A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

Abstract

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.

Figure 1

Active Ca²⁺ uptake in subcellular fractions of etiolated pea epicotyls. A, ⁴⁵Ca²⁺ uptake by subcellular fractions measured in a reaction mixture containing 1 μm free Ca²⁺ in the presence (▪) and in the absence (□) of ATP. B, NADH cytochrome c (Cyt c) reductase activity insensitive to antimycin A. C, XG-FucTase activity. D, PM-ATPase activity measured in the presence (♦) and in the absence (⋄) of 70 μm vanadate. E, UDPase activity measured in native gels. F, Cyt c oxidase activity. G, Detection of γ-tonoplast intrinsic protein (γ-TIP) in subcellular fractions using western blots. H, Suc concentration and total protein across the gradient, determined by refractometry and the bicinchoninic acid method, respectively. The results are the average of three independent gradients measured by triplicates. The average relative activity and its deviation are plotted. The relative activity was standardized by setting the highest value obtained from the measurements at 100% relative activity. All subsequent values were then adjusted accordingly and the deviation was calculated. The highest value on each case correspond to: A, 1.95 pmol min⁻¹ μL⁻¹; B, 196 nmol min⁻¹ μL⁻¹; C, 0.52 pmol min⁻¹ μL⁻¹; D, 6.13 nmol min⁻¹ μL⁻¹; and F, 3.92 μmol min⁻¹ μL⁻¹.

Figure 2

Separation of Golgi apparatus membranes and active Ca²⁺ uptake from tonoplast. A, ATP-dependent Ca²⁺ uptake in subcellular fractions measured in the presence of 1 μm free Ca²⁺. B, Suc concentration across the gradient determined by refractometry. C, XG-FucTase activity. D, Total protein across the gradient. E, UDPase activity measured in native gels. F, Detection of γ-TIP using western blots. The measurements were done in triplicate. The average relative activity and its deviation were plotted. The relative activity was standardized by setting the highest value obtained from the measurements at 100% relative activity. All subsequent values were then adjusted accordingly and the deviation was calculated. The highest value on each case correspond to: A, 0.314 pmol min⁻¹ μL⁻¹; and C, 0.13 pmol min⁻¹ μL⁻¹.

Figure 3

Active Ca²⁺ uptake by Golgi vesicles. A, Time course of Ca²⁺ incorporation into Golgi apparatus vesicles measured in the incubation buffer containing 300 nm free Ca²⁺ in the presence (▪) and in the absence (□) of ATP. B, Time course of Ca²⁺ uptake and release by the Ca²⁺ ionophore A23187. ▪, Control; ○, ionophore. Free Ca²⁺ concentration was 210 nm. Values are mean ± se. The arrows indicate the time when the ionophore was added. C, Ca²⁺ dependence of active Ca²⁺ uptake in Golgi apparatus-enriched fraction. Ca²⁺ uptake initial rates were measured at different free Ca²⁺ concentrations that were estimated with the WinMaxC 2.05 computer program (Chris Patton, Hopkins Marine Station, Stanford University, CA). The experimental points were fitted by a nonlinear fitting program (GraphPad Prism 2.0, GraphPad Software, Inc., San Diego). Apparent K_m value of 0.21 μm (pCa 6.68) was obtained from by interpolation in nonlinear fit. Experiments were performed in triplicate. Values are mean ± se.

Figure 4

Ca²⁺ uptake in the presence of other divalent cations. The ⁴⁵Ca²⁺ uptake by the pea Golgi vesicles was measured at 25°C for 1.5 min, in an incubation buffer containing 870 nm free Ca²⁺ (4 times pump K_m), 2 mm ATP, and 3 mm MgCl₂. Experiments were carried out in the absence and in the presence of increasing concentrations (870 nm, 1.47 μm, 4.35 μm, and 8.70 μm) of each of the following divalent cations: Cu²⁺, Co²⁺, Mn²⁺, Fe²⁺, and Cd²⁺. Values of the Ca²⁺ uptake at 1.5 min in the presence of different concentrations of the divalent cations are expressed as a percentage of the Ca²⁺ uptake measured in the absence of other cations (6.3 ± 0.2 nmol mg⁻¹).

Figure 5

Effect of thapsigargin on the Ca²⁺ uptake by subcellular fractions from etiolated pea stems. Organelles were separated as described in Figures 1A or 2B, and the uptake of ⁴⁵Ca²⁺ by subcellular fractions was measured in a reaction mixture containing 1 μm free Ca²⁺ in the presence (□) and absence of 2 μm thapsigargin (▪). The lines named Golgi in A and B indicate the distribution in the gradient of the Golgi marker XG-FucTase. The line named ER in A indicates the distribution in the gradient of the ER marker, NADH Cyt c reductase activity insensitive to antimycin A. The activity of the ER marker was negligible in B. THG, Thapsigargin.

Figure 6

Thapsigargin inhibits the uptake of Ca²⁺ in Golgi but not in ER vesicles. Uptake of Ca²⁺ was measured using Golgi apparatus-enriched vesicles (A) and ER-enriched vesicles (B). After 1 min of incubation, 2 μm thapsigargin (THG; □) or the vehicle (▪) were added to the incubation medium and the Ca²⁺ uptake was determined for another 4 min. The free Ca²⁺ concentration in the medium was 300 nm. Values are mean ± se.

Figure 7

A thapsigargin-sensitive Ca²⁺ pump is present in Golgi-enriched cauliflower subcellular fractions. Membrane fractions (1–5, depicted in I) were obtained from cauliflower as described in “Materials and Methods.” Ca²⁺ uptake was measured in the presence and absence of thapsigargin (A). The thapsigargin-sensitive Ca²⁺ uptake component is shown in C. The Golgi markers Latent UDPase and XG-FucTase are shown in E and G, respectively. B, NADH Cyt c reductase activity insensitive to antimycin A (ER marker). D, Vanadate-sensitive PM-ATPase activity (PM marker). F, Detection of γ-TIP in membrane fractions using western blots. H, Cyt c oxidase activity. I, Suc concentration across the gradient determined by refractometry. The lines named 1 through 5 indicate the interfaces from where the membrane fractions were collected. J, Total protein measured in the membrane interfaces.