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Arabidopsis thaliana plasma membrane Ca2+-ATPase isoform 8: molecular analysis of the autoinhibitory mechanism using chimeras
No full textACA8 is a plasma membrane (PM) Ca2+-ATPase of A. thaliana which has an extended N-te containing both an autoinhibitory domain and a calmodulin (CaM) binding site: CaM binding suppresses autoinhibition. This regulatory mechanism is similar to that of animal PM Ca2+ ATPase and plant PM H+-ATPase, but in these proteins the regulatory site is localised in the C-te. To analyse in details the autoinbitory mechanism, mutants in which the N-te of ACA8 is inserted at the C-te of the protein have been constructed using the cDNA of CaM insensitive mutant ï „74-ACA8. Moreover, chimeras in which ï „74-ACA8 is fused to the C-te domains of AHA1 (isoform 1 of A. thaliana PM proton pump) or of PMCA4b (isoform 4b of human PM Ca2+-ATPase) have been produced. All mutants expressed in the S. cerevisiae strain K616 are functional. Results show that repositioning the N-te region of ACA8 to the C-te of the protein does not interfere with its ability to autoinhibit the pump, thus the regulatory function of the terminal domain is independent from its position in ACA8. A detailed characterisation of the chimeras analysing their activity in presence of 14-3-3 and fusicoccin or CaM is in progres
Effect of phosphorylation of serine residues in the N-te region of ACA8 on the enzyme activity
No full textFunctional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant
No full textA constitutively active form of At-ACA8, a
plasma membrane Ca2+-ATPase from Arabidopsis thaliana
(L.) Heynh., from which the first 74 amino acids
containing the calmodulin-binding domain (D74-At-
ACA8) had been deleted, was expressed in Saccharomyces
cerevisiae strain K616, which lacks the main
endogenous active Ca2+ transport systems. D74-At-
ACA8 complemented the K616 phenotype, making it
able to grow in a calcium-depleted medium. D74-At-
ACA8 protein, which co-migrated with the endoplasmic
reticulum marker BiP in a sucrose-density gradient,
catalyzed MgATP-dependent Ca2+ uptake and Ca2+-
dependent MgATP hydrolysis, and retained the biochemical
characteristics of the native plant plasma
membrane Ca2+-ATPase (low specificity for nucleoside
triphosphate, high sensitivity to inhibition by the fluorescein
derivatives erythrosin B and eosin Y), thus
confirming that it is correctly folded and functional.
Substitution of the 794HE residues (numbers refer to fulllength
At-ACA8) following the highly conserved
TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece
with two lysine residues generated an hyperactive
protein, with a catalytic activity 2-fold higher than that
of D74-At-ACA8. The 794HE fi KK mutant was also
about 6-fold more sensitive than D74-At-ACA8 to
inhibition by vanadate, indicating that the mutation
determines an increase in the proportion of enzyme in
the E2 state during the catalytic cycle
Plasma membrane Ca2+-ATPase for crystallisation: Production and purification of the deleted mutant D109ACA8
No full textPlant and animal type 2B Ca2+-ATPases: Evidence for a common auto-inhibitory mechanism
No full textAbstractPlant auto-inhibited Ca2+-ATPase 8 (ACA8) and animal plasma membrane Ca2+-ATPase 4b (PMCA4b) are representatives of plant and animal 2B P-type ATPases with a regulatory auto-inhibitory domain localized at the N- and C-terminus, respectively. To check whether the regulatory domain works independently of its terminal localization and if auto-inhibitory domains of different organisms are interchangeable, a mutant in which the N-terminus of ACA8 is repositioned at the C-terminus and chimeras in which PMCA4b C-terminus is fused to the N- or C-terminus of ACA8 were analysed in the yeast mutant K616 devoid of endogenous Ca2+-ATPases. Results show that the regulatory function of the terminal domain is independent from its position in ACA8 and that the regulatory domain belonging to PMCA4b is able to at least partially auto-inhibit ACA8
LA Ca2+-ATPasi della membrana plasmatica di Arabidopsis thaliana : purificazione di un'isoforma espressa in lievito
No full textCalmodulin/Ca2+-ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform-specific Ca2+ dependencies
No full textArabidopsis thaliana plasma membrane (PM) Ca2+-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At-ACA8, the first cloned A. thaliana PM Ca2+-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At-ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide I-41-T-63 had the same affinity of the native protein [apparent dissociation constant (KD) at 10 mu M free Ca2+ about 25 nM], thus localizing At-ACA8 CaM-binding site within this sequence. The interaction of At-ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca2+-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca2+-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca2+ concentration, suggesting that enzyme conformation is affected by Ca2+. Binding of CaM isoforms to At-ACA8 N-terminus was affected differently by free Ca2+ concentration, suggesting that plant CaMs may have different affinities for Ca2+
The plant Ca2+-ATPases repertoire : biochemical features and physiological functions
No full textCa2+ ATPases are P-type ATPases which use the energy of ATP hydrolysis to pump Ca2+ from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca2+ pumping ATPases, named respectively ECAs (for ER-type Ca2+ ATPase) and ACAs (for auto-inhibited Ca2+ ATPase). Each type comprehends different isoforms, localised on different membranes. Here we summarise the available knowledge of the biochemical characteristics and of the physiological role of plant Ca2+ ATPases, greatly improved after gene identification which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants
Unravelling the molecular mechanisms of regulation of plant type 2B Ca2+-ATPases using Arabidopsis thaliana plasma membrane isoform ACA8 as a model system
No full textType 2B Ca2+-ATPases of plants (ACAs) have an extended cytosolic N-terminus containing an auto-inhibitory domain which by interacting with the catalytic head hampers pump activity. Using the Arabidopsis thaliana plasma membrane isoform ACA8 as a model, we have shown that fine-tuning of plant type 2B Ca2+-ATPases depends on multiple molecular mechanisms. Calmodulin (CaM) is the best known regulator of type 2B Ca2+-ATPases: CaM-binding to ACA8 at two sites in the N-terminus suppresses auto-inhibition and determines both an increase of Vmax and a decrease of the K0.5 for free Ca2+. Beside CaM, acidic phospholipids (APL) – as e.g. phosphatidylinositol-4P – stimulate ACA8 activity via a dual mechanism, involving different APL binding sites. APL binding to the N-terminus suppresses its auto-inhibitory action similarly to CaM, while binding to another, yet unidentified, site further increases the enzyme affinity for Ca2+. In addition, the N-terminus of ACA8 contains several Ser residue
Auto-inhibition of Arabidopsis thaliana plasma membrane Ca2+-ATPase involves an interaction of the N-terminus with the small cytoplasmic loop
No full textAbstractType IIB Ca2+-ATPases have a terminal auto-inhibitory, domain the action of which is suppressed by calmodulin (CaM) binding. Here, we show that a peptide (6His-1M-I116) corresponding to the first 116 aminoacids (aa) of At-ACA8, the first cloned isoform of Arabidopsis thaliana plasma membrane Ca2+-ATPase, inhibits the activity of the enzyme deprived of the N-terminus by controlled trypsin treatment 10-fold more efficiently than a peptide (41I-T63) corresponding only to the CaM-binding site. A peptide (268E-W348) corresponding to 81 aa of the small cytoplasmic loop of At-ACA8 binds peptide 6His-1M-I116 immobilized on Ni–NTA agarose. Peptide 268E-W348 stimulates Ca2+-ATPase activity. Its effect is not additive with that of CaM and is suppressed by tryptic cleavage of the N-terminus. These results provide the first functional identification of a site of intramolecular interaction with the terminal auto-inhibitory domain of type IIB Ca2+-ATPases
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