A Ca²⁺-ATPase and a Mg²⁺/H⁺-antiporter are present on tonoplast membranes from roots of Zea mays L.

Abstract

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.