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Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2+ )-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca 2+ and Mg 2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2+ /Mg 2+ mixed binding sites and two low-affinity Ca 2+ -specific sites. Binding of Ca 2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca 2+ or Mg 2+ , stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca 2+ -ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca 2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2+ pumps., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ca2+ signals are transient, hence, upon a stimulus-induced increase in cytosolic Ca2+ concentration, cells have to re-establish resting Ca2+ levels. Ca2+ extrusion is operated by a wealth of transporters, such as Ca2+ pumps and Ca2+/H+ antiporters, which often require a rise in Ca2+ concentration to be activated. Here, we report a regulatory fine-tuning mechanism of the Arabidopsis thaliana plasma membrane-localized Ca2+-ATPase isoform ACA8 that is mediated by calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) complexes. We show that two CIPKs (CIPK9 and CIPK14) are able to interact with ACA8 in vivo and phosphorylate it in vitro. Transient co-overexpression of ACA8 with CIPK9 and the plasma membrane Ca2+ sensor CBL1 in tobacco leaf cells influences nuclear Ca2+ dynamics, specifically reducing the height of the second peak of the wound-induced Ca2+ transient. Stimulus-induced Ca2+ transients in mature leaves and seedlings of an aca8 T-DNA insertion line exhibit altered dynamics when compared with the wild type. Altogether our results identify ACA8 as a prominent in vivo regulator of cellular Ca2+ dynamics and reveal the existence of a Ca2+-dependent CBL-CIPK-mediated regulatory feedback mechanism, which crucially functions in the termination of Ca2+ signals., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant auto-inhibited Ca²⁺-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca²⁺-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues--highly conserved in other ACA isoforms--localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca(2+)-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D--and to a lesser extent also mutants S22D and S27D--are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-(1)M-I(116)) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (K(D) ≈ 10-fold higher than wild-type ACA8) and S99D (K(D) about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-(1)M-I(116) peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.

Plant auto-inhibited Ca(2+)-ATPase 8 (ACA8) and animal plasma membrane Ca(2+)-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 Ca(2+)-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., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

ACA8 is a type 2B Ca(2+)-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu(252) to Asn(345) sequence. Mutation to Ala of any of six tested acidic residues (Glu(252), Asp(273), Asp(291), Asp(303), Glu(302), or Asp(332)) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca(2+)-ATPase plays a role in the attainment of the auto-inhibited state.

The 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 approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-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 Delta74-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.

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca(2+)-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K(0.5) value for the complex of 15 +/- 1 microg ml(-1), which is unaffected by free [Ca(2+)]. Heparin increases V(max) up to 3-fold while it does not significantly affect the apparent K(m) for free Ca(2+) and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain (Delta74-ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin-agarose gel show that heparin directly interacts with ACA8. Binding to the heparin-agarose gel occurs also with a peptide reproducing ACA8 sequence (1)M-I(116). Several single-point mutations within ACA8 sequence A56-T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca(2+)-ATPase and its animal counterpart, which is inhibited by heparin.

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.

Type 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.

A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-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 (794)HE residues (numbers refer to full-length 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 delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.