At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus

A Ca2+-ATPase was purified from plasma membranes (PM) isolated from Arabido psis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryp tic fragments from the protein were microsequenced and the corresponding cD NA was amplified by polymerase chain reaction using primers designed from t he microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibi ted Ca2+-ATPase, isoform 8, accession no. AJ249352) enco des a 1,074 amino acid protein with 70 putative transmembrane domains, which contains all of the characteristic motifs of Ca2+-transporting P-type Ca2+-ATrases. The ide ntity of Af-ACA8p as the PM Ca2+-ATPase was confirmed by immunodetection wi th an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca2+-ATPases. Confocal fluo rescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM loc alized Ca2+-ATPase to be cloned and is clearly distinct from animal PM Ca2-ATPases due to the localization of its CaM-binding domain. CaM overlay ass ays localized the CaM-binding domain of At-ACA8p to a region of the N termi nus of the enzyme around tryptophan-47, in contrast to a C-terminal localiz ation for its animal counterparts. Comparison between the sequence of Af-AC A8p and those of endomembrane-localized type IIB Ca2+-ATPases of plants sug gests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca2+-ATrases.