Planta (1983) 159 : 84-90
9 Springer-Verlag 1983
ATP-dependent CA2 + transport in endoplasmic retieulum isolated from roots of Lepidium sativum L. Thomas J. Buckhout* Botanisches Institut der Universitfit, Venusbergweg 22, D-5300 Bonn 1, Federal Republic of Germany
Abstract. Endoplasmic reticulum membranes were isolated from roots of garden cress ( L e p i d i u m satir u m L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca 2+ uptake activity was measured using the filter technique described by Gross and Marm6 (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated C a 2 + uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitoc h o n d r i a l C a 2+ transport (ruthenium red, LaC13 and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanide m-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5'-triphosphate, respectively. The results indicate a Ca 2 + transport function located on the endoplasmic reticulum of garden cress roots. * Present address and address f o r correspondence: Lehrstuhl fiir Pflanzenphysiologie, Ruhr-Universit/it, D-4630 BochumQuerenburg, Federal Republic of Germany Abbreviations: CCCP = carbonylcyanide m-chlorophenylhydrazone; ER = endoplasmic reticulum; P M = plasma membrane
Key words: Endoplasmic reticulum - L e p i d i u m Membrane isolation - Root (endoplasmic reticulure) - Transport (calcium).
Introduction Calcium ions can function as regulatory ions in cells (Rasmussen 1970). This has been clearly demonstrated for animal cells and appears also true for plant cells (Gross and Marm6 1978; Dieter and Marm6 1980a, b; Marm6 and Dieter 1982). The transport of C a 2 + across membrane barriers and its subsequent compartmentation is a critical step in regulating the free cytoplasmic Ca 2 + level. To better understand these transport steps, it is important to identify and characterize membranes involved in calcium transport. In animal cells, a variety of organelles apart from the well studied sarcoplasmic reticulum show ATP-dependent accumulation of Ca 2 + in vitro. These include endoplasmic reticulum (ER) (Moore et al. 1975; Bruns et al. 1976; Walz 1979; Colca et al. 1982), golgi apparatus (Hodson 1978), plasma membrane (PM) (Waisman et al. 1981 ; Gimble et al. 1982; Kraus-Friedmann et al. 1982) and mitochondria (Lehninger et at. 1967). C a 2+ transport in ER membranes often requires a Ca 2 + trapping agent such as oxalate or phosphate while the golgi and PM membranes do not (Moore et al. 1975; Bruns et al. 1976; Colca et al. 1982). In plant cells, there appear to be two Ca 2+ transport systems, a microsomal and a mitochondrial (Dieter and Matin6 1980b). These two systems can be distinguished from each other most easily through the use of the specific inhibitors of mitochondrial Ca 2 + transport ruthenium red, lanthanides (Reed and Bygrave 1974) or in the case of ATP-dependent transport, oligomycin (Hodges
T.J. Buckhout: Ca 2+ transport in endoplasmic reticulum membranes
and Hanson 1965). The mitochondrial system has been extensively studied and characterized (Hodges and Hanson 1965; Wilson and Graesser 1976) while the microsomal system has not been so extensively studied. Reports of in vitro microsomal Ca z § transport in plant plasma membrane (Gross and Marm6 1978; Dieter and Marm~ 1980a, b; Kubowicz et al. 1982), tonoplast (Gross 1982) and endoplasmic reticulum (Gross 1982) are found in the literature; however, by the authors own admission, the origin of the vesicles is not known. ATP-dependent, microsomal C a 2+ movement is directly coupled to the hydrolysis of ATP. This type of transport is found on the plasma membrane, sarcoplasmic reticulum and ER (Carafoli and Zurini 1982). Alternately, Ca z+ transport can be coupled to the transport of a second ion down an electrochemical gradient by an antiport-like mechanism. This is the case for Na+/Ca 2+ exchange in animal cell plasma membrane (Gill et al. 1 9 8 1 ) o r H + / C a 2+ exchange in Neurospora (Stroobant and Scarborough 1979). The two transport mechanisms can be distinguished by using specific ionophores or protonophores to eliminate the ion gradient. Recent results indicate that both a H+/ Ca 2 + antiporter and a Ca 2 + pump may be present in plant cells (Hager and Hermsdorf 1981; RasiCaldogno et al. 1982a, b). As a beginning to the identification of the membranes involved in and the mechanism of Ca 2§ transport in higher plants, we have isolated and purified ER from roots of garden cress. We report here the details of that procedure and the characterization of in vitro Ca z § transport in this membrane fraction. Material and methods Plant material. Seeds of Lepidium sativum L. cv Krause were soaked in tap water for 30 min and sown on wire mesh racks as described by Buckhout et al. (1982). Roots with an in situ length of l0 15 mm were harvested and stored on ice (maximum storage time 30 min). Isolation o f endoplasmic reticulum. Roots (60 g fresh weight) were homogenized in 45 ml of homogenization buffer (50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol - 2-(N-morpholino)ethanesulfonic acid (Tris-Mes), pH 8.0, 5 0 m M KC1, 0.1 m M ethylenediaminetetraacetic acid, 5 mM MgC1 z and 1% dextran) containing 0.5 M sucrose and i4 m M mercaptoethanot. Industrial grade dextran was purchased from Sigma Chemical Co. (St. Louis, Mo., USA) with an average molecular weight of 63,200 daltons. The homogenate was filtered through a single layer of nylon cloth and the tenate pressed until the filtrate volume reached 70 ml. The filtrate was centrifuged at 10,000 g for 20 min (Sorvall HB4 rotor, Bad Nauheim, FRG) and 10 ml of the resulting supernatant was layered onto a discontinuous sucrose gradient with steps of 0.6 M (2.0 ml), 1.0 M (1.5 ml), 1.14 M (1.5 ml) and 1.2 M (1.5 ml) sucrose. The su-
85 Homogenate 10000 • g 20 rain
Pellet (discard } cell walls , nuclei, plastids mil'ochondria , sl'arch
-0.6M 1.0M -
-1.2M g0 000 • _g 90 rain
FAraction B C
30 O00xg " 30 min-
- Supernatant "
/resuspend D .~'~n homogenization J / buffer P e l l e t - -
Endoplasmic reticulum pellet
Fig. 1. Schematic illustration of the procedure for endoplasmic reticulum isolation from roots of Lepidium sativum L. For details see Material and methods crose solutions were prepared in homogenization buffer. The gradients were centrifuged at 90,000 g for 90 min (Spinco SW 27 rotor, Palo Alto, Calif., USA). The resulting pellet was resuspended in homogenization buffer containing 0.5 M sucrose and pelleted at 30,000 g for 30 min. The resulting pellet is referred to as the endoplasmic reticulum fraction. A summary of this procedure is given in Fig. 1. A total membrane pellet was obtained by diluting 5 ml of filtered homogenate in 15 ml homogenization buffer containing 0.5 M sucrose and centrifuging this solution for 90 min at 90,000 g (Spinco SW 27 rotor). Enzyme assays. All enzyme tests were conducted as described by Buckhout et al. (1982). The individual tests were succinate-piodonitrotetrazolium violet reductase (Pennington 1961), NADH-cytochrome c reductase (Hodges and Leonard 1972), acid phosphatase and c~-mannosidase (Boller and Kende 1979), inosine diphosphate (IDP)ase (Bowles and Kauss 1976) and ATPase (Buckhout et al. 1982). Protein concentration was measured following trichloroacetic acid precipitation by the method of Lowry et al. (1951) with bovine serum albumin as standard. Electron microscopy. Membrane fractions were fixed with 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide both in 0.1 M cacodylate buffer, pH 7.2. The fixed material was en bloc stained for 2 h with 1% uranyl acetate. Subsequent dehydration and embedding were performed as described by Sievers and Volkmann (1972). Silver-gold sections were cut and stained with alkaline lead citrate. Morphometric analysis of electron micrographs was conducted by the method of Ovtracht et al. (i 973) from randomly selected samples taken from five independently isolated ER fractions. Organelles were identified by their characteristic morphology and membrane vesicles of unidentified origin were labelled "smooth membrane organelles". Vesicles with " c a p p e d " ribosomes (see below) were considered rough ER. Ca 2 + transport assay. Endoplasmic reticulum membranes were resuspended in 50 m M Tris-Mes, pH 7.0, buffer containing 100 mM KC1, 5 mM MgC12, 3 mM N a N 3 (except where otherwise indicated) and 20 mM ammonium oxalate. The suspension was chromatographed through a shortened PD-J0 column (Pharmacia, Uppsala, Sweden) (void volume of 1.5 ml) equili-
T.J. Buckhout: Ca 2+ transport in endoplasmic reticulum membranes
Fig. 2 A-D. Electron micrographs of the endoplasmic reticulum (ER) fraction. A A representative micrograph from the ER fraction showing rough ER vesicles with the cytoplasmic surface oriented outwards and an occasional protein body-like vesicle (v). B The ribosomes are frequently " c a p p e d " forming a rough appendage (arrowheads)and a smooth vesicle. C When sectioned at an angle approx. 90 ~ to that shown in B, the rough appendages appear as rough vesicles within vesicles (arrowheads).D Membranes taken from samples found at the bottom of the ER pellet show contamination by mitochondria (m). Fixation in glutaraldehyde and post-fixation in osmium tetroxide. Bars equal 1 gm in A and D and 0.5 gm in B and C
brated in the above buffer. The void volume fraction was collected. Calcium transport was determined by the filtration technique described by Gross and Marmb (1978) in a total volume of 500 gl containing 50 mM Tris-Mes, pH 7.0, 100 mM KCI, 5 mM MgC12, 3 mM NaN 3 (except where otherwise indicated), 50 gM CaC12 plus 45CAC12 (0.32 kBq) and 50-75 ~tg membrane protein. The reaction was started by the addition of ATP to a final concentration of 5 mM. Following the desired reaction time, the test mixture was filtered through 0.22 gm GSWP Millipore filters (Millipore, Molsheim, France) and washed with
three 1 ml-volumes of buffer containing 2 mM (ethylene-bis(oxyethylenenitrile))tetraacetic acid in the absence of MgC12. The filters were dried and the radioactivity determined by scintillation spectroscopy.
Isolation of ER membranes. T h e p r o c e d u r e f o r i s o lation of ER membranes
is d e s c r i b e d in d e t a i l i n
T.J. Buckhout : Ca z + transport in endoplasmic reticulum membranes Table 1. Morphometric analysis of the isolated endoptasmic reticulum fraction composition. Membranes were isolated as described in Material and methods. Morphometric analysis was conducted on randomly selected samples taken from 5 independently isolated ER fractions by the method of Ovtracht et al. (1973). The numbers in parentheses are standard deviations Organelle
Rough endoplasmic reticulum Mitochondria Protein body-like vacuoles Smooth-membrane organelles
77.8 8.4 3.4 10.2
(8.0) (7.2) (2.3) (7.7)
Material and methods and is summarized in Fig. 1. Electron micrographs prepared from this fraction show that it is composed of rough ER vesicles and that the vesicles are oriented with the cytoplasmic surface directed outward (Fig. 2A). The ribosomes are frequently "capped" on one side of the vesicle forming a rough appendage and a dilated smooth vesicle (Fig. 2B, arrowheads). When sectioned at an angle 90 ~ to that in Fig. 2B, the constricted regions appear as a vesicle within a vesicle (Figs. 2A, C, arrowheads). The membrane pellet is somewhat heterogeneous with a number ofmitochondria located in the lower region of the pellet (Fig. 2D). Detailed morphometric analyses of micrographs from this fraction indicate that it is composed of 78% rough ER vesicles with 10% smooth membrane organelles, 8% mitochondria and 3% protein body-like vesicles (Table 1). Since the ribosomes tend to cap (Figs. 2B, C), a part of the smooth membranes seen here are likely smooth portions of rough vesicles sectioned as to exclude the rough portion. Analysis of marker enzymes has been conducted, comparing the specific enzyme activity in the ER fraction to the specific activity of a total membrane pellet. The results of this analysis support those obtained from the morphometric analysis. Only the marker enzyme for ER, cytochrome c reductase, was enriched in this fraction, while all other enzymes tested had specific activities less than those found in the total membrane pellet (Fig. 3). A significant mitochondrial contamination was indicated from the INT-reductase activity and was confirmed by the morphometric analysis in Table 1. Although IDPase, ATPase and c~-mannosidase are thought to be marker enzymes for Golgi apparatus, PM and tonoplast, respectively, the activities have not been excluded from ER. In fact, certain 0~-mannosidase isozymes have been identified in ER (Van der Wilden and Chrispeels 1983). All electron microscopic, morphometric and enzymologic data obtained thus far indicate that
C t.c- Acid cI-Ivlann.IDPase Mg2-~ K§ 2t Red Phosphat. ATPase ATPas
Fig. 3. Marker enzyme analysis of the endoplasmic reticulum fraction. The specific enzyme activity is compared between the ER fraction and a total membrane fraction. The markers and the specific activities for the total membrane fraction are mitochondria (succinate-p-iodonitrotetrazolium violet-reductase (INT Red.), 0.024nkat.mg protein-Z), ER (NADH-cytochrome c reductase (Cyt. c-Red.), antimycin A resistant, 1.01 nkat.mg protein-I), tonoplast (acid phosphatase and c~mannosidase, 1.07 and 0.02 nkat.mg protein-~, respectively), Golgi apparatus (inosine diphosphate (IDP)ase, 6.22 nkat.mg protein-X) and PM (MgZ+-ATPase and K +, MgZ+-ATPase, 1.43 and 1.59 nkat-mg protein -1, respectively). The results are an average of three separate ER preparations and in all cases the standard error is less than 10%
E _u o 0
Time (min) Fig. 4. C a 2 + uptake into endoplasmic reticulum-fraction vesicles. Ca 2+ transport measured as described in Material and methods using 50 pg of ER membrane protein with (o,o) or without (re,n) 20 mM oxalate in the presence (o,n) or absence (o,n) of 5 mM ATP. The results are taken from a single, representative experiment
T.J. Buckhout: Ca 2 + transport in endoplasmic reticulum membranes
This accumulation was never more than 20% of the plus oxalate value, and Ca 2 § uptake was only observed in the first 15-20 min of the incubation (data not shown). Membrane vesicles accumulated between 2 and 10 nmol Ca 2 + .mg protein- 1.45 m i n - 1. Although in a large number of experiments the accumulation was near 8 n m o l . m g protein- ~.45 min-~ (see Figs. 5, 6), lower values were not uncommon. The vesicles continued to accumulate C a 2 + for at least 90 min although the tests were routinely stopped after 45-60 min (data not shown). The data in Fig. 4 indicated an oxalate-stimulated, ATP-dep e n d e n t C a 2 + uptake in this ER fraction.
-6 E,n4 c
2 E u w
Nucleotide Fig. 5. An analysis of nucleoside di-and triphosphates on Ca 2 + transport activity in endoplasmic reticulum fraction membranes. Ca 2 + uptake was measured as described in Material and methods with 5 m M (final concentration) of the nucleoside di- and triphosphates of adenine, guanine (GDP, GTP), inosine (IDP, ITP), cytosine (CDP, CTP) and uracil (UDP, UTP) added. The results are an average of three experiments with the standard errors indicated
Table 2. The effect ofinhibitors on Ca 2+ transport in the endoplasmic reticulum fraction. Ca 2 + uptake was measured as described in Material and methods without (control) or with inhibitors at the final concentrations indicated in parentheses. The results were taken from a single, representative experiment Treatment
Control Ethanol (0.05%) N a N 3 (3 raM) CCCP (20 IxM) CCCP + N a N 3 Ruthenium red (5 gM) LaC13 (5 gM) Oligomycin (4 gg. m l - 1 )
8.9 8.8 9.6 8.7 9.5 6.9 6.2 6.5
100 99 112 98 107 78 70 73
The activity is expressed as nmoles Ca z + . m g -1 protein 45 min - 1
the membrane fraction described is highly enriched ER membranes.
In vitro Ca 2+ uptake. Ca z+ transport was measured by the method of Gross and Marm6 (1978). In the presence of ATP and oxalate (Gross 1982), the ER fraction accumulated Ca z+ (Fig. 4). The C a 2 + uptake was ATP-dependent and was greatly stimulated by oxalate. The data in Fig. 4 show little Ca 2+ uptake in the absence of oxalate. However, in several experiments, a small amount of Ca 2+ uptake was observed in the absence of oxalate.
Effects of inhibitors on Ca 2+ uptake. To better understand the nature of C a 2 + uptake in the ER fraction, the effect of several inhibitors on this uptake was tested. The concentrations used were 3 mM for N a N 3 (Dieter and Marm6 1980b), 20 gM CCCP (Rasi-Caldogno et al. 1982b), 5 gM for ruthenium red and LaC13 (Reed and Bygrave 1974) and 4 gg. ml-1 for oligomycin (Hodges and Hanson 1965). The results of these experiments are shown in Table 2. Ethanol, azide, carbonylcyanide m-chlorophenylhydrazone (CCCP) and azide plus CCCP showed no effect. Ruthenium red, LaC13 and oligomycin all inhibited C a 2 + uptake by approximately 25%. Increasing the concentration of ruthenium red or LaC1 a to 10 gM resulted in no increased inhibition (data not shown). LaC13 and ruthenium red were shown to be specific inhibitors o f C a 2+ transport in mitochondria (Reed and Bygrave 1974). That these inhibitors inhibited a portion of the C a 2 + uptake, suggested that mitochondria were responsible for 25% of the Ca 2+ uptake. This was also implied by the data observed in Figs. 2 D and 3 and Table 1. Although a portion of the CaZ+-uptake activity could be attributed to mitochondria, the majority of the activity was clearly of microsomal origin9 Nucleotide specificity of C a 2+ uptake. To test the specificity of C a 2+ uptake for ATP, a series of tests were conducted comparing the nucleoside diand triphosphates of adenine, guanine, inosine, cytosine and uracil. These results showed that ATP was the preferred nucleotide with inosine triphosphate and guanosine 5'-triphosphate having 36 and 29% of the ATP activity, respectively, and uridine 5'-triphosphate and cytosine 5'-triphosphate showing little or no activity (Fig. 5). A D P showed an activity 49% that of ATP; however, this is likely an artifact resulting from the conversion of A D P to ATP and A M P by adenylate kinase. This phenomenon has been previously reported (Gross and
Ca 2 +
transport in endoplasmic reticulum membranes
Marm6 1978 ; Kubowicz et al. 1982). Other nucleoside diphosphates supported little or n o Ca 2 + uptake. Thus, the C a 2 + uptake is specific for ATP. Discusssion The data in this report present evidence for active Ca 2+ transport in isolated membrane vesicles. Ca 2§ transport is inferred from the ATP-dependent uptake of Ca 2 + into membrane vesicles. Only a portion of the transport can be attributed to mitochondria while the major portion is microsomal. The membrane fraction has been characterized and shown to be composed of nearly 80% rough E R membranes. Thus, the data presented here strongly suggest that the ER membranes are responsible for the transport activity. The fact that Ca 2 § transport in E R membranes requires oxalate as a Ca 2 § trap in liver cells (Moore et al. 1975), adipocytes (Bruns et al. 1976), pancreatic islet-cells (Colca et al. 1982) and in this study, is further evidence for the localization of Ca 2 + transport in ER. The membrane fraction used here contains 10% unidentified smooth membrane contamination. The possibility cannot be excluded that only these membranes are involved in Ca 2 § transport. However, it seems unlikely that these membranes could account for the total microsomal Ca 2§ transport, since the amount of transport over a 45-60 rain period in this study is in close agreement to that reported for plant Ca 2 ~ transport in isolated membrane pellets (Gross and Marm6 1978; Dieter and Marm6 1980 a, b; Rasi-Caldogno et al. 1982a, b) but somewhat lower than that reported by Kubowicz et al. (1982) in a P M fraction. As far as is known, only one other report has shown Ca 2§ transport in plant ER membranes (Gross 1982). In that study, E R transport was indicated by a similar sedimentation rate during centrifugation of ER marker enzyme and Ca 2 § uptake activity, and an increase in E R buoyant density following incubation with oxalate, presumably resulting from formation of a calcium oxalate, precipitate in the vesicles. The conclusions of that report are in agreement with those reported here. As described earlier, ATP-dependent, Ca 2+ transport can occur by a direct coupling of Ca 2 + transport to ATP hydrolysis or by an indirect H + / Ca 2 § antiporter. The former mechanism seems to be involved here. First, Ca 2 § uptake is not inhibited by CCCP, a protonophore which dissipates a proton gradient needed for a H + / C a 2+ antiporter. And, the specificity for ATP resembles closely that reported for direct Ca 2 + transport and not for the antiporter (Gross and Marm6 1978; Rasi-Caldogno et al. 1982a, b). Thus, it seems rea-
sonable to conclude that the Ca 2 § transport reported here occurs by a direct transport mechanism. A physiological role for Ca 2 § transport in ER membranes is not known, although the presence of such a transport function in itself implies a physiological significance. The specific and intense staining of E R by O s F e C N a Ca 2 § stain (Hepler 1982) and the large number of Ca 2§ binding proteins in E R membranes (Brummer and Parish 1983) also suggest a role for the E R in Ca 2+ metabolism. Inevitably, the regulation of cell Ca 2 § must occur at the PM since the cell maintains an inner Ca 2 § concentration several orders of magnitude lower than the environmental Ca 2 + concentration and the cell has a limited Ca z+ storage capacity. However, in analogy to the function of the sarcoplasmic reticulum in muscle cells, ER in plant cells could function in the rapid alteration of cytoplasmic Ca 2 + concentration in response to hormonal or environmental signals. The stimulation of Ca 2 + transport following tissue pretreatment with indole-3-acetic and inhibition following pretreatment with zeatin may be significant in this regard (Kubowicz et al. 1982). Similarly, the proposed function of phytochrome, Ca 2 § and microfilaments in light-induced chloroplast rotation in Mougeotia is likely an example of transduction of an environmental signal by Ca 2+ (reviewed by Britz 1979). In this regard, the interaction of the amyloplasts with the statocyte E R following gravity stimulation could induce a Ca 2+ release from ER and account for the rapid gravity-induced plasmalemma depolarization observed in statocytes (Sievers and Hensel 1982) and function in graviperception (Volkmann and Sievers 1979). This example remains purely speculative; however, the presence of a Ca 2 + pump on E R membranes may be an important first step in elucidating such complex phenomena as stimulus tranduction. This work was supported by funds from the Deutsche Forschungsgemeinschaft to Professor A. Sievers and Dr. G. Scherer. The author wishes to thank Professor A. Sievers for his support and criticism during this study and to Professor N. Amrhein and Dr. G. Scherer for their critical reading of the manuscript. The author thanks as well Mrs. Gudrun Buckhout for typing the final version of the manuscript.
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