From the Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI), a high capacity low affinity Ca2+-binding protein in the endoplasmic reticulum of sea urchin eggs (Lebeche, D., and Kaminer, B. (1992) Biochem. J. 287, 741-747), shares 55% sequence identity with mammalian PDI and has PDI activity (Lucero, H., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119). We report on ECaSt/PDI functioning as a Ca2+ storage protein in the endoplasmic reticulum (ER) of a living cell and compare it with calsequestrin and calreticulin, high capacity low affinity Ca2+-binding proteins in the sarcoplasmic reticulum and ER, respectively. Stably transfected Chinese hamster ovary cell clones expressed these proteins, which were localized in the ER of the cell. Microsomes from cells expressing ECaSt/PDI, calreticulin, and calsequestrin accumulated 17.2 ± 0.27, 20.0 ± 0.82, and 38.0 ± 0.28 nmol of Ca2+/mg of protein, respectively; control microsomes accumulated from 2.6 ± 0.17 to 2.9 ± 0.14 nmol of Ca2+/mg of protein. The initial rate of Ca2+ uptake was similar in microsomes from transfected and control cells. Microsomes containing an ECaSt/PDI mutant in which 45% of the acidic residue pairs in the C terminus were truncated had a reduced Ca2+ storage capacity. This supports our previous hypothesis that the degree of low affinity Ca2+ binding is dependent on the number of pairs of carboxyl groups in the molecule. The maximal Ca2+ accumulation by microsomes containing the expressed ECaSt/PDI, C-terminally truncated ECaSt/PDI, calreticulin, or calsequestrin correlates approximately with the Ca2+ binding capacity of the respective proteins.
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INTRODUCTION |
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Intracellular Ca2+ plays an important role in signal transduction mechanisms, motility, secretory processes, and enzyme activity. The cytoplasmic Ca2+ concentration is finely regulated by calcium pumps and channels in the plasma membrane and the endoplasmic reticulum (ER),1 the lumen of which stores Ca2+ (1, 2). Muscle sarcoplasmic reticulum, a specialized form of the ER, is known to regulate cytoplasmic Ca2+ (3), and the proteins involved were first well characterized in this organelle (4, 5). One of these proteins, calsequestrin, a high capacity low affinity calcium-binding protein (6), is concentrated in the cisternae of the sarcoplasmic reticulum (7) and is linked by triadin to the cisternal membrane (8, 9), which contains the Ca2+ release channel, the ryanodine receptor (10). Ikemoto et al. (11) postulated that Ca2+-induced conformational changes in calsequestrin affect the junctional face membrane proteins, which in turn regulate the opening and closing of the Ca2+ channel. Calsequestrin's location in the sarcoplasmic reticulum and its Ca2+ binding properties in vitro strongly suggest that it functions as a Ca2+ storage protein, but as yet, no direct evidence has been reported on such a role in a living cell. The ER of smooth muscle and many other cells contains calreticulin, a high capacity low affinity Ca2+-binding protein postulated to be a calcium storage protein (12, 13).
We identified a 58-kDa protein, characterized as calsequestrin-like, in a microsomal fraction of sea urchin eggs (14), in which Ca2+ is released following fertilization (15, 16) and subsequently during phases of the cell cycle (17). By immunocytology, we showed its presence in the ER of sea urchin eggs and also showed, for the first time, the ER undergoing dynamic changes in structure and location, following fertilization, with aggregation occurring around the mitotic apparatus (18). Such movement of the ER was corroborated later by Terasaki and Jaffe (19). Subsequently, we determined the Ca2+ binding properties of this egg protein (23 mol of Ca2+/mol of protein at low affinity) and identified a unique N-terminal sequence and other features distinct from calsequestrin and calreticulin (20). The two latter proteins have been identified immunocytologically in the egg (21). The amino acid sequence of the 58-kDa protein deduced from cDNA surprisingly shows a 55% identity to mammalian protein-disulfide isomerase (PDI) (22), an enzyme of importance in disulfide bond formation of nascent proteins within the ER (23-25). PDI may also act as a chaperone (26-28). The sea urchin protein also has PDI activity, and we designated it ERcalcistorin/PDI (ECaSt/PDI), alluding to its putative bifunctional role as a Ca2+ storage protein and an isomerase in the ER (22).
Macer and Koch (29), using a 45Ca2+ overlay technique that identifies both high and low affinity Ca2+-binding proteins (30), illustrated binding of Ca2+ by PDI, but no quantitative measurements were made. Following our quantitation of the Ca2+ binding properties of ECaSt/PDI by equilibrium dialysis (20), we found, for the first time, that mammalian PDI also bound 19 mol of Ca2+/mol of protein with low affinity (31), close to the parameters of ECaSt/PDI from the sea urchin egg.
In this work, we report on experiments demonstrating directly the calcium storage role of ECaSt/PDI in the ER of cells and compare it with calreticulin and calsequestrin. Purified microsomes representing the ER from CHO cells expressing ECaSt/PDI, calreticulin, or calsequestrin cDNA took up considerably more Ca2+ than those from control cells. The microsomes from CHO cells expressing an ECaSt/PDI mutant in which 45% of the acidic pairs in the C terminus were truncated took up less Ca2+ than did those from cells expressing the complete protein. This finding is in keeping with a previously enunciated hypothesis on paired acidic residues being low affinity Ca2+-binding sites (22, 32). Interestingly, the maximal Ca2+ accumulation by microsomes containing each expressed protein showed an approximate correlation with the Ca2+ binding capacity of the respective protein. Since ECaSt/PDI from sea urchins can function as a Ca2+ storage protein in the ER, mammalian PDI, now known to be a high capacity low affinity Ca2+-binding protein (31), should presumably also be regarded as another Ca2+ storage protein in the ER of cells.
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EXPERIMENTAL PROCEDURES |
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Materials-- Ham's F-12 culture medium and G418 were obtained from Life Technologies, Inc. 45Ca2+ was from NEN Life Science Products. Fetal bovine serum was from Intergen Co. (Purchase, NY). Ion-exchange resin AG-501-X8 was from Bio-Rad, and thapsigargin was from LC Laboratories (Woburn, MA).
Constructs for Expression-- Plasmids expressing the precursors of 1) ECaSt/PDI, 2) C-terminally truncated ECaSt/PDI, 3) calreticulin, 4) calsequestrin, and 5) lyso-KDEL as a control were constructed according to standard procedures (33).
1) For ECaSt/PDI, the full-length cDNA in pBlueScript (Stratagene, La Jolla, CA) (22) was digested with NotI and ApaI and ligated into the same sites of the mammalian expression vector pRc/CMV (Invitrogen, San Diego, CA). 2)Cell Culture and DNA Transfection-- CHO K-1 cells were routinely maintained in Ham's F-12 medium supplemented with 10% fetal calf serum, 250 units/ml penicillin, and 250 µg/ml streptomycin (complete medium) in a 37 °C incubator with a humidified environment of 5% CO2 and 95% air. DNA transfection was carried out by the calcium phosphate method. About 106 CHO cells/flask, plated the day before transfection, were transfected with 10 µg of DNA in 800 µl of calcium phosphate solution. After 30 min at room temperature with occasional gentle swirling, 8 ml of complete medium were added per flask, and the cells were incubated at 37 °C. After 5 h, the DNA-containing medium was replaced with fresh complete medium, and the cells were cultured for 24-48 h.
Selection, Growth, and Maintenance of Cell Lines-- Cells were passaged on day 3 after transfection and selected for resistance to Geneticin (G418 sulfate; a concentration of >600 µg of active antibiotic/ml of complete medium was used). G418-containing medium was changed every 4 days for a period of 16 days. Colonies were subcloned by dilution and grown in 96-well plates. Selected derivatives were propagated in the presence of G418 to maintain selective pressure and screened for protein expression by RNA dot-blot hybridization. Positive clones with the strongest signals were selected. The following clones were identified by immunoblotting for further studies: clones C5 and T15, expressing full-length and C-terminally truncated ECaSt/PDI, respectively; clone CRT-21, expressing calreticulin; clone CSQ-15, expressing calsequestrin; and clone 43, expressing lyso-KDEL. Clone 33, isolated from CHO cells transfected with the vector pRc/CMV bearing no cDNA (referred to as mock cells), was included as a control.
RNA Preparation and Dot Hybridization-- Total RNA from transfected and nontransfected CHO cells was isolated directly from tissue culture flasks by the guanidium thiocyanate/phenol extraction procedure (37). For RNA dot-blot analysis, 10 µg of RNA from the cultures were denatured for 15 min at 65 °C in 150 mM NaCl and 15 mM sodium citrate, pH 7.0 (1× SSC buffer), containing 50% formamide and 7% formaldehyde. Samples, briefly quenched on ice, received 2 volumes of 20× SSC and were then blotted onto nitrocellulose membranes, soaked with 10× SSC, using a dot-blot apparatus. After prehybridization for 2 h at 42 °C in a solution containing 750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA, pH 7.4 (5× SSPE solution), 0.4 mg/ml polyvinylpyrrolidone, 0.4 mg/ml bovine serum albumin, 0.4 mg/ml Ficoll 400 (2× Denhardt's reagent), 50% formamide, and 0.1% SDS, the filters were hybridized in the same solution containing the corresponding full-length 32P-labeled cDNA for 24 h at 42 °C. The filters were washed extensively and then autoradiographed (33).
Immunofluorescent Staining of Cultured CHO Cells-- Transfected and nontransfected CHO cells were seeded onto polylysine-coated chamber glass slides and grown overnight in complete medium. The slides were washed twice with PBS, and the cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde in 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.3 (PBS), for 10 min at room temperature. After washing twice with PBS, cells were permeabilized by incubation in 1% Nonidet P-40 and 0.1% bovine serum albumin in PBS for 10 min. Cells, washed as described above, were incubated for 2 h at 4 °C with the corresponding antibodies and then for 1 h with rhodamine-conjugated IgG diluted 1:200. Cells were washed extensively with PBS, mounted in an anti-bleaching glycerin/p-phenylenediamine solution (90% glycerol in PBS containing 0.1% p-phenylenediamine), and then viewed by laser scanning confocal fluorescence microscopy (using a Leica CLSM). Images, displayed on a video monitor, were then photographed.
Preparation of Microsomal Vesicles from CHO
Cells--
Microsomes were prepared as described by Maruyama and
MacLennan (38). Briefly, the cells were washed twice with 5 ml of PBS/flask and harvested in 2 ml of PBS/flask and 5 mM EDTA.
They were suspended in 2 ml of a hypotonic solution (10 mM
Tris-HCl, pH 7.5, and 0.5 mM MgCl2) and left
swollen for 15 min on ice. A mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml antipain) was
added to the suspensions, and the cells were homogenized with 40 strokes in a Teflon-glass Dounce homogenizer with an A-type
tight-fitting pestle. An equal volume of a solution containing 10 mM Tris-HCl, pH 7.5, 500 mM sucrose, 300 mM KCl, 40 µM CaCl2, and 6 mM 2-mercaptoethanol was added to the homogenates and again
homogenized with 20 strokes on ice. The suspensions were centrifuged at
10,000 × g for 20 min to remove nuclei and mitochondria. The supernatants, with additional KCl to a concentration of 0.6 M, were centrifuged at 100,000 × g
for 60 min to sediment the microsomal fraction. The pellets were
resuspended in 250 mM sucrose, 150 mM KCl, 3 mM 2-mercaptoethanol, 20 µM
CaCl2, and 10 mM Tris-HCl, pH 7.5, and
centrifuged again at 100,000 × g for 60 min. Fresh
pellets resuspended in the same buffer were used for electron
microscopy and Ca2+ uptake assays, and the rest were frozen
at 70 °C.
Electron Microscopy and Marker Enzyme Activities of Microsomal Fractions-- Microsomal pellets, prepared as described above, were resuspended in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3, for 30 min and then centrifuged at 10,000 × g for 1 h (39). The resulting pellets were rinsed in cacodylate buffer and post-fixed in 2% osmium tetroxide and 1.5% potassium ferrocyanide for 2 h. The microsomal specimens were dehydrated in a graded series of ethanol and embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate, mounted on nickel grids, and examined in a Phillips 300 electron microscope.
Glucose-6-phosphatase was assayed, in microsomal fractions, as an ER marker by measuring the release of Pi from glucose 6-phosphate (39), and the Pi was quantitated by the Fiske-SubbaRow method (40). Cytochrome c oxidase was assayed, as a marker for mitochondrial contamination of the microsomal preparations, following the procedures of Wharton and Tzagoloff (41) and Silver et al. (42).SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- Samples of microsomal preparations and purified ECaSt/PDI, CRT, and CSQ were electrophoresed on SDS gels according to Laemmli (43) and transferred electrophoretically onto nitrocellulose membranes. The blots were immunostained using 1:200 dilutions of rabbit anti-ECaSt/PDI (14), rabbit anti-human CRT (Stressgen Biotech Corp., Victoria, British Columbia, Canada), and rabbit anti-chicken cardiac CSQ (from K. P. Campbell) antisera and a 1:1500 dilution of monoclonal antibody 9E10 (Calbiochem) against a c-Myc epitope for detection of lyso-KDEL. The appropriate alkaline phosphatase-conjugated secondary antibody was used to detect the primary antibodies.
Immunoblots were quantitated to assess the level of expression of ECaSt/PDI and C-terminally truncated ECaSt/PDI in the microsomes from the respective clones. Microsomes (20 µg of protein) from cells expressing full-length ECaSt/PDI and a series of concentrations (60-600 ng) of purified recombinant ECaSt/PDI (produced in Escherichia coli) were immunoblotted. Similarly, microsomes (20 µg of protein) from cells expressing C-terminally truncated ECaSt/PDI and a series of concentrations (60-600 ng) of purified recombinant C-terminally truncated ECaSt/PDI were immunoblotted. Five immunoblots, reproduced for each type of molecule, were subjected to gel transmission densitometry and blot reflectance measurements using SigmaScan ProTM (Jandel Scientific, San Rafael, CA) using the principles outlined by Haselgrove et al. (44) and Stephens (45). The optical densities of relative reflectance values were obtained from the 8-bit video intensity scans of Western blot lanes by applying Beer's law: mathematical transform OD = log(average background intensity/data point intensity). The concentration of the protein expressed in each microsomal preparation was determined from the standard curve generated in each immunoblot. To assess whether transfection influenced the expression of endogenous ER proteins, the relative quantity of calreticulin in microsomes from all the cell clones used in this study was determined. Microsomes (20 µg of protein) were immunoblotted against anti-calreticulin, and the resulting blots were scanned with an ASTRA 1200S (UMAX) scanner. Using Sigma Gel (Jandel Scientific), digital images of immunoblots were converted to relative reflectance (log(255/pixel intensity)) and quantified in terms of integrated band densities above the average blot background with the software "flood-fill" feature as reported (46).Ca2+ Uptake Assay of Microsomal Preparations-- Ca2+ uptake by the microsomal preparations was assayed as described previously (38) with some modifications. The reaction medium (1 ml) contained microsomes (10 µg of protein), 150 mM KCl, 20 mM MOPS/NaOH, pH 6.8, 5 mM MgCl2, 0.5 mM EGTA, and 0.49 mM CaCl2 (containing 45Ca2+ at a specific activity of 105 cpm/nmol). The uptake process was started by the addition of 5 mM ATP to the reaction mixture at room temperature. At different time periods, 150-µl aliquots were withdrawn; mixed thoroughly with 150 µl of a medium containing 150 mM KCl, 3 mM MgCl2, 40 mM Tris/MES, pH 7.5, and 5 mM LaCl3 (47); and placed on 4-ml ion-exchange Tris/Dowex columns (48) that were pre-equilibrated with a medium containing 250 mM sucrose, 20 mM Tris-HCl, pH 7.5, and 2 mg/ml albumin. After elution with 2.7 ml of a medium containing 250 mM sucrose and 20 mM Tris-HCl, pH 7.5, samples were mixed thoroughly, and aliquots were counted in Liquiscint in a Packard 1900CA Tri-Carb liquid scintillation analyzer. Background counts, obtained in aliquots prior to the addition of ATP, were subtracted from subsequent counts. The effects of the Ca2+ ionophore A23187 and the sarco/endoplasmic reticulum Ca2+ATPases-specific inhibitor thapsigargin on Ca2+ uptake were tested at concentrations of 1 µM and 100 nM, respectively.
ATPase Assay of Microsomal Preparations-- Ca2+-dependent ATPase activity was determined at 30 °C using an enzyme-coupled spectrophotometric assay (49) in a medium (0.5 ml) containing 20 mM MOPS/NaOH, pH 6.8, 115 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 0.49 mM CaCl2, 1.5 mM phosphoenolpyruvate, 1 mM dithiothreitol, 0.3 mM NADH, 5 units of lactate dehydrogenase, 2.5 units of pyruvate kinase, 2 µM A23187, and microsomes containing 30-50 µg of protein. The assay medium was preincubated for 5 min, and the reaction was started by the addition of 2 mM ATP. The ATPase activity measured in the absence of Ca2+ was subtracted.
Ca2+ Content in CHO Cells-- CHO cells were cultured in monolayers, as described above, for 54 h in the presence of 10 µCi/ml 45Ca2+, and their cellular Ca2+ content was determined at equilibrium (50, 51). The time required to reach isotopic equilibrium was ~40 h in all cell clones studied. Monolayers were then washed three times with the culture medium (without fetal calf serum), detached by trypsinization (0.15% trypsin and 0.02% EDTA in Ca2+/Mg2+-free PBS), and resuspended to a concentration of 106 cells/ml in Ca2+-free medium (51) containing 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 20 mM glucose, and 20 mM HEPES, pH 7.4. Aliquots (200 µl) of suspended cells were placed on 4-ml ion-exchange Tris/Dowex columns (48) that were pre-equilibrated with Ca2+-free medium containing 20% dialyzed fetal calf serum. Cells were eluted with 2.7 ml of Ca2+-free medium, and 45Ca2+ was monitored as described above for the Ca2+ uptake assay. Cell counting before and after passage through the Dowex columns indicated an elution of >97% of the cells. The radioactivity that bound to cells that were suspended in 45Ca2+ medium and immediately passed through an ion-exchange column was taken as background and subtracted from the radioactivity measured in cells incubated for 54 h in the presence of 45Ca2+. Total cellular calcium content was calculated taking into account the cell-associated radioactivity at equilibrium and the specific activity of Ca2+. Calcium concentration in the culture medium was determined using a Ca2+-sensitive microelectrode as described (52).
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RESULTS |
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Localization of Expressed Proteins in CHO Cells-- The localization of the expressed Ca2+-binding proteins was detected by indirect immunofluorescence microscopy in the ER network, which was densely stained in the perinuclear region as reported for CRT (13). Fig. 1A shows diffuse localization of the expressed ECaSt/PDI (panel i) and the truncated mutant (panel ii) molecules in the ER. Control cells showed no immunostaining (panel iii). Expressed calreticulin also localized in the ER (Fig. 1B, panel ii), as did endogenous calreticulin (panel i). Differences in staining intensities of these two preparations could not be visualized. On the other hand, expression of CRT in the transfected cells above the endogenous CRT content was detected by immunoblotting of microsomal preparations (see Figs. 3C and 4). CHO cells transfected with CSQ cDNA also showed diffuse immunofluorescent staining of the ER (Fig. 1C). Control cells did not stain (data not shown).
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Microsomal Fractions-- Microsomes, fractionated from clones and control cells, contained membranous vesicles with no obvious mitochondrial contamination, as seen on the electron micrograph in Fig. 2. Mitochondrial contamination of the microsomes, as evaluated by the activity of cytochrome c oxidase, was negligible; it was <7% of the specific activity of the enzyme in mitochondrial pellets. On the other hand, glucose-6-phosphatase activity (an ER marker) was high, ~4.4 µmol of Pi/mg of protein/min.
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Expressed Proteins in Purified Microsomal Fractions-- The expression of full-length and C-terminally truncated ECaSt/PDI in clones C5 and T15, respectively, was determined by immunoblotting extracts of purified microsomes (Fig. 3A). In lane 2 is shown a major protein band from microsomal preparations of CHO cells transfected with full-length ECaSt/PDI cDNA (clone C5) with a molecular size (58 kDa) similar to that of purified ECaSt/PDI (lane 1). The expressed protein comigrated with the native molecule, the two bands being superimposed, when extracts of microsomes from transfected cells were mixed with the native protein and separated in the same lane (data not shown). Lane 3 shows, as expected, a 45-kDa protein band from microsomal preparations of cells transfected with the C-terminally truncated ECaSt/PDI molecule (clone T15). When T15 microsomal extracts were mixed with native ECaSt/PDI in the same lane, the two proteins separated and ran at their respective molecular masses (data not shown). No protein bands were detected in preparations from control cells (lane 4). Although equal amounts of microsomal proteins were loaded in lanes 2 and 3, the differing densities of the respective bands indicate a greater expression of the truncated protein compared with full-length ECaSt/PDI; quantitation of the densities of the respective bands gave expression values averaging 10.8 and 9.6 µg/mg of total microsomal proteins, respectively (Fig. 3B).
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Levels of Endogenous CRT in Transfected Cells-- The possibility that the expression of exogenous proteins in the ER of CHO cells could alter the level of endogenous ER resident proteins was tested by comparing the quantities of CRT in microsomes from nontransfected and transfected cells. Microsomes from mock-transfected cells and from cells expressing lyso-KDEL, ECaSt/PDI, C-terminally truncated ECaSt/PDI, and CSQ showed similar levels of endogenous CRT (Fig. 4). As expected, cell transfection with the CRT cDNA led to an ~2-fold increase in the amount of CRT due to expression over and above endogenous CRT.
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Ca2+ Uptake by Microsomes-- The main purpose of our investigation was to determine if ECaSt/PDI, a high capacity low affinity Ca2+-binding protein isolated from the ER, contributed to the Ca2+ storage capacity of the ER in a living cell. We therefore determined the effect of expression of ECaSt/PDI and the C-terminally truncated mutant in the ER of transfected CHO cells on the Ca2+ uptake and storage capacity of their isolated ER microsomes. We then compared these measurements with those obtained in similar preparations from cells transfected with calreticulin and calsequestrin cDNAs.
Microsomes from cells expressing ECaSt/PDI, C-terminally truncated ECaSt/PDI, CRT, and CSQ attained initial Ca2+ uptake rates of 1.32 ± 0.04, 1.41 ± 0.05, 1.32 ± 0.07, and 1.50 ± 0.1 nmol of Ca2+/mg of protein/min, respectively (Fig. 5, A and B). The maximal Ca2+ levels (expressed in nmol of Ca2+/mg of microsomal protein) reached after 30 min were 17.2 ± 0.27 (for ECaSt/PDI), 12.3 ± 0.32 (for truncated ECaSt/PDI), 20.0 ± 0.82 (for CRT), and 38.0 ± 0.38 (for CSQ) (Fig. 5, A and C).
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ATPase Activity in the Microsomal Fraction-- We excluded the possibility (although unlikely) that transfection of the cells with the putative Ca2+ storage proteins could influence the Ca2+-ATPase of the respective microsomes and be responsible for the increased Ca2+ uptake. The Ca2+-ATPase activities in microsomes from mock-transfected cells and from cells expressing lyso-KDEL, ECaSt/PDI, C-terminally truncated ECaSt/PDI, CRT, and CSQ were essentially the same, ranging from 7.0 ± 0.57 to 7.8 ± 0.53 nmol of ATP/mg of protein/min (Fig. 6). The Ca2+-ATPase activities were inhibited by 100 nM thapsigargin (data not shown).
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Ca2+ Content in CHO Cells-- Confirming the findings in the microsomal preparations, expression of the high capacity Ca2+-binding proteins in the cell led to significant increases in Ca2+ content compared with the controls. ECaSt/PDI accumulated 114 ± 5 pmol of Ca2+/106 cells; C-terminally truncated ECaSt/PDI accumulated 90 ± 4.7 pmol of Ca2+/106 cells; CRT accumulated 120 ± 5 pmol of Ca2+/106 cells; and CSQ accumulated 159 ± 4 pmol of Ca2+/106 (Fig. 7). The Ca2+ content in control cells was significantly lower; in mock-transfected cells, it was 57 ± 6.2 pmol of Ca2+/106 cells (Fig. 7), similar to values reported for other cell types (50, 51), and in cells transfected with lyso-KDEL, it was 65 ± 6.8 pmol of Ca2+/106 cells.
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DISCUSSION |
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ECaSt/PDI, previously identified as a high capacity low affinity Ca2+-binding protein (20), actually acts, as shown in this investigation, as a Ca2+ storage protein in microsomes, membranous vesicles of the ER of a living cell in which the protein was expressed. Similarly, the Ca2+ storage capacity of expressed CRT and CSQ was determined in microsomes and compared with that of ECaSt/PDI. Differences in Ca2+-ATPase activity were ruled out as being responsible for the Ca2+ uptake findings. Also ruled out was the possibility that the expression of exogenous ER proteins may have increased the endogenous level of a Ca2+ storage protein such as CRT.
We have presented, for the first time, direct evidence that overexpression of calsequestrin in the ER of a living cell increases the Ca2+ uptake of its isolated microsomes and the total Ca2+ content of the cell. This confirms all the strong putative evidence and the recent indirect evidence based on Ca2+ release studies; vasopressin-induced release of Ca2+ by differentiated myoblasts transfected with CSQ cDNA was inferred as being due to an increase in Ca2+ storage (53). Such inferences from release studies were also made with regard to CRT. Reduced expression of CRT in cultured NG-108-15 cells resulted in a decreased Ca2+ release on application of bradykinin (54). HeLa cells transfected with a molecularly tagged CRT cDNA showed an increase in Ca2+ release on treatment with thapsigargin (55). Overexpression of CRT in Xenopus oocytes inhibited repetitive inositol 1,4,5-trisphosphate-induced Ca2+ waves due apparently to the high affinity Ca2+-binding site and not to the Ca2+ storage domain of CRT (56). Recently, while our work was in progress, Mery et al. (51) obtained direct evidence that an increase in Ca2+ uptake in whole fibroblasts overexpressing CRT was mostly due to an increase in the size of thapsigargin-sensitive stores. Our findings show that CRT stores calcium in fractionated microsomes representing the ER of living cells.
Precise correlations of the maximal Ca2+ uptake, in the various microsomal preparations, with the Ca2+ binding capacities of the respective expressed proteins would require quantitation of the amounts of expressed proteins in the microsomes, which is beyond the scope of this investigation. Nevertheless, the approximate correlations are worth examining. ECaSt/PDI and CRT bind 23 (20) and 20 (57) mol of Ca2+/mol of protein, and the maximal Ca2+ uptake levels of microsomes due to expressed ECaSt/PDI and CRT were 14.3 and 17.1 nmol of Ca2+/mg of protein, respectively (values obtained after subtracting the value measured in the control microsomes; see legend to Fig. 5). CSQ binds about twice the amount of calcium (43 mol of Ca2+/mol of protein), and microsomes with expressed CSQ likewise took up about twice the maximal amount of Ca2+ compared with ECaSt/PDI and CRT (see legend to Fig. 5). Also worthy of note is the reduced uptake of Ca2+ by microsomes containing C-terminally truncated ECaSt/PDI (9.4 nmol of Ca2+/mg of protein) compared with microsomes containing the whole molecule (14.3 nmol/mg of protein) (values again obtained by substraction of the control value; see legend Fig. 5). This 34% reduction could be adjusted to 41% if the expressed truncated molecule/whole molecule ratio of 10.8:9.6 were taken into account; the adjusted value would then approach more closely the 45% reduction of acidic residue pairs in the truncated molecule. This 45% reduction of binding sites correlates well with a 46% reduction in the Ca2+ binding of the purified truncated protein compared with the complete ECaSt/PDI molecule2 and is supportive of the hypothesis made previously by Lucero et al. (22) that pairs of acidic residues are low affinity Ca2+-binding sites. The tertiary structure determination of the three Ca2+ storage proteins will lead to clearer insights into the nature and location of low affinity Ca2+-binding sites. Since ECaSt/PDI acts as a Ca2+ storage protein in the ER, mammalian PDI, now known to be a high capacity low affinity Ca2+-binding protein (31), should presumably also be regarded, like calreticulin, as a Ca2+ storage protein in the ER.
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ACKNOWLEDGEMENTS |
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We thank Drs. Douglas Gowenbuck and Guillermo Taccioli for the CHO cells; Dr. Kevin Campbell for an antibody; Drs. David MacLennan, Marek Michalak, and Hugh Pelham for cDNAs; and Drs. Richard Fine and Robin Johnson for a sample of calreticulin. We are grateful to Dr. Kathy Svoboda for assistance with confocal microscopy and Rozanne Richman for doing the electron microscopy. Drs. Gregor Jones and Ray Stephens offered valuable advice.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These two authors contributed equally to this work.
§ To whom correspondence and reprint requests should be addressed: Dept. of Physiology L-708, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4392; Fax: 617-638-4273.
1 The abbreviations used are: ER, endoplasmic reticulum; PDI, protein-disulfide isomerase; ECaSt/PDI, ERcalcistorin/protein-disulfide isomerase; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; CRT, calreticulin; CSQ, calsequestrin; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid.
2 H. A. Lucero and B. Kaminer, unpublished data.
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REFERENCES |
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