Maria Ida De Michelis

3-O-Methyl glucose uptake stimulation by auxin and by fusicoccin in plant materials and its relationships with proton extrusion.

Auxin and fusicoccin (FC) stimulate the active uptake of 3-O-methyl glucose (3-O-MG) in those materials in which they have been shown to activate an electrogenic proton extrusion (Pisum sativum L. stems, Zea mays L. coleoptiles and roots). In maize roots the curve relating 3-O-MG influx to external concentrations indicated that the values of the apparent Km increase in the 3-O-MG concentration range between 2×10-5 mol l-1 and 2×10-2 mol l-1. FC did not alter the Km values and its stimulating effect was nearly constant at all 3-O-MG concentrations tested. Basal and FC-induced uptake of 3-O-MG appeared associated with a transient proton influx suggesting that also in maize roots a sugar-proton contransport occurs. Diethyl stilbestrol, which inhibits proton extrusion, inhibited also basal and FC-induced 3-O-MG uptake. The data support the view that the stimulation by FC of 3-O-MG uptake is closely related to that of proton extrusion. The stimulation by FC of 3-O-MG uptake cannot be replaced by increasing extracellular proton concentration, nor may be explained only by the FC-induced hyperpolarization of transmembrane potential difference. The hypothesis is proposed that the effect of FC on 3-O-MG uptake depends on an increase of cytoplasmic pH, following the activation of the proton extruding system.

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

In plant Ca2+ pumps belonging to the P2B 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 (Arg43 to Lys68) 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.

Active transport of Ca2+ in membrane vesicles from pea: Evidence for a H+/Ca2+ antiport

Two non mitochondrial systems involved in ATP-dependent Ca2+ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca2+ accumulation system, which shows the characteristics of an ATP-dependent H+/Ca2+ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO4-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg2+ or Mn2+. A second system of ATP-dependent Ca2+ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO4-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg2+ or Mn2+. This system is interpreted as corresponding to the (Ca2+ + Mg2+)-ATPase described by Dieter, P. and Marme, D. ((1980) Planta 150, 1–8).

H+/Ca2+ exchange driven by the plasma membrane Ca2+-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification.

The plasma membrane Ca2+‐ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)‐affinity chromatography and reconstituted in proteoliposomes by the freeze‐thaw sonication procedure. The reconstituted enzyme catalyzed CaM‐stimulated 45Ca2+ accumulation and H+ ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca2+ uptake, but it is a primary event linked to Ca2+ uptake in the form of countertransport.

Single point mutations in the small cytoplasmic loop of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, generate partially deregulated pumps.

ACA8 is a type 2B Ca2+-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 Glu252 to Asn345 sequence. Mutation to Ala of any of six tested acidic residues (Glu252, Asp273, Asp291, Asp303, Glu302, or Asp332) 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 Ca2+-ATPase plays a role in the attainment of the auto-inhibited state.

Ca2+-dependent phosphoregulation of the plasma membrane Ca2+-ATPase ACA8 modulates stimulus-induced calcium signatures

The plasma membrane Ca2+-ATPase ACA8 is a novel target of Ca2+-dependent CIPK–CBL complexes which tunes the pump activity affecting a stimulus-induced cytosolic Ca2+ transient in planta.

Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-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-1M-I116) 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 (KD ∼10-fold higher than wild-type ACA8) and S99D (KD 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-1M-I116 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.

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

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≅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 Δ74-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.

Ca2+ Pumps and Ca2+ Antiporters in Plant Development

Calcium (Ca2+) efflux transporters remove Ca2+ from the cytosol of the cell either by transporting it out of the cell across the plasma membrane or into internal organelles. These transporters, which include Ca2+-ATPases and Ca2+/H+ antiporters, have a critical role in preventing Ca2+ toxicity, maintaining cytosolic Ca2+ at a low resting level, and transferring Ca2+ to specific cellular locations where it is required. Many genes encoding plant Ca2+-ATPases and Ca2+/H+ antiporters have now been identified and characterised to elucidate their biochemical and genetic features. Furthermore, the use of gene knockouts has begun to provide evidence for an involvement of these Ca2+ transporters in Ca2+-signaling networks and in various aspects of plant development.

Reconstitution of proton pumping activity of a plasma membrane ATPase purified from radish

Abstract Plasma membrane ATPase partially purified from radish seedlings ( Raphanus sativum L.) (2.4–3.5 μmol P i min −1 mg −1 protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg:ATP. Mg:ATP-dependent proton pumping is prevented by N , N ′-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase.