Vacuolar Ca2+-ATPases in Plants. Regulation by calmodulin binding to a N-terminal autoinhibitory domain.

The calcium ion (Ca2+) is essential for all plant and animal life. One important function of Ca2+ is as a second messenger in the responses of the cell to environmental and hormonal signals as well as in some intrinsic developmental processes. The intracellular concentration of Ca2+ must be strictly regulated, since a high concentration of free cytosolic Ca2+ is toxic. All eukaryotic cells carry calcium-transporting enzymes, Ca2+-ATPases, that pump Ca2+ out from the cytosol, across the plasma membrane or into the endo(sarco)plasmic reticulum and the vacuole. The Ca2+-ATPases have a high affinity for Ca2+ and keep the cytosolic free Ca2+ concentration at 100 — 200 nM. In mammalians, seven Ca2+-ATPase genes have been identified, while in the model plant Arabidopsis thaliana eleven Ca2+-ATPases have been identified. Ca2+-ATPases can be divided into two major groups: those that bind and are stimulated by calmodulin (CaM) and those that are not. The cDNA, BCA1 (Brassica oleracea Ca2+-ATPase 1), for a vacuolar, CaM-stimulated Ca2+ -ATPase from cauliflower was cloned by PCR, based on sequence information from tryptic peptides obtained from the CaM-affinity-purified protein. The location to the vacuolar membrane was confirmed by confocal immunomicroscopy on sections from cauliflower inflorescence. The CaM-binding domain was identified within the first 43 amino acids in the N terminus, which is in contrast to the animal plasma membrane Ca2+-ATPases which have their CaM-binding domain in the C terminus. The N terminus of BCA1 was expressed as a fusion protein and shown to bind CaM in a Ca2+-dependent fashion. Results from inhibition studies with a peptide corresponding to the CaM-binding domain of BCA1, indicate that the N terminus also functions as an autoinhibitor. The autoinhibitory domain overlaps at least partially with the CaM-binding domain. In vitro phosphorylation studies and controlled proteolysis suggest that the BCA1 N terminus probably also is a target for phosphorylation and regulatory proteolysis. Taken together, the results demonstrate the regulatory importance of the N terminus of BCA1 as a target for CaM binding, trypsin cleavage, and possibly phosphorylation, as well as its importance as an autoinhibitory domain. BCA1 was the first plant Ca2+-ATPase cloned for which a Ca2+-pumping activity had been shown for the corresponding gene product, and also the first Ca2+-ATPase shown to have a CaM-binding domain at its N terminus. The N-terminal location of the CaM-binding regulatory domain has later turned out to be a typical feature of CaM-stimulated Ca2+-ATPases in plants. The cloning and characterization of a vacuolar CaM-stimulated Ca2+-ATPase from Arabidopsis, ACA4 (Arabidopsis Ca2+-ATPase 4) is also described. ACA4 is 84% identical to BCA1, contains an N-terminal CaM-binding domain, and was shown to localize to small vacuoles in Arabidopsis protoplasts. Overexpression of activated ACA4 protected Arabidopsis seedlings against elevated NaCl in the growth medium, indicating a role for ACA4 in response to salt stress and one possible physiological function of a Ca2+-ATPase in planta