS-PAGE. The marker proteins are shown in lane M.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IPurification of ABP57 from rice seedlingsStep 1-aCX, crude extracts; AS, ammonium sulfate fractionation at 30–60% saturation; IA, immunoaffinity chromatography using anti-BSA-agarose; CL, cleanup by passing through a DEAE-Sepharose CL-6B column.ProteinTotal activitySpecific activitymgunitsunits/mgCX97.21091.1AS12.180.16.6IA0.03468.32009CL0.03064.72157Purification was from 250 g of fresh shoots.1-a CX, crude extracts; AS, ammonium sulfate fractionation at 30–60% saturation; IA, immunoaffinity chromatography using anti-BSA-agarose; CL, cleanup by passing through a DEAE-Sepharose CL-6B column. Open table in a new tab Purification was from 250 g of fresh shoots. Because ABP57 functions as an activator of PM H+-ATPase (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar), a relevant question is whether the ABP interacts directly with the target enzyme or indirectly via a specific protein, i.e. a docking protein in the membrane. If they interact directly to form a complex, a certain functional group, such as a sulfhydryl or primary amine, on the ABP molecule and on the H+-ATPase molecule might be in close proximity in the complex; this could be tested by chemical cross-linking (19Mattson G. Conklin E. Desai S. Nielander G. Savage D. Morgensen S. Mol. Biol. Rep. 1993; 17: 167-183Crossref PubMed Scopus (227) Google Scholar). SDS-PAGE analysis of the mixture of isolated H+-ATPase and ABP57 incubated with DMS (Fig.3) revealed that the proteins were covalently linked through reaction with the bifunctional cross-linker, which has a spacer arm 11 Å long. Under these conditions, diminution of the H+-ATPase (102 kDa) and ABP57 (57 kDa) bands is accompanied by the appearance of a new protein band with molecular mass of ∼162 kDa, virtually equal to the combined mass of the interacting proteins. In contrast to experiments with isolated H+-ATPase, H+-ATPase associated with PM was not cross-linked to ABP by DMS. This led us to infer that the quaternary structure of the complex formed between the membrane-associated enzyme and ABP57 may be somewhat different from that of the isolated enzyme-ABP57 complex. If such is the case, the primary amines responsible for the cross-linking may be situated beyond the range of the spacer arm of the cross-linker, being unable to react concurrently with the two imidoester groups of DMS. The hyperbolic dependence of PM H+-ATPase activity on the amount of ABP57 was studied in the presence of IAA at various concentrations (0–1 mm). The maximal stimulation of the target enzyme by the ABP as an effector (Smax) and the concentration of the effector required for one-half Smax(S0.5) were determined from the respective hyperbolic curves and plotted as functions of IAA concentration. This revealed that S0.5 values changed biphasically with increasing concentrations of IAA, as did Smax, with both the lowest S0.5 value and the highest Smax value appearing at 5 μ m IAA, as illustrated (Fig.4). The S0.5 value is often considered to inversely represent the affinity in an effector-enzyme interaction (21Verghese G.M. Johnson J.D. Vasulka C. Haupt D.M. Stumpo D.J. Blackshear P.J. J. Biol. Chem. 1994; 269: 9361-9367Abstract Full Text PDF PubMed Google Scholar). Therefore, such an effect of IAA on S0.5may be taken as an indication that auxin binding affects the intrinsic affinity of ABP57 for the H+-ATPase in a biphasic mode, maximizing it at about 5 μ mIAA. Our earlier study has shown that there are two different kinds of IAA-binding sites on the ABP molecule, a primary site with a smaller dissociation constant (Kd) value and a secondary site with a larger Kd value (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). In the case of the isoform from rice shoots, the primary site is fully occupied by IAA at 5 μ m, above which further binding of IAA to the secondary site takes place. Taking the results together, it may be concluded that the binding of IAA to the primary site enhances the affinity of ABP57 for the H+-ATPase, which, in turn, causes an increase in catalytic efficiency of the enzyme. Meanwhile, additional IAA binding to the secondary site diminishes the enhanced affinity of the ABP as well as the increased catalytic efficiency. Three different tests were performed to test whether IAA binding induces changes in the structure of the ABP57 molecule. In the first test, light scattering of the ABP in solution was assessed in the presence of 0–1 mmIAA. Because the angular dependence of light scattering by the solution was not measured, it was impossible to obtain significant quantitative information about the shape and size of the molecule. However, plotting the relative intensity of the scattered light measured at 90o against IAA concentration gave a titration curve that still appeared informative with respect to overall conformational alteration, as shown (Fig.5 A). The occurrence of IAA binding was manifested by a sharp increase in light scattering. This may indicate that, upon IAA binding, significant changes took place in some physical parameters involved in the process of light scattering, such as the polarizability, the refractive index, or the radius of gyration, that are closely linked to the overall structural property of a protein (22Pittz E.P. Lee J.C. Bablouzian B. Townsend R. Timasheff S.N. Methods Enzymol. 1971; 27: 209-256Crossref Scopus (34) Google Scholar). The results of CD experiments also appeared to conform to the concept of IAA-induced conformational alteration. CD data in the short UV region give some information on the secondary structure of proteins. For instance, a negative band at about 222 nm and a negative and positive couplet at about 208 and 190 nm are assigned toα-helix (23Johnson W.C. Tinoco I. J. Am. Chem. Soc. 1972; 94: 4389-4390Crossref PubMed Scopus (140) Google Scholar), and the CD for a β-strand has a negative band at about 215 nm and a positive band at about 198 nm (24Brahms S. Spach G. Brack A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3208-3212Crossref PubMed Scopus (162) Google Scholar). The spectra of ABP57 were not measured in wavelengths shorter than 200 nm because of limited sensitivity of the instrument with a limited amount of sample. Yet, the appearance of a negative band at 208 nm with a shoulder at around 220–225 nm indicates the presence of both α-helix and β-strand in ABP57. The binding of IAA gave rise to an increase in the content of these secondary structures, as shown (Fig. 5 B), which should affect the tertiary structure. Virtually the same pattern of structural response to IAA binding was observed in the other experiment, where the degree of protein digestion by trypsin was measured with the incubation of ABP57, IAA, and trypsin. The binding of IAA apparently made the ABP more susceptible to tryptic digestion, probably by inducing a conformational change as illustrated (Fig. 6).Figure 6Tryptic digestion of ABP57incubated with IAA at various concentrations. Data are presented as means ± S.D. (n = 3). The insetshows a typical SDS-PAGE result.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An intriguing feature of the above results was that, despite a significant difference in the regulatory effect between ABP57 with IAA bound only to the primary site and ABP57 with IAA saturation, no difference in overall conformation was detected between these two states. Therefore, the binding of IAA to the secondary site, without inducing further conformational change, was thought to somehow influence the interaction of the ABP with PM H+-ATPase, reducing it to the level of the interaction between IAA-free ABP57 and PM H+-ATPase. To investigate this effect, an experiment was conducted. The H+-ATPase of PM vesicles was activated to the highest degree by the optimal concentration of IAA (5 μ m) in the presence of the ABP, and excess IAA (1 mm) was subsequently added to the reaction mixture. It was then tested whether the additional, excess supply of IAA affects the H+-ATPase activity, revealing that the enzyme remained fully activated for both ATP hydrolysis and H+ translocation even under conditions of supraoptimal auxin concentrations. This was a clear contrast to the assay in which IAA was added first at 1 mm to the assay system (Fig.7). An incubation experiment was also conducted in which samples of PM vesicles plus ABP57 were incubated with IAA, added first at 5 μ m and then at 1 mm, and assayed for ATP hydrolysis at fixed time intervals for 20 h. As it turned out, the maximum activation of the H+-ATPase attained by 5μ m IAA via the ABP was not dissipated at all by the presence of excess IAA during the prolonged incubation (data not shown). A slight decrease in the H+-ATPase activity, which started to occur about 8 h after incubation at 4 °C (3 h at 10 °C), was thought to be due probably to thermal denaturation of the proteins. From these observations the following inference could be drawn. ABP57 by itself binds only loosely to the H+-ATPase, and free IAA can thus have easy access to both the primary and the secondary binding sites. On the other hand, the ABP with IAA only at the primary site binds tightly to the H+-ATPase so that the secondary IAA-binding site is effectively shielded, limiting the accessibility of IAA. The presence of IAA at the secondary site on the ABP causes interference, either sterically or electrostatically, with the protein-protein interaction. It is apparent that ABP57 shares a common epitope with BSA. However, it remains to be confirmed whether the identified partial sequence of 18 amino acids shared by both proteins is directly involved in the binding of IAA. Whatever the structural characteristics of ABP57 are, as compared with those of BSA, the cross-reactivity of the ABP with anti-BSA antibody provides the basis for large scale preparation of the ABP through practically a single chromatographic step. ABP57 was formerly identified and isolated based solely on a biochemical functional assay, rather than on ligand binding assays. For this, at least four chromatographic separation steps involving anion exchange, cation exchange, size exclusion, and tryptophan probe binding were needed for purifying to homogeneity with yields of about 3% for the ABP isoform from rice roots and about 6% for that from the shoots (10Kim Y.-S. Kim D. Jung J. FEBS Lett. 1998; 438: 241-244Crossref PubMed Scopus (21) Google Scholar, 11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). Compared with this earlier protocol of ours, the new protocol demonstrates a drastic improvement of purification in terms of overall yield, simplicity, and rapidity. ABP57 has intriguing biochemical properties related to regulating plant PM H+-ATPase. It may first be mentioned that the ABP interacts directly with the H+-ATPase, enhancing the catalytic efficiency by altering kinetic parameters, such as decreasing Km for the substrate and increasingVmax of ATP hydrolysis (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). Because PM H+-ATPase has an autoinhibitory domain in its cytoplasmic C-terminal region that is removed either reversibly or permanently when the enzyme is stimulated (25Palmgren M.G. Askerlund P. Fredrikson K. Widell S. Sommarin M. Larsson C. Plant Physiol. ( Bethesda ). 1990; 92: 871-880Crossref PubMed Scopus (172) Google Scholar, 26Palmgren M.G. Sommarin M. Serrano R. Larsson C. J. Biol. Chem. 1991; 266: 20470-20475Abstract Full Text PDF PubMed Google Scholar), ABP57 binding to the H+-ATPase is thought to displace the inhibitory domain in some manner. The effect of the regulatory domain displacement is typified by the result of tryptic digestion, in which the truncated enzyme with the C terminus deleted shows an increased activity with decreased Km and increasedVmax (25Palmgren M.G. Askerlund P. Fredrikson K. Widell S. Sommarin M. Larsson C. Plant Physiol. ( Bethesda ). 1990; 92: 871-880Crossref PubMed Scopus (172) Google Scholar). A protein modulator of plant PM H+-ATPase may be found in the 14–3-3 protein that interacts directly and reversibly with the H+-ATPase, in which a fungal toxin, fusicoccin, plays a role in stabilizing the complex formed between 14–3-3 and the enzyme (27Jahn T. Fuglsang A.T. Olsson A. Bruntrup I.M. Collinge D.B. Volkmann D. Sommarin M. Palmgren M.G. Larsson C. Plant Cell. 1997; 9: 1805-1814PubMed Google Scholar, 28Oecking C. Piotropski M. Hagemeier J. Hagemann K. Plant J. 1997; 12: 441-453Crossref Scopus (117) Google Scholar, 29Fullone M.R. Visconti S. Marra M. Fogriano V. Aducci P. J. Biol. Chem. 1998; 273: 7698-7702Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 30Baunsgaard L. Fuglsang A.T. Jahn T. Korthout H.A. de Boer A.H. Palmgren M.G. Plant J. 1998; 13: 661-671Crossref PubMed Scopus (186) Google Scholar). Recent evidence also indicates that phosphorylation of the penultimate threonine residue of the C terminus of the H+-ATPase facilitates 14–3-3 binding regardless of the presence of fusicoccin (31Svennelid F. Olsson A. Piotropski M. Rosenquist M. Ottman C. Larsson C. Oecking C. Sommarin M. Plant Cell. 1999; 11: 2379-2391PubMed Google Scholar, 32Fuglsang A.T. Visconti S. Drumm K. Jahn T. Stensballe A. Mattei B. Jensen O.N. Aducci P. Palmgren M.G. J. Biol. Chem. 1999; 274: 36774-36780Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 33Maudoux O. Batoko H. Oecking C. Gevaert K. Vadekerckhove J. Boutry M. Morsomme P. J. Biol. Chem. 2000; 275: 17762-17770Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In a sense, ABP57 is another kind of intrinsic protein modulator of PM H+-ATPase in plant cells. However, its major physiological relevance may rather be found in a role as a mediator of IAA action on plant PM, which has not been identified with the 14–3-3 protein. Another important property of ABP57 is believed to have connection with the presence of the primary and secondary binding sites with different affinities for IAA. Numerous results presented herein as well as in an earlier report (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar) are consistent with the concept that the ABP can exist in three different states depending on the degree of IAA binding, in the presence of a wide range of IAA concentrations. The second state (State II) is the most “activated” in terms of the ability to stimulate PM H+-ATPase through a direct protein-protein interaction, and the first and third states (State I and State III) are activated to a much lesser extent. The isoform of ABP57 from rice shoots remains in State I at IAA concentrations up to 0.1 μ m, in State III at concentrations greater than 100 μm, and in State II at ∼5 μ m IAA. In the case of the isoform from the roots, State I and State III were found to be attained at [IAA] < 0.5 nm and [IAA] > 100 nm, respectively, and State II was found to be attained at [IAA] = 5 nm(data not shown). Evidence is provided here that IAA binding indeed induces a change in the conformation of ABP57, which appears related to the ABP-mediated IAA effect in plant PM as far as stimulation of the H+-ATPase is concerned. A model of the two-step conformational change caused by stepwise binding of IAA to the primary and secondary sites would provide a rather feasible explanation as to the bell-shaped dose response of the enzyme to IAA, as suggested earlier (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). However, the present study shows that this is not the case, because conformational change occurs only once as IAA concentration increases. We therefore propose a mechanism, schematically represented herein (Fig.8), involving a putative ABP57-docking site on the ABP molecule that is able to interact intramolecularly with the autoinhibitory domain when it is docked by the ABP. As a result of such communication, the regulatory domain may be displaced from the catalytic region of the enzyme. According to this model, the ABP in State II shows the highest affinity for PM H+-ATPase because its structure fits well with the docking site. Meanwhile, the ABP in State I and State III may have low affinity for the docking site due to poor structural compatibility and hindrance arising from the presence of IAA at the secondary binding site, respectively. The proposed mechanism for describing both the stimulation and destimulation phases of the IAA effect on PM H+-ATPase activity is rather simple and straightforward. In this respect, ABP57 is distinct from other proteins claimed to possess auxin receptor function, most importantly including ABP1, which perceives auxin signals on the cell surface but is predominantly localized to the lumen of the endoplasmic reticulum (3Napier R.M. Venis M.A. New Phytol. 1995; 129: 167-201Crossref Scopus (83) Google Scholar, 34Barbier-Brygoo H. Crit. Rev. Plant Sci. 1995; 14: 1-25Crossref Scopus (42) Google Scholar). To explain auxin signal transfer between ABP1 and the H+-ATPase in PM, complex mechanistic schemes need to be formulated, involving the export of ABP1 from the endoplasmic reticulum (35Cross J.W. New Biol. 1991; 8: 813-819Google Scholar), the existence of a hypothetical ABP1-docking protein in plant PM (34Barbier-Brygoo H. Crit. Rev. Plant Sci. 1995; 14: 1-25Crossref Scopus (42) Google Scholar, 36Klämbt D. Plant Mol. Biol. 1990; 14: 1045-1050Crossref PubMed Scopus (65) Google Scholar), and signaling intermediates, such as phospholipase A2 and protein kinase (37Scherer G.F.E. André B. Planta. 1993; 191: 515-523Crossref Scopus (42) Google Scholar, 38Yi H. Park D. Lee Y. Physiol. Plant. 1996; 96: 359-368Crossref Scopus (37) Google Scholar), etc. Furthermore, despite evidence that ABP1 is involved in hyperpolarization of plant PM by auxin (36Klämbt D. Plant Mol. Biol. 1990; 14: 1045-1050Crossref PubMed Scopus (65) Google Scholar, 39Barbier-Brygoo H. Maurel C. Shen W.H. Ephritikhine G. Delbarre A. Guern J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 891-895Crossref PubMed Google Scholar, 40Barbier-Brygoo H. Ephritikhine G. Klämbt D. Maurel C. Palme K. Schell J. Guern J. Plant J. 1991; 1: 83-94Crossref Scopus (144) Google Scholar), it remains unclear whether auxin-induced variations of transmembrane potential in the presence of exogenous ABP1 result from the modulation of the H+-ATPase. The expression and activity of ion channels may also regulate the electrochemical gradient of protons (41Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-689PubMed Google Scholar). In conclusion, the present study not only discloses a peculiar immunological property of ABP57 that provides the basis for developing a simple protocol for purification, but also, more importantly, provides insights into the underlying mechanism for IAA-regulated H+ efflux in plant cells. The biphasic mode of the ABP-mediated IAA action observed with in vitrosystems may be correlated with the bell-shaped dose-response curves forin vivo IAA effects that have long been seen in the elongation growth of the coleoptiles and apical root segments of plants (20Radermacher E. Klämbt D. J. Plant Physiol. 1993; 141: 698-703Crossref Scopus (14) Google Scholar, 42Cleland R. Planta. 1972; 104: 1-9Crossref PubMed Scopus (54) Google Scholar, 43Shen W.H. Petit A. Guern J. Tempe J. Proc. Natl. Acad. Sci. U. S. A. 1988; 5: 3417-3421Crossref Scopus (178) Google Scholar, 44Karz W. Stolarek J. Piettruszka M. Malkowski E. Physiol. Plant. 1990; 80: 257-261Crossref Scopus (25) Google Scholar). In addition, it is worth mentioning that other active auxins, notably including synthetic auxins, such as 1-naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid, fail to elicit the same response of plant PM H+-ATPase in the presence of ABP57 as seen with the main natural auxin, IAA (11Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press.Google Scholar). This failure may conversely implicate that there are some other receptors that, together with various active auxins, play a role in electrochemically energizing plant PM." @default.
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• W2042009962 cites W1970763447 @default.
• W2042009962 cites W1971474078 @default.
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• W2042009962 cites W1980900314 @default.
• W2042009962 cites W1982309895 @default.
• W2042009962 cites W1983241185 @default.
• W2042009962 cites W1985111316 @default.
• W2042009962 cites W1985568006 @default.
• W2042009962 cites W1987762347 @default.
• W2042009962 cites W1989060957 @default.
• W2042009962 cites W1994646819 @default.
• W2042009962 cites W1998440852 @default.
• W2042009962 cites W1998750365 @default.
• W2042009962 cites W2003093819 @default.
• W2042009962 cites W2015206392 @default.
• W2042009962 cites W2027835533 @default.
• W2042009962 cites W2035148202 @default.
• W2042009962 cites W2049708690 @default.
• W2042009962 cites W2062273766 @default.
• W2042009962 cites W2073548573 @default.
• W2042009962 cites W2074011490 @default.
• W2042009962 cites W2077716409 @default.
• W2042009962 cites W2082362306 @default.
• W2042009962 cites W2087752405 @default.
• W2042009962 cites W2104146932 @default.
• W2042009962 cites W2110398660 @default.
• W2042009962 cites W2117094398 @default.
• W2042009962 cites W2142257645 @default.
• W2042009962 cites W2469005906 @default.
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