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.

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 ) 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 wildtype 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؉

Protein bodies of cereal seeds consist of ordered, largely insoluble heteropolymers formed by prolamin storage proteins within the endoplasmic reticulum (ER) of developing endosperm cells. Often these structures are permanently unable to traffic along the secretory pathway, thus representing a unique example for the use of the ER as a protein storage compartment. In recent years, marked progress has been made in understanding what is needed to make a protein body and in formulating hypotheses on how protein body formation might have evolved as an efficient mechanism to store large amounts of protein during seed development, as opposed to the much more common system of seed storage protein accumulation in vacuoles. The major key evolutionary events that have generated prolamins appear to have been insertions or deletions that have disrupted the conformation of the eight-cysteine motif, a protein folding motif common to many proteins with different functions and locations along the secretory pathway, and, alternatively, the fusion between the eight-cysteine motif and domains containing additional cysteine residues.

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.