Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots

plan cienc e ELSEVIER SCIENTIFIC PUBLISHERS IRELAND

Plant Science 92 (1993) 143-150

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots Olivier Gavin, Paul-Emile Pilet, Alain Chanson* Institute of Plant Biology and Physiology, University of Lausanne, Biology Building, CH-IOI5 Lausanne, Switzerland (Received 5 April 1993; revision received 17 May 1993; accepted 17 May 1993)

Abstract The subcellular localization of a calmodulin-stimulated calcium (Ca2÷)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca 2÷ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure 45Ca 2÷ transport in sealed vesicles. Most of the ATP-dependent Ca 2÷ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H÷/Ca 2÷ antiport) and the plasma membrane. When the Ca 2÷ transport was measured in the presence of exogenous calmodulin (1 ttM), a 3-5fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H÷/Ca 2÷ antiport. In conclusion, four different Ca 2÷ transporters are present in young maize root cells. A Ca2÷/H ÷ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulininsensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast. Key words: Ca2+-ATPase; Calmodulin; Marker enzymes; Tonoplast; Endoplasmic reticulum; Root; Zea mays L.

* Corresponding author. Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; BTP, bis-tris-propane; CCCP, carbonyl cyanide m-chlorophenyl-hydrazone; DCCD, N, N'-dicyclohexylcarbodiimide; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disuifonate; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; PATPase, plasma membrane ATPase; PPase, pyrophosphatase; UDPase, uridine diphosphatase; V-ATPase, vacuolar ATPase; 1 KS, 1000 x g supernatant; 6 KS, 6000 x g supernatant; 120 KP, 120 000 x g pellet; 148 KP, 148 000 x g pellet.

1. Introduction Calcium (Ca 2÷) plays an extremely important role in the regulation o f growth and developmental processes in higher plant cells [1,2]. The cytosolic free Ca 2÷ concentration is maintained at a low level (about 0.1 gM), against an electrochemical gradient favoring its passive influx, to avoid cytotoxic interactions [3]. Excess Ca 2÷ can be extrud-

0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03657-H

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ed across the plasma membrane by a primary ATP-dependent pump or sequestered into intracellular organelles [1,4]. Mitochondria and plastids can accumulate Ca 2+ from the cytosol, but it would appear unlikely that they play an important role in the regulation of Ca 2+ concentration [1]. A Ca2+/H + antiport, depending on a protonmotive force, is located at the tonoplast [2,5]. Demonstration of calcium transport in 'purified' endoplasmic reticulum (ER) vesicles suggests that a calcium-pumping ATPase is associated with this membrane [6]. Transport is postulated to be from the cytoplasm into the lumen of the ER. This ATPase is inhibited by vanadate and is insensitive to nitrate, N,N'-dicyclohexylcarbodiimide (DCCD), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). All these results suggest that the ER transport system is a Ca2+-Mg2+-ATPase that directly couples ATP hydrolysis to Ca 2+ transport. Interestingly, a primary Ca2+-ATPase has sometimes been reported to be present on the tonoplast [2]. A transient increase in the cytoplasmic level of Ca 2÷ (e.g., by opening Ca 2+ channels) in response to external stimuli is a mechanism by which Ca 2÷ exerts its function as second messenger [7]. The activity of key regulatory enzymes, such as protein kinases, is modulated directly by these transient increases of cytosolic Ca 2+ [2,8]. In addition, calmodulin, an ubiquitous protein in higher plant cells, is thought to be a fundamental component of calcium signal transduction pathways, in modulating the activities of several enzymes (NAD and protein kinases, the plasma membrane Ca 2+ATPase, etc.) and some non-enzymic proteins in a calcium-dependent manner [8]. So far, the results on the stimulation by calmodulin of these different Ca 2+ transport activities have been puzzling and often contradictory (2,8,9). The presence of a calmodulin-stimulated Ca2÷-ATPase in maize root microsomes has been shown by different laboratories [6,10-16]. However, the subcellular origin of this activity is still a matter for debate [2]. The aim of the present study was to analyze the subcellular distribution, in maize root cells, of

O. Gavin et al. / Plant Sci. 92 (1993) 143-150

Ca 2+ transport activities (in the presence and absence of exogenous calmodulin), using marker enzymes to localize the different membranes. The long term objective of this research is to understand the role played by the different Ca 2+ transporters in maize root Ca 2+ homeostasis. 2. Materials and methods 2.1. Plant material

Maize (Zea mays L., cv LG 11, Association Suisse des S61ectionneurs, Lausanne) roots (10-20 mm in length) were grown (48 h in darkness), harvested and homogenized in buffer A (250 mM sucrose, 2 mM EDTA, 1 mM DTT, 0.1% BSA, 50 mM Tris, pH adjusted to 7.8 with solid Mes) as previously described [17]. For the Mg2+-sucrose gradients, 4 mM MgC12 and 1 mM EDTA were present in buffer A. The homogenate was centrifuged for 5 min at 1000 x g (Beckman J2-21, JA-20 rotor) to sediment the starch grains, unbroken cells, wall fragments and nuclei. The supernatant (1 KS) was collected and centrifuged for 20 min at 6000 × g (Beckman J2-21, JA-20 rotor) to sediment the majority of the mitochondria [17]. The supernatant (6 KS) was collected and centrifuged for 30 min at 148 000 × g (Kontron Centrikon T-2080, TST 28.17 rotor) and the microsomal pellet (148 KP) resuspended in 10% sucrose (in gradient buffer B: 1 mM DTT, 2.5 mM Tris, adjusted to pH 7.5 with solid Mes). For the Mg2+-sucrose gradients, 3 mM MgCI2 were present in the gradient buffer. 2.2. Linear sucrose gradients MgCI2

with or without

The 148-KP fraction was layered onto a linear gradient consisting of a 2-ml cushion of 45% (w/w) sucrose, 20 ml 10-45% sucrose and a 1-ml overlay of 10% sucrose (in gradient buffer B with or without MgC12). The gradients were centrifuged at 80 000 × g for 3 h (Kontron Centrikon T-2080, rotor TST 28.38) and fractionated into 16 portions of 1.5 ml each. All the fractions were frozen in liquid nitrogen and stored at -70°C until subsequent use.

145

O. Gavin et al./ Plant Sci. 92 (1993) 143-150 2.3. Preparation of sealed plasma membrane vesicles Maize roots were homogenized using buffer C (250 mM sucrose, 2 mM EGTA, 2 mM MgSO4, 2 mM ATP.Na2, 1 mM PMSF, 2 mM DTT, 10% (w/v) glycerol, 0.5% (w/v) BSA, 25 mM bis-trispropane (BTP), pH adjusted to 7.8 with solid Mes) and a 6-KS was prepared, as previously decribed. The 6-KS was centrifuged for 45 min at 120 000 x g (Kontron Centrikon T-2080, TST 28.17 rotor). The microsomal pellet (120 KP) was resuspended in buffer D (10% (w/v) glycerol, 250 mM sucrose, 2 mM BTP, pH adjusted to 7.2 with solid Mes). 2.4. Linear dextran gradient The 120-KP fraction was layered onto a linear gradient consisting of a 2-ml cushion of 12% (w/w) dextran, 20 ml 1-12% dextran and a 1-ml overlay of 1% dextran (in buffer D). The gradient was centrifuged at 80 000 × g for 3 h (Kontron Centrikon T-2080, rotor TST 28.38) and fractionated into 16 portions of 1.5 ml each. All the fractions were frozen in liquid nitrogen and stored at -70°C until subsequent use. 2.5. Measurement of Ca 2+ uptake Ca 2÷ uptake was assayed by the membrane filtration technique as previously described [5,18]. Standard incubation medium contained 50 mM KCI, 25 mM BTP-Mes (pH 7.5), 10% sucrose, 1 mM NaN 3, 3 mM MgC12, 3 mM ATP.Na2 (or 0.1 mM PPi.Na4) and 10 /~M CaCI2+ 45CAC12 (50 000 counts/min). The reaction was initiated by the addition of vesicles (20-50 t~g protein) at the appropriate time. After 10 min, the membranes were collected by filtration on 0.45 #m filters (Gelman GN-6). The filters were washed three times with 1 ml of washing medium (50 mM KC1, 10 #M CaCI2, 10% sucrose and 25 mM BTP-Mes at pH 7.5). Radioactivity associated with the filters was determined by liquid scintillation spectrometry in 4 ml of Filter-Count TM (Packard Instruments). 2.6. Measurement of proton-pumping The method of Chanson and Pilet [19] was used with minor modifications. Membrane vesicles, 3 mM MgSO4, 50 mM KC1 (vacuolar ATPase [V-

ATPase]) or KNO3 (plasma membrane ATPase [P-ATPase] and pyrophosphatase [PPase]) and 9amino-6-chloro-methoxyacridine (ACMA, 2 ~M final concentration) were added to an assay buffer of BTP-Mes (P-ATPase: pH 6.5; V-ATPase and PPase: pH 7.5) to a final volume of 0.6 ml. Fluorescence was measured at room temperature with a Perkin Elmer LS-3 Luminescence Spectrometer (excitation, 430 nm; emission, 500 nm). After temperature equilibration, the reaction was initiated by the addition of 3 mM ATP.Na2 (pH 7.5 or 6.5 with BTP) or 0.1 mM PP • iNa 4. At the end of the experiment, the pH gradient was collapsed using 3 tA of 1 mM monensin (in ethanol). The initial rate of ATP- or PPi-dependent fluorescence quenching was measured (%/min). 2.7. Marker enzyme assays and protein determination The activities of different marker enzymes were determined as previously described [20]. Vanadate-sensitive P-ATPase was selected as a plasma membrane marker; PPase as a tonoplast marker; latent UDPase as a Golgi marker; antimycin-A insensitive NADH-cytochrome c reductase as an ER marker and cytochrome c oxidase for the mitochondria. Protein determinations were carried out using the method of Bradford [21] with BSA as a reference standard. 2.8. Data presentation All experiments were performed at least three times and results are from one representative experiment. 2.9. Chemicals 45CAC12 (10-40 mCi/mg of calcium) was obtained from the Radiochemical Centre, Amersham, UK. Tris, Mes and BTP were bought from Calbiochem-Behring. Calmodulin (P 2277) and BSA were from Sigma Chemical Company. All the other chemicals were purchased from Fluka AG, Buchs, Switzerland. 3. Results

Density gradient centrifugations and marker assays were used in order to determine the distri-

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O. Gavin et a l . / Plant Sci. 92 (1993) 143-150

bution of Ca 2+ transport activities in maize roots. Two types of density gradients were employed (sucrose and dextran) so as to ensure a correct separation and identification of membrane vesicles.

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3.1. Distribution of calcium uptake after linear sucrose gradient centrifugation Linear sucrose gradients, in the presence or absence of Mg 2+, were used to separate the different membranes from a microsomal fraction (148 KP) [20]. Direct measurements of Ca 2+ uptake in sealed vesicles were done using 45Ca2+ and a filtration technique [5,18]. The transport experiments were carried out in the presence of 10 #M monensin (an electroneutral proton-sodium ionophore), to avoid any interference due to the tonoplast Ca2+/H + antiport activity [5]. NADH-cytochrome c reductase was used as an ER marker and PPase as a tonoplast marker. In the absence of Mg 2+ (Fig. 1), the two activities were recovered at a density of about 1.11 g/cm 3 (Fig. IB) together with a peak of protein (Fig. IA) corresponding to light membranes (tonoplast, ER and dictyosomes mainly) [17,19]. The ATP-dependent Ca 2+ uptake activities, in the presence or the absence of calmodulin (1 t~M), were also associated with these low density membranes (Fig. 1C). A very strong stimulation of Ca 2+ uptake was observed in the presence of calmodulin. When Mg 2+ was present in the homogenization and gradient buffers, the NADH-cytochrome c reductase activity was effectively shifted to higher densities (1.16 g/cm 3) (Fig. 2), as previously reported [20]. Under these conditions, most of the PPase activity was separated from the NADHcytochrome c reductase (Fig. 2B). The distribution of the ATP-dependent Ca 2+ uptake was similar to the ER distribution (Fig. 2C). When calmodulin (1/xM) was added to the incubation medium, a very strong stimulation of Ca 2÷ uptake was observed, with a distribution similar to the PPase activity. These experiments indicate that at least two ATP-dependent Ca2÷-pumps are present in maize root membranes. The first Ca2+-ATPase is associated with the ER membranes and is not (or only slightly) stimulated in vitro by calmodulin. The second Ca2+-ATPase is almost inactive in vitro in the absence of calmodulin, and has a dis-

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alternative separation of membrane vesicles (as compared to sucrose gradient centrifugation) with an improved recovery of sealed vesicles [20]. The latter were important in studies of H +- and C a 2+ uptake by the different vesicle populations. For

this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and C a 2+ transport activities. The maize roots were homogenized in the presence of glycerol, PMSF and ATP, to protect the ATPases, and a microsomal pellet was prepared (120-KP fraction) by differential centrifugation. A linear dextran gradient was used to separate the different membranes from the 120-KP fraction (Fig. 3) [20]. The PPase (Fig. 3B) and the NADH-cytochrome c reductase (Fig. 3C) activities floated on the top of the gradient. The plasma membrane (P-ATPase) (Fig. 3B), the dictyosomes (latent UDPase; Fig. 3D) and a small amount of mitochondria (cytochrome c oxidase; Fig. 3C) were recovered in the gradient. Using a dextran gradient, the tonoplast (PPase) distribution was separated from the bulk of the ER, plasma membrane, dictyosomes and mitochondria. The presence in the gradient of sealed plasma membrane vesicles was confirmed by the measurement of proton transport by the P-ATPase (H+-PATPase; Fig. 3D), using a fluorimetric technique. The proton-pumping activities of the PPase (H +PPase) and the V-ATPase (H+-V-ATPase) were also measured (Fig. 3E). The distributions of the H+-PPase and H+-V-ATPase were identical to the PPase distribution. The H+-PPase was much more active in vitro than the H+-V-ATPase and the H +P-ATPase. After linear dextran density gradient fractionation, the ATP-dependent Ca 2+ uptake (in the absence of monensin) was associated mainly with the ER (Fig. 3F). However, a small amount of the C a 2+ uptake was recovered with the tonoplast (corresponding to the activity of the H+/Ca 2+ antiport) and with the plasma membrane. When PPi was used to specifically induce a protonmotive force in sealed tonoplast vesicles, the distribution of C a 2+ uptake (due to the activity of the Ca2+/H + antiport [5]; Fig. 3F) was identical to the tonoplast distribution. When calmodulin (1/~M) was present in the incubation medium, a strong stimulation of ATP-dependent Ca 2÷ uptake was observed (Fig. 3G), whereas the uptake in the presence of PPi was not changed (Fig. 3H). The distribution of the calmodulin-stimulated activity (Fig. 31) was identical to the PPase distribution (Fig. 3B).

O. Gavin et al./Plant Sci. 92 (1993) 143-150

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O. Gavin et al./ Plant Sci. 92 (1993) 143-150

These experiments indicate that a primary Ca2÷-ATPase, which is extremely calmodulindependent, is associated with the tonoplast of young maize root cells. This activity is almost undetectable in the absence of calmodulin and cannot be derived from a stimulation of the Ca2+/H + antiport. 4. Discussion Four different Ca 2÷ transport activities are present in two day old maize roots. A Ca2÷-trans locating ATPase is known to be associated with the ER [2,6]. The presence of a primary Ca 2+ATPase on the plasma membrane has also been clearly demonstrated using maize roots [15] or other plant materials [2,22-25]. In this study, when an experimental protocol was specifically developed to prepare microsomal membranes containing sealed plasma membrane vesicles, a shoulder of ATP-dependent Ca 2+ uptake activity was clearly associated with this membrane (Fig. 3F). A Ca2+/H + antiport is present on the tonoplast of maize root cells [5]. When the transport experiments were carried out in the absence of monensin, the tonoplast Ca2+/H + antiport activity was clearly detectable, using the V-ATPase or the PPase to energize the vesicles [5]. So far the results on the stimulation by calmodulin of the plasma membrane and ER Ca 2+ATPases from higher plant cells have been puzzling and often contradictory [2,81. The lack of calmodulin effect using membrane fractions from different plant species could be related to the presence of sufficient endogenous (bound) calmodulin to mask any effect of added exogenous calmodulin [26]. These conflicting results may also suggest that the plant Ca2+-ATPases are regulated by calmodulin only in some tissues or in special instances and not in others [27]. In the present study, a very strong calmodulin stimulation of Ca 2+ transport can be observed in light membrane vesicles after linear sucrose gradient centrifugation (in the absence of Mg2+). Furthermore, after Mg 2÷ shift, most of the calmodulin-stimulated activity co-migrates with the tonoplast vesicles and is no longer associated with the ER. These results indicate that a primary Ca2+-pumping ATPase is present on the tonoplast of maize root cells.

149

Indeed, for the first part of our experiments, any calmodulin stimulation of the Ca2÷/H + antiport activity can be ruled out by the presence of monensin in the incubation medium [5]. Consequently, the calmodulin stimulation of Ca 2÷ transport activity cannot be due to a direct effect of this substance on the tonoplast Ca2÷/H + antiport or an indirect effect on the V-ATPase. A primary Ca2÷-ATPase has already been reported to be present on the tonoplast of different plant materials [2]. When vacuoles were isolated from apple fruits, a carbonyl cyanide m-chlorophenylhydrazone (CCCP, a proton ionophore) insensitive, calmodulin-stimulated Ca 2+ transport system was associated with tonoplast vesicles [28]. When tonoplast-enriched fractions were prepared from spinach leaves by free-flow electrophoresis, an ATP-dependent Ca 2÷ transport was associated with these vesicles [29]. The complete inhibition by CCCP indicated that most of the transport was achieved by the tonoplast Ca2+/H + antiport. However, the Ca 2+ transport was also strongly inhibited by vanadate and stimulated by calmodulin, arguing for the presence of a calmodulinstimulated Ca2+-ATPase on the tonoplast of spinach leaves [29]. An ATP-dependent Ca 2+ pump was also detected at the same density as the tonoplast in maize roots [15], barley roots [30] and cauliflower inflorescences [31]. The identification of a calmodulin-stimulated Ca2+-ATPase on the tonoplast complements the previous description of a CaZ+/H+ antiport associated with this membrane [5]. The presence on the tonoplast of young maize root cells of two Ca 2+ tranport systems (a primary Ca2+-ATPase and a Ca2÷/H + antiport) is puzzling and might be developmentally regulated. The uptake of Ca 2+ into the vacuole may be of particular significance in providing pools for rapid fluxes of Ca 2+ in response to different stimuli [2]. The present studies should thus allow future investigation of the roles of these two Ca 2÷ transporters in cellular Ca 2+ homeostasis and in growth and development. 5. Acknowledgments We would like to thank Jo611e Testuz for expert technical assistance and Prof. Edward E. Farmer for critically reading the manuscript. This work

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O. Gavin et al./Plant Sci. 92 (1993) 143-150

was supported by grant 31-28893.90 from the Swiss National Foundation. 17

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