Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant.
A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-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 (794)HE 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 delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.
Bonza
Maria Cristina
MC
Dipartimento di Biologia "L Gorini", Università di Milano, CNR Istituto di Biofisica-Sezione di Milano, via G Celoria 26, 20133, Milano, Italy.
Luoni
Laura
L
De Michelis
Maria Ida
MI
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D000252:Calcium-Transporting ATPases