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Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant

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Abstract

A constitutively active form of At-ACA8, a plasma membrane Ca2+-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (Δ74-At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca2+ transport systems. Δ74-At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Δ74-At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca2+ uptake and Ca2+-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca2+-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the 794HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of Δ74-At-ACA8. The 794HE→KK mutant was also about 6-fold more sensitive than Δ74-At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E2 state during the catalytic cycle.

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Abbreviations

aa :

Amino acids

CaM :

Calmodulin

EB :

Erythrosin B

ER :

Endoplasmic reticulum

EY :

Eosin Y

FITC :

Fluorescein isothiocyanate

PM :

Plasma membrane

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Acknowledgements

We thank Dr. N.E. Hoffman (Department of Plant Biology, Carnegie Institution of Washington, Stanford), Dr. C.W. Slayman (Departments of Genetics and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven) and Dr. A. Vitale (CNR, Istituto di Biologia e Biotecnologia Agraria, Milano, Italy) for the generous gifts of the antisera against At-ACA1, N. crassa H+-ATPase and BiP, respectively. We also acknowledge Dr. K.W. Cunningham (Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts) for providing S. cerevisiae K616 and K601 strains. We are grateful to Dr. M. Beltrame and Dr. L. Popolo (Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Italy) for suggestions about yeast handling and to Prof. R. Jennings (Dipartimento di Biologia, Università di Milano, Italy) for kindly revising the English manuscript. This project was supported by the Italian Ministry for Instruction, University and Research in the COFIN 2000 frame.

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  1. Dipartimento di Biologia “L. Gorini”, Università di Milano, CNR Istituto di Biofisica—Sezione di Milano, via G. Celoria 26, 20133, Milano, Italy

    Maria Cristina Bonza, Laura Luoni & Maria Ida De Michelis

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  1. Maria Cristina Bonza
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  2. Laura Luoni
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  3. Maria Ida De Michelis
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Correspondence to Maria Ida De Michelis.

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Bonza, M.C., Luoni, L. & De Michelis, M.I. Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant. Planta 218, 814–823 (2004). https://doi.org/10.1007/s00425-003-1160-y

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