Cloning and characterization of a gene encoding ABP57, a soluble auxin-binding protein

Auxin-binding protein 57 (ABP₅₇), a soluble auxin-binding protein, acts as a receptor to activate plasma membrane (PM) H⁺-ATPase. Here, we report the cloning of abp57 and the biochemical characterization of its protein expressed in E. coli. The analysis of internal amino acid sequences of ABP₅₇ purified from rice shoots enabled us to search for the corresponding gene in protein DB of NCBI. Further BLAST analysis showed that rice has four abp57-like genes and maize has at least one homolog. Interestingly, Arabidopsis seems to have no homolog. Recombinant ABP₅₇ expressed in E. coli caused the activation of PM H⁺-ATPase regardless of the existence of IAA. Scatchard analysis showed that the recombinant protein has relatively low affinity to IAA as compared to natural ABP₅₇. These results collectively support the notion that the cloned gene is responsible for ABP₅₇.     

A major isoform of the maize plasma membrane H(+)-ATPase: characterization and induction by auxin in coleoptiles.

The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.     

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.     

Heterologously expressed protein phosphatase calcineurin downregulates plant plasma membrane H+-ATPase activity at the post-translational level

To investigate the effects of calcineurin expression on cellular ion homeostasis in plants, we have obtained a transgenic cell culture of tomato, expressing constitutively activated yeast calcineurin. Transgenic cells exhibited reduced growth rates and proton extrusion activity in vivo. We show that reduction of plasma membrane H+-ATPase activity by expression of calcineurin is the basis for the observed phenotypes. Transgenic calli and cell suspensions displayed also increased salt tolerance and contained slightly higher Ca2+ and K+ levels. This demonstrates that calcineurin can modulate ion homeostasis in plants as it does in yeast by affecting the activity of primary ion transporters.     

Two isoforms of soluble auxin receptor in rice (Oryza sativa L.) plants: Binding property for auxin and interaction with plasma membrane H+-ATPase

An auxin receptor protein, isolated from the soluble fractionsof rice shoots and roots, was characterised in terms of the affinity andspecificity for IAA and the modulating effect onH⁺-ATPase of plant plasma membrane. The receptor proteingives a biphasic binding isotherm for IAA, indicating the existence ofthe primary and secondary binding sites. The predominant isoform of thereceptor in roots shows much higher affinity to IAA compared with thatin shoots. Being monomeric protein with about the same molecular mass(57–58 kDa) and showing a similar chromatographic behaviour, bothisoforms mediate IAA-induced modulation of the plasma membraneH⁺-ATPase in the respective IAA concentration rangesseparated by ca. 3 orders of magnitudes(10^-10–10^-7 M vs.10^-7–10^-4 M). Analysis of kinetic data ofthe H⁺-ATPase activity revealed that the receptor perse functions as an effector of the enzyme, causing a decrease inK_m and an increase in V_max through protein-proteininteraction at a 1:1 ratio. Further, it appeared that, while IAAdoes not affect by itself the kinetic parameters of theH⁺-ATPase, the auxin exerts its effect via thereceptor, biphasically regulating the efficiency of the effectormolecule probably by inducing two-phase conformational changes thatinvolve IAA binding to two separate binding sites. It was also foundthat other active auxins examined, such as indole-3-propionic acid,1-naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid, do notwork together with the receptor to elicit the same response of theH⁺-ATPase as seen with IAA.     

A novel immunological approach establishes that the auxin-binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma membrane.

Interactions of a collection of monoclonal antibodies (mAbs) to the recombinant Nicotiana tabacum auxin-binding protein 1 (Nt-abp1) were extensively characterized using surface plasmon resonance. Dynamic interaction studies using combinations of Nt-abp1, synthetic peptides corresponding to conserved sequences within auxin-binding proteins, and the mAbs have shown that a number of the mAbs recognized discontinuous epitopes revealing the junction of distinct domains in the folded protein. In particular, the two putative auxin binding domains and the C terminus of the protein were shown to interact with each other in the folded protein. Using the auxin-induced electrical response of tobacco protoplasts as a functional assay, all the mAbs exhibited either auxin antagonist or hormonomimetic properties. These effects, measured for the first time in homologous conditions, confirm that Nt-abp1 is present at the plasma membrane and is involved in the activation of the auxin-dependent electrical response of tobacco protoplasts. Based on our surface plasmon resonance data, we propose that the key event leading to the activation of this auxin electrical response consists of a conformational change in Nt-abp1.     

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.     

Isolation, purification and kinetic characterization of plasma membrane H(+)-ATPase of Candida albicans.

The plasma membrane ATPase of Candida albicans was solubilized by Tween 40 and purified to homogeneity on glycerol step gradient. The purified protein appeared as a single band of 100 +/- 4 KDa, represented greater than 98% of the total pure protein on densitometer scan. The purified PM-ATPase which was very specific to MgATP, had Km of about 0.77 mM and a sharp pH optimum at 6.6. Orthovanadate was able to inhibit the enzyme in a non-competitive manner, however, at higher concentrations the nature of inhibition changed to uncompetitive type. Based on molecular size, immuno cross-reactivity and sensitivity to different inhibitors, PM-ATPase of C. albicans appears to be similar to other ion pumps.     

A critical review on natural compounds interacting with the plant plasma membrane H+-ATPase and their potential as biologicals in agriculture

The plant plasma membrane (PM) H⁺-ATPase is an essential enzyme controlling plant growth and development. It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation. We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H⁺-ATPase is a potential target for these. In this review, we have evaluated the known non-protein natural compounds with regulatory effects on the PM H⁺-ATPase, focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.     

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.     

Bafilomycin A1 inhibits lysosomal,phagosomal, and plasma membrane H+-ATPase and induces lysosomal enzyme secretion in macrophages

Bafilomycin A₁, a specific inhibitor of H⁺-ATPases of the vacuolar type, was in the present study shown, at similar concentrations, to induce secretion of lysosomal enzyme and to elevate lysosomal pH in mouse macrophages. These results lend support to the previous suggestion of a triggering role for an increase in lysosomal pH and a permissive role for cytosolic pH in the exocytosis of macrophage lysosomal enzyme. Vacuolar H⁺-ATPases are present in the macrophage plasma membrane as well as in intracellular membranes, for example, those of the lysosomal and phagosomal compartments. Phagosomal acidification was shown to be achieved in part by a mechanism with a similar sensitivity to bafilomycin A₁ as lysosomal H⁺ transport and in part by an early, bafilomycin A₁-insensitive mechanism. We found a lesser sensitivity towards bafilomycin A₁ of the lysosomal and phagosomal H⁺-ATPase than that localized in the plasma membrane, indicating differences among H⁺-ATPases at the subcellular level. Also, by attempts to mobilize lysosomal H⁺-ATPase to the plasma membrane, support was obtained for the notion that subcellular H⁺-ATPase populations differ and thus possibly could be differentially regulated. © 1995 Wiley-Liss, Inc.     

Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence. Homology to Na+/K+-, Ca2+-, and K+-ATPase

The gene for the Neurospora crassa plasma membrane H+-ATPase has been cloned and sequenced. The gene encodes for a protein of 920 amino acids with a molecular weight of 100,002. The coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus. The deduced amino acid sequence of the N. crassa plasma membrane H+-ATPase exhibits 75% homology to the amino acid sequence of the Saccharomyces cerevisiae plasma membrane H+-ATPase. Also, an amino acid comparison with the Na+/K+-ATPase from sheep kidney, Ca2+-ATPase from rabbit muscle, and K+-ATPase from Escherichia coli reveals that certain regions are highly conserved and suggest that these regions may serve essential functions which are common to the various cation-motive ATPases. This observation suggests that the phosphorylatable, cation-motive ATPases may function via a similar energy transduction mechanism.     

Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.     

Stalk segment 4 of the yeast plasma membrane H+-ATPase. Mutational evidence for a role in the E1-E2 conformational change

In the P(2)-type ATPases, there is growing evidence that four alpha-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P(2)-ATPases. When site-directed mutants in this region of the yeast plasma membrane H(+)-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC(50) values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K(m) for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E(2) conformation of the ATPase toward the E(1) conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an alpha-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane.     

A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.     

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.     

A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins

The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.     

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.