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Plant Physiol, August 2000, Vol. 123, pp. 1495-1506

At-ACA8 Encodes a Plasma Membrane-Localized Calcium-ATPase of Arabidopsis with a Calmodulin-Binding Domain at the N Terminus1

Maria Cristina Bonza, Piero Morandini, Laura Luoni, Markus Geisler,2 Michael Gjedde Palmgren, and Maria Ida De Michelis*

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 (M.C.B., P.M., L.L., M.I.D.M.); and Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark (M.G., M.G.P.)

A Ca2+-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 Ca2+-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 Ca2+-transporting P-type Ca2+-ATPases. The identity of At-ACA8p as the PM Ca2+-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 Ca2+-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 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 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 Ca2+-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca2+-ATPases.

1 This project was supported by the Italian Ministry for University and Scientific Technology Research in the COFIN 99 frame and by the European Union Biotechnology Program FP-4.

2 Present address: Institut de Botanique, Physiologie Végétale, Université de Neuchâtel, CH-2007 Neuchâtel, Switzerland.

* Corresponding author; e-mail midemic{at}mailserver.unimi.it; fax 39-02-26604399.

© 2000 American Society of Plant Physiologists


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