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
Articolo
Data di Pubblicazione:
2004
Citazione:
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 / M.C. Bonza, L. Luoni, M.I. De Michelis. - In: PLANTA. - ISSN 0032-0935. - 218:5(2004), pp. 814-823.
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 (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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Ca2+-ATPase; Plasma membrane;
Arabidopsis; Heterologous expression;
Saccharomyces; Site-directed mutagenesis
Elenco autori:
M.C. Bonza, L. Luoni, M.I. De Michelis
Link alla scheda completa: