At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus.
A Ca(2+)-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca(2+)-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca(2+)-transporting P-type Ca(2+)-ATPases. The identity of At-ACA8p as the PM Ca(2+)-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca(2+)-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca(2+)-ATPase to be cloned and is clearly distinct from animal PM Ca(2+)-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembrane-localized type IIB Ca(2+)-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca(2+)-ATPases.
Dipartimento di Biologia, Università di Milano, Centro di Studio del Consiglio Nazionale delle Ricerche per la Biologia Cellulare e Molecolare delle Piante, via G. Celoria 26, 20133 Milano, Italy.
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MC
Bonza
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Morandini
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Luoni
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Geisler
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Palmgren
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De Michelis
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000201:enzymology; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D002147:Calmodulin; D018076:DNA, Complementary; D010940:Plant Proteins; D000252:Calcium-Transporting ATPases