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Arabidopsis 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 41I-T63 had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µM 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+. [ABSTRACT FROM AUTHOR]

The effect of phospholipids on the activity of the plasma membrane (PM) Ca2+-ATPase was evaluated in PM isolated from germinating radish (Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca2+ concentrations (10 μM) acidic phospholipids did not affect the Ca2+-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca2+-ATPase activity on free Ca2+ concentration showed that acidic phospholipids increased Vmax and lowered the apparent Km for free Ca2+ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent Km for free Ca2+ of the Ca2+-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca2+-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca2+-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed. [ABSTRACT FROM AUTHOR]