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 Ca2+-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (D74-At- ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca2+ transport systems. D74-At- ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. D74-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 fulllength 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 D74-At-ACA8. The 794HE fi KK mutant was also about 6-fold more sensitive than D74-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.