A NOVEL CALMODULIN-REGULATED CA2-ATPASE (ACA2) FROM ARABIDOPSIS WITH AN N-TERMINAL AUTOINHIBITORY DOMAIN()

To study transporters involved in regulating intracellular Ca2+, we is olated a full-length cDNA encoding a Ca2+-ATPase from a model plant, A rabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). AC A2p is most similar to a ''plasma membrane-type'' Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membra ne, as shown by aqueous-two phase fractionation of microsomal membrane s. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p ) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and P MC1, Although basal Ca2+-ATPase activity of the full-length protein wa s low, it was stimulated 5-fold by calmodulin (50% activation around 3 0 nM). In contrast, the truncated pump was fully active and insensitiv e to calmodulin, A calmodulin binding sequence was identified within t he first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins, Thus, ACA2 encodes a novel calmodulin -regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.