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

A constitutively active form of At-ACA8, a plasma membrane Ca^sup 2+^-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 Ca^sup 2+^ 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 Ca^sup 2+^ uptake and Ca^sup 2+^-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca^sup 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 ^sup 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 Δ74-At-ACA8. The ^sup 794^HE[arrow right]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 E^sub 2^ state during the catalytic cycle.[PUBLICATION ABSTRACT]