ACA8, a type 2B Ca ATPase, has a regulatory N-terminus with autoinhibitory action suppressed by binding of calmodulin (CaM). ACA8 N-terminus binds a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To define the role of this interaction in autoinhibition we have analysed a number of single point mutants of ACA8 E252-N345 sequence. Mutation to Ala of any of 6 acidic residues (E252, D273, D291, D303, E302, D332) originates an enzyme with normal activity in presence of CaM, but less CaM-stimulated. These results highlight the relevance in autoinhibition of a negative charge in the small cytoplasmic loop of ACA8. The most deregulated mutant is D291A, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the D291A mutant is stronger than that of D291N suggesting a more direct involvement of this residue in autoinhibition. Of the other mutants (I284A, N286A, P289A, P322A, V344A, N345A), only P322A has a basal activity higher than that of the WT. These results provide the first evidence that the small cytoplasmic loop of a type 2B Ca ATPase plays a role in the attainment of the autoinhibited state.
Mutations in the actuator of ACA8, a Ca-ATPase of A. thaliana, generate partially deregulated pumps / T. Fusca, M.C. Bonza, L. Luoni, S. Meneghelli, C. Marrano, M.I. De Michelis. ((Intervento presentato al 1. convegno Congress SIBV tenutosi a Verona nel 2009.
Mutations in the actuator of ACA8, a Ca-ATPase of A. thaliana, generate partially deregulated pumps
T. FuscaPrimo
;M.C. BonzaSecondo
;L. Luoni;S. Meneghelli;C. MarranoPenultimo
;M.I. De MichelisUltimo
2009
Abstract
ACA8, a type 2B Ca ATPase, has a regulatory N-terminus with autoinhibitory action suppressed by binding of calmodulin (CaM). ACA8 N-terminus binds a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To define the role of this interaction in autoinhibition we have analysed a number of single point mutants of ACA8 E252-N345 sequence. Mutation to Ala of any of 6 acidic residues (E252, D273, D291, D303, E302, D332) originates an enzyme with normal activity in presence of CaM, but less CaM-stimulated. These results highlight the relevance in autoinhibition of a negative charge in the small cytoplasmic loop of ACA8. The most deregulated mutant is D291A, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the D291A mutant is stronger than that of D291N suggesting a more direct involvement of this residue in autoinhibition. Of the other mutants (I284A, N286A, P289A, P322A, V344A, N345A), only P322A has a basal activity higher than that of the WT. These results provide the first evidence that the small cytoplasmic loop of a type 2B Ca ATPase plays a role in the attainment of the autoinhibited state.Pubblicazioni consigliate
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