1,721,001 research outputs found
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
Functional 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
Characterization of the interaction between the plasma membrane H+-ATPase of Arabidopsis thaliana and a novel interactor (PPI1)
No full textProton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (EC 3.6.3.6) (Morandini P, Valera M, Albumi C, Bonza MC, Giacometti S, Ravera G, Murgia I, Soave C & De Michelis MI (2002) Plant J 31, 487-497). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H +-ATPase. PPI1-induced stimulation of H+-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H+-ATPase. These results indicate that PPI1 can bind the H+-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action
LA Ca2+-ATPasi della membrana plasmatica di Arabidopsis thaliana : purificazione di un'isoforma espressa in lievito
No full textDual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids : evidence for a phospholipid binding site which overlaps the calmodulin-binding site
No full textThe effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine=phosphatidylethanolamine=0. Acidic phospholipids increased Vmax-Ca2+ and lowered the value of K0.5-Ca2+ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K0.5-Ca2+ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant D74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein
Calmodulin/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+
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
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