Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H(+)-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H(+)-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H(+)-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H(+)-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H(+)-ATPase.
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PMID:Phenylarsine oxide inhibits the fusicoccin-induced activation of plasma membrane H(+)-ATPase. 1067 39

The plasma membrane H(+)-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H(+)-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b, vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H(+)-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H(+)-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H(+)-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H(+)-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H(+)-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H(+)-ATPase in guard cells.
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PMID:Specific binding of vf14-3-3a isoform to the plasma membrane H+-ATPase in response to blue light and fusicoccin in guard cells of broad bean. 1116 Oct 66

A fungal phytotoxin fusicoccin (FC) causes irreversible opening of stomata by activation of the plasma membrane H+-ATPase in guard cells. However, the mechanism by which FC activates the H+-ATPase is not fully understood with respect to the event of phosphorylation. In this study, we provide quantitative evidence that FC-dependent activation of H+-ATPase requires the phosphorylation of the C-terminus, and that FC maintains the activated state by preventing the dephosphorylation. The plasma membrane H+-ATPase in guard cells was phosphorylated on serine and threonine residues in the C-termini of both VHA1 and VHA2 by FC, and the phosphorylation level paralleled the rates of H+-pumping and ATP hydrolysis. An endogenous 14-3-3 protein was co-precipitated with the H+-ATPase, and the amount of 14-3-3 protein was proportional to the phosphorylation level of H+-ATPASE: The recombinant 14-3-3 protein bound to the C-terminus only when it was phosphorylated, even in the presence of FC. The phosphorylated C-terminus was dephosphorylated by alkaline phosphatase, and the dephosphorylation was completely prevented when the C-terminus had been incubated with both FC and 14-3-3 protein. The results suggest that FC activates the H+-ATPase by accumulating the complex of phosphorylated H+-ATPase and 14-3-3 protein through inhibition of the dephosphorylation in guard cells.
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PMID:Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin. 1133 14

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.
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PMID:Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase. 1137 Nov 65

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H+ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 microM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H+ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H+ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen.
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PMID:Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth. 1152 49

Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.
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PMID:Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant. 1174

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHAI), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H+-ATPase. These effects are seen specifically in the inoculated epidermis and not in the whole leaf. We propose that 14-3-3 proteins are involved in an epidermis-specific response to the powdery mildew fungus, possibly via an activation of the plasma membrane H+-ATPase.
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PMID:Do 14-3-3 proteins and plasma membrane H+-AtPases interact in the barley epidermis in response to the barley powdery mildew fungus? 1199 70

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.
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PMID:Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase. 1219 5

The 14-3-3 protein family is a family of regulatory proteins involved in diverse cellular processes. In a previous study of regulation of individual 14-3-3 isoforms in the germinating barley embryo, we found that a post-translationally modified, 28 kDa form of 14-3-3A was present in specific cell fractions of the germinated embryo. In the present study, we identify the nature of the modification of 14-3-3A, and show that the 28 kDa doublet is the result of cleavage of the C-terminus. The 28 kDa forms of 14-3-3A lack ten or twelve amino acid residues at the non-conserved C-terminus of the protein, respectively. Barley 14-3-3B and 14-3-3C are not modified in a similar way. Like the 30 kDa form, in vitro produced 28 kDa 14-3-3A is still capable of binding AHA2 H+-ATPase in an overlay assay. Our results show a novel isoform-specific post-translational modification of 14-3-3 proteins that is regulated in a tissue-specific and developmental way.
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PMID:Post-translational modification of barley 14-3-3A is isoform-specific and involves removal of the hypervariable C-terminus. 1236 28

Blue light (BL) activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus with concomitant binding of 14-3-3 protein to the terminus in stomatal guard cells. However, the binding site and role of 14-3-3 protein in this physiological response have not been elucidated. We investigated the above using synthetic phosphopeptides designed from the C-terminus of Vicia H(+)-ATPase (isoform 1; VHA1). The presence of KGLDIDTIQQHYphospho-T(950)V peptide (P-950) prevented binding of 14-3-3 protein to the phosphorylated H(+)-ATPase. Dephosphorylated P-950 and other phosphopeptides, including typical phosphorylation sites in the C-terminus, had no effect on the binding. Incubation of BL-activated plasma membrane H(+)-ATPase with P-950 dissociated the 14-3-3 protein from the H(+)-ATPase without affecting phosphorylation levels and decreased the H(+)-ATPase activity. By contrast, incubation of P-950 with the activated H(+)-ATPase from fusicoccin-treated guard-cell protoplasts neither dissociated the 14-3-3 protein nor decreased the H(+)-ATPase activity. These results indicate that BL induces phosphorylation on threonine residue (Thr(950)) in the C-terminus of H(+)-ATPase, and that the binding of 14-3-3 to this site is required for the activation of H(+)-ATPase in stomatal guard cells.
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PMID:Biochemical evidence for the requirement of 14-3-3 protein binding in activation of the guard-cell plasma membrane H+-ATPase by blue light. 1246 Nov 36


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