ATP-Dependent Ca<sup>2+</sup> Transport in Tonoplast Vesicles from Apple Fruit

Abstract

Protoplasts and vacuoles were isolated from immature apple fruit (Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca2+ uptake was identified in both protoplast vesicles and tonoplast vesicles. The apparent Km for Ca2+ of the tonoplast transport system was 43.4 μM. The pH optima were 7.2 and 6.7 for Ca2+ transport by protoplast and tonoplast vesicles, respectively. Ca2+ transport in tonoplast vesicles was strongly inhibited by the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamide hydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamide hydrochloride (W-5) was relatively ineffective. Addition of exogenous calmodulin stimulated transport by 35%. Ca2+ uptake was inhibited by vanadate, but not by the ionophores carbonylcyanide m-chlorophenyl hydrazone (CCCP) or valinomycin. The results indicate that apple tonoplasts have a Ca2+ transport system that is driven by the direct hydrolysis of ATP, and may be calmodulin dependent.