From the
Stable Chinese hamster ovary (CHO) cell lines overexpressing the
human plasma membrane Ca
The plasma membrane Ca
To study the physiological function of the
isoforms, it was necessary to develop systems which would allow their
expression and eventually purification. Successful overexpression of
the PMCA4CI (for the isoforms nomenclature, see Carafoli(1994)) has
been achieved in different systems (Adamo et al., 1992a; Heim et al., 1992; Enyedi et al., 1993). They have
permitted the study of the properties of this isoform and provided the
tool for creating mutated variants (Enyedi et al., 1993,
1994). They were all based on the transient overexpression of the pump
in COS cells or in viral systems (Baculovirus, Heim et al.,
1992), i.e. they were unsuitable for the study of the long
term physiological effects of pump overexpression.
To overcome the
problem, stable CHO cells overexpressing the PMCA4CI isoform were
generated after transfection with a glutamine synthetase-based vector
(Cockett et al., 1990). Of the different clones which were
isolated, three expressed high amounts of active PMCA pump and
correctly delivered it to the plasma membrane. The cells were able to
survive with a 15-20-fold excess of PMCA activity as compared
with the parental cells. Interestingly, when grown to confluence they
displayed a decreased level (4-5-fold) of the endogenous SERCA
pump as compared with nontransfected cells. Although the duplication
time of the transfected cells was similar to that of the nontransfected
counterparts, they had a 12-16 h longer lag phase.
Glasgow minimal Eagle's medium, dialyzed FCS (fetal
calf serum), antibiotics, and other cell culture media supplements were
purchased from Life Technologies, Inc. (Basel, Switzerland). Other
reagents were of the highest quality commercially available.
Genomic DNA was prepared from
cells according to standard procedures (Sambrook et al.,
1989). It was digested twice overnight with the desired restriction
enzyme, precipitated, and its concentration determined
spectrophotometrically. Southern blotting was performed according to
Sambrook et al.(1989) and hybridization was performed with a
randomly primed labeled EcoRI-EcoRI 1200-bp fragment
(corresponding to the nucleotides 2177-3403 of PMCA4CI, Strehler et al., 1990) under stringent conditions.
Microsomes were prepared as follows: after
homogenization of the cells in 10 mM Tris-HCl, pH 7.4 (40
strokes of a Dounce homogenizer on ice), sucrose and NaCl were added to
a final concentration of 10% and 150 mM, respectively, and
centrifuged 5 min at 750
The coverslips were overlaid with the primary antibody
(antiserum p94.2) diluted 1:50 in blocking buffer. After five washes
with the blocking buffer, the cells were treated with the secondary
antibody for 1 h (swine anti-rabbit fluorescein-conjugated antibodies
diluted 1:30 to 1:50 in blocking buffer, Dako A/S, Glostrup, Denmark).
The coverslips were washed five times with blocking buffer prior to
mounting in a medium containing 80% glycerol, 2.5% DABCO
(2,4-diazabicyclo-[2,2,2]-octan) in PBS, pH 8.0. The cells
were observed in an Axiovert 10 microscope (Carl Zeiss, Oberkochen,
Germany) equipped with epifluorescence illumination and a
The Ca
All three stable CHO cell clones described expressed high
levels of the PMCA4CI. Numerous attempts were made to obtain cell
clones expressing still higher levels of PMCA, but the upper limit was
apparently that of the CHO-10-150 clone, whose level of expression was
at least 15 times higher than that of the parental CHO cells; similar
levels of pump expression had been previously obtained by transfecting
CHO cells with the SERCA pump cDNA (Hussain et al., 1992),
although a different selection system had been used. The three clones
were isolated independently, i.e. they resulted from the
incorporation of the cDNA at different sites as indicated also by the
fact that no correlation was found between the copies of PMCA4CI cDNA
incorporated and the amount of expressed pump. The experiments
presented here and those on the SERCA pump (Hussain et al.,
1992) thus demonstrate that CHO cells can tolerate high amounts of
active Ca
Trypsinized cells loose all the attachment points to the
plastic bottles. After replating, they attach to the surface and begin
to crawl, a process in which Ca
The overexpressed pump was correctly
delivered to the plasma membrane. This was in agreement with earlier
results by Heim et al. (1992) and Zvaritch et
al.(1992), but at variance with those by Adamo et al. (1992a). The very high level of pump expression in the latter
study may have overloaded the protein targeting system, leading to the
trapping of part of the overexpressed pump in the endoplasmic
reticulum. In the experiments reported here the enrichment of the PMCA
pump in the stable CHO cell lines was about 15-fold, whereas in the
transiently transfected COS cells, the overexpression factor (assuming
a transfection efficiency of 10% and a total overexpression of six
times) was probably much higher.
An interesting, but intriguing,
property of the stable transfected cells was their lower amount of
active SERCA pump. Whether the latter was specifically down-regulated
at the level of expression or whether the observed decrease of activity
was related to other unknown mechanisms cannot be decided at the
moment. However, it may be added that the decrease was observed under
different experimental conditions and was independent of the method
used to prepare the membranes. The regulation of the expression of the
SERCA genes is an open problem; non-muscle tissues express mostly the
SERCA2 and SERCA3 genes (Bobe et al., 1994; Wuytack et
al., 1994), and indications have been presented that the SERCA3
gene can be up-regulated in platelets of hypertensive rats (Bobe et
al., 1994). Experiments with CHO cells transfected with the SERCA
DNA have shown that they were able to grow in the presence of higher
concentration of thapsigargin than nontransfected cells (Gutheil et
al., 1994). When exposed for long times to increasing
concentrations of thapsigargin, they expressed higher amount of the
SERCA pump (and also of the P-glycoprotein). This indicated that cells
can regulate the expression of the SERCA pump in response to external
influences. It would be tempting to propose that the factor regulating
the pump expression processes is Ca
Attempts to determine the in vivo membrane
topology of the PMCA pump in red blood cells were hampered by the low
amount of endogenous pump protein (between 0.1 and 0.01% of the total
membrane protein). It was nevertheless possible to experimentally
demonstrate the existence of the first predicted cytosolic loop between
transmembrane domains 1 and 2 (Feschenko et al., 1992). The
stable transfected CHO cells overexpressed the PMCA pump in a form
which apparently was topologically correct, e.g. the protein
could be labeled from the extracellular side with high efficiency.
These cells will thus be a useful tool in experiments on the membrane
topology of the PMCA. It may be added that CHO cells can be adapted to
grow in suspension cultures (Cockett et al., 1990), i.e. it would in principle be possible to scale up of the production
the PMCA4CI isoform.
We thank Dr. J. T. Penniston (Rochester, MN) for the
gift of the 5F10 monoclonal antibody, Dr. F. Wuytack (Leuven, Belgium)
for the polyclonal antibody against the SERCA2b, and Thees Breyhan
(ETH, Zürich, Switzerland) for the help in performing some of the
experiments. We would also like to thank Celltech Ltd. for providing
the pEE14 vector.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase (PMCA) were
generated, and three independent cell clones were characterized in
details. They overexpressed high amounts of active PMCA pump
(15-20 times over the amount of endogenous PMCA) as indicated by
experiments in which the formation of the phosphoenzyme intermediate
and the uptake of Ca
by microsomes were measured.
Immunocytochemistry experiments coupled to the biotinylation of the
pump in the intact cells indicated the correct delivery of the
expressed pump to the plasma membrane. The expressed pump was purified
by affinity chromatography on calmodulin sepharose. The PMCA of
transfected CHO cells promoted an increase of Ca
into
the medium, after induction of Ca
release from the
internal stores by activation of a purinergic receptor. An evident
decrease of the activity of the endogenous sarcoplasmic reticulum
Ca
-ATPase pump was observed, probably related to the
down-regulation of its expression. The cells overexpressing the PMCA
pump had delayed recovery after trypsinization and plating. Their
doubling time was, however, the same as CHO cells.
-ATPase (PMCA)
(
)(Carafoli, 1991, 1992) and the
Na
/Ca
-exchanger are responsible for
the ejection of Ca
from eucaryotic cells (Carafoli,
1991). At variance with the
Na
/Ca
-exchanger, which is
predominantly present in excitable tissues (Blaustein et al.,
1991), the plasma membrane Ca
pump is present in all
tissues studied (Carafoli and Guerini, 1993). An important difference
between the two systems is the affinity for Ca
. That
of the pump is much higher, i.e. the pump is likely to be
responsible for the fine regulation of intracellular Ca
(Carafoli, 1991). An exact quantification of the respective role
of the two systems is difficult, mostly because specific inhibitors are
not available, as they are, for example for the sarcoplasmic reticulum
Ca
-ATPase (SERCA); thapsigargin has been important in
the definition of the role of the system in the contraction of myocytes
(Wrzosek et al., 1992) and of other systems. Most of the
studies on the physiological role of the PMCA pump were performed on
erythrocytes (Schatzmann, 1982), i.e. on a system lacking
internal organelles and in which the PMCA is the only enzyme that pump
Ca
across the plasma membrane. The pump was first
purified from erythrocytes, characterized biochemically (Carafoli,
1992), and eventually cloned (Shull and Greeb, 1988; Verma et
al., 1988). It is encoded by four human genes (Strehler, 1991;
Carafoli and Guerini, 1993), the number of possible isoforms being
increased by a complex pattern of alternative splicing (Brandt et
al., 1992; Keeton et al., 1993; Stauffer et al.,
1993). The different isoforms are expressed in a striking
tissue-specific way (Stahl et al., 1992; Stauffer et
al., 1993).
Materials
Methods
DNA Manipulations
The PMCA4CI was cloned in
pTZ18/U between the BamHI and the KpnI sites. The
resulting vector was cut with KpnI, blunt-ended with Klenow
polymerase, and cut with XbaI (this site is present in the
polylinker of pTZ18/U). The 3600-bp PMCA4CI cDNA fragment was ligated
into the XbaI-SmaI cut pEE14 vector (Celltech Ltd.,
4EN Berkshire, United Kingdom). The pEE14 vector contains the glutamine
synthetase gene, which can be used for the selection of stable
transfected cells (Cockett et al., 1990). The plasmid DNA was
purified by CsCl gradient centrifugation prior to transfection
(Sambrook et al., 1989).
Isolation of Stable Transfected Cells
CHO.K1 cells
(ATCC CCL 61) were maintained in Glasgow minimal Eagle's medium,
10% dialyzed FCS, 100 µg/ml gentamicin supplemented with amino
acids, nucleotides, and pyruvate as described earlier (Cockett et
al., 1990). Transfections were performed by the
Ca(PO
)
method (Cockett et
al., 1990) on semiconfluent CHO cells. One day after transfection,
25 µM MSX (methionine sulfoximide, Sigma) was added to the
medium. Clones were collected and analyzed for the integration of the
PMCA4CI gene. Positive clones were subjected to additional
selection-amplification rounds at 150, 300, and 500 µM MSX.
Preparation of Membranes
Crude membranes were
prepared as follows: cells were resuspended at 5-10 10 cells/ml in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA,
5 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin
after two washings with TBS (25 mM Tris-HCl, pH 7.4, and 150
mM NaCl), and subjected to three cycles of freeze and thaw.
The particulate fraction was sedimented at 15,000
g for 15 min. In some case the supernatant was centrifuged for 1 h
at 110,000
g. The resulting protein pellet was
resuspended in 4 mM Tris-HCl, pH 8.0, 10% sucrose and frozen
at -70 °C.
g. The postnuclear pellet was
sedimented by 20-min centrifugation at 10,000
g. The
microsomes were collected from the postnuclear supernatant by
centrifuging it twice for 45 min at 100,000
g. The
relative content of plasma membrane was determined by measuring the
Na
/K
-ATPase activity in the
preparation essentially as described by Jewell and Lingrel(1991).
Following assay buffer was used: 50 mM Tris-HCl, pH 7.4, 100
mM NaCl, 10 mM KCl, 0.2 mM EGTA, 2
mM [
-
P]ATP (10-30 Ci/mmol)
in the presence or in the absence of 5 mM ouabain.
Western Blotting
Proteins were separated by
SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and transferred
to nitrocellulose membranes (Towbin et al., 1979). The
membranes were incubated with the polyclonal antibody 94.2 (diluted
1/500) or with the monoclonal antibody 5F10. After incubation with
alkaline phosphatase-coupled secondary antibodies (Bio-Rad), the
immunocomplexes were visualized with 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium (Bio-Rad).
Formation of the Phosphoenzyme Intermediate from
ATP
Membrane proteins were resuspended in 20 mM MOPS-KOH, pH 6.8, 100 KCl in the presence of 100 µM Ca, 100 µM Ca
plus 100 µM La
, or 1 mM EGTA as described in the legend for the figures. The reaction was
started by adding 0.2-2 µM [
P]ATP (50-500 Ci/mmol) on ice
and stopped 30 s later by the addition of 7% trichloroacetic acid. The
proteins in the washed pellet were separated on acidic gels (Sarkadi et al., 1986), stained with Coomassie Brilliant Blue, dried,
and exposed for 1-3 days at -70 °C.
Immunoprecipitation
200-250 µg of
membranes were phosphorylated as described above in the presence of 0.2
µM ATP, 200 µM CaCl, and 200
µM LaCl
. After precipitation with
trichloroacetic acid, the pellet was washed with 200 µl of cold
H
O and resuspended in 200 µl of MEN buffer (50 mM MES-NaOH, pH 6.4, 150 mM NaCl, 1 mM EDTA, 0.2%
gelatin, 0.1% NaN
, 0.1% SDS, 0.1% Nonidet P-40). The
resuspended proteins were centrifuged at 10,000
g for
30 min at 4 °C and divided in three aliquots: 20 µl were kept
aside as control for the phosphoenzyme intermediate stability, two
aliquots of 90 µl were diluted with 1 ml of MEN buffer and mixed
with 2 µl of the monoclonal antibody 5F-10 (specific for the PMCA,
Borke et al., 1989) or 4 µl of polyclonal serum of rabbits
immunized against the SERCA2b pump (Wuytack et al., 1989). The
samples were incubated under gentle rocking for 1 h at 4 °C, before
addition of 15 µl of packed protein A-Sepharose (protein
A-Sepharose CL-4B, Pharmacia Fine Chemicals, Uppsala, Sweden). After
another 2-h incubation at 4 °C, the immunocomplexes bound to the
protein A-Sepharose were recovered by centrifugation. The pellet was
washed four times with MEN buffer, twice with 20 mM MOPS-KOH,
pH 6.8, 0.1% Nonidet P-40, and finally twice with 20 mM MOPS-KOH, pH 6.8. The pellet was resuspended at room temperature
for 30 min in 30-40 µl of 70 mM Tris-PO
,
pH 6.4, 5% SDS, 5% dithiothreitol and 8 M urea, separated on
acidic gels (Sarkadi et al., 1986) and further processed as
described above.
Ca
Microsomes were
prepared as described above (see ``Preparation of
Membranes''). The uptake of Ca Uptake
was measured by
the Millipore fast filtration method (Enyedi et al., 1993):
30-36 µg of microsomal proteins were resuspended in the
uptake buffer (25 mM TES-KOH, pH 7.2, 100 mM KCl, 7
mM MgCl
, 40 mM
KH
PO
, 5 mM NaN
, 4
µg/ml oligomycin, 0.5 mM ouabain, 200 nM thapsigargin and 200 nM calmodulin). The free
Ca
concentration was adjusted using the program of
Fabiato and Fabiato (1979). Immediately after the addition of
Ca
(2-3
10
cpm/ml), the uptake was initiated by the addition of 6 mM ATP. At given times, 100 µl of the mixture were rapidly
filtered through 0.45-µm nitrocellulose filters (Millipore SA,
Molsheim, France), which were washed four times with cold 150 mM KCl and 1 mM CaCl
. The filters were then
counted in a Beckman scintillation counter.
Indirect Immunofluorescence Microscopy
The cells
were plated on coverslips and analyzed by indirect immunofluorescence
microscopy 48-60 h after plating. The cells were washed twice
with PBS (phosphate-buffered saline, 150 mM NaCl, 20 mM NaHPO
, pH 7.4, 0.1 mM CaCl
, 0.1 mM MgCl
), fixed for 20
min in 3% paraformaldehyde, washed four times with PBS, and then
incubated for 30 min in 0.1 M glycine. After four washes with
PBS, the cells were permeabilized in 0.1% Triton X-100 for 3 min,
washed four times with PBS, and incubated in blocking buffer (5% FCS,
0.1% bovine serum albumin, 5% glycerol, 0.04% NaN
in PBS)
for 1 h.
63
oil immersion plan-neofluar objective, and photographed with HP5 Ilford
films.
Biotinylation
Cells were metabolically labeled
with [S]methionine for 2 h in MEM, supplemented
with nonessential amino acids and 150
µCi[
S]methionine/ml. After the labeling the
cells were rinsed twice with TBS and once with borate buffer, pH 9.0
(10 mM sodium borate, 154 mM NaCl, 12 mM KCl, 2.25 mM CaCl
). Biotinylation with
NHS-SS-Biotin
(sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate,
Pierce, 0.5 mg/ml in borate buffer), was carried out twice for 15 min
(1.5 ml/100 mm plate). After two washes with borate buffer, free biotin
was blocked with 50 mM NH
Cl/PBS for 10 min. The
cells were rinsed twice again with borate buffer and solubilized for 1
h in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% bovine serum
albumin, 75 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml
antipapain, 5 µg/ml pepstatin, 5 µg/ml leupeptin). The cell
lysate was centrifuged for 30 min at 15,000
g and the
supernatant was rocked overnight with 2 µl monoclonal antibody
5F10. 50 µl of protein A-Sepharose CL-4B were added to the mixture,
and the incubation was carried on for 8 additional h before washing the
beads six times with lysis buffer. To recover the immunoprecipitated
biotinylated antigens, the beads were boiled twice for 3 min with 20
µl of 10% SDS, diluted with lysis buffer (1000 µl/tube), and
centrifuged 2 min at 15,000
g. An aliquot of the
supernatant (one-half of the recovered material) was taken as the
immunoprecipitated, non-avidin-precipitated material. The remainder of
the recovered material was incubated overnight with 50-µl
avidin-agarose beads (50% aqueous slurry, 1-2 mg of avidin/ml of
gel, Pierce). The beads were washed as described above and boiled in
SDS-PAGE sample buffer. Immunoprecipitates and avidin precipitates were
analyzed by gel electrophoresis and autoradiography.
Purification of the ATPase
The
Ca-ATPase was purified by affinity chromatography on
calmodulin-Sepharose, according to the procedure described by Niggli et al.(1979).
Ca
Cells were
plated on 12-well plates (Costar Corp., Cambridge, MA) and let recover
for 2-3 days at 37 °C until they reached a density of 2 to 5
Efflux Experiments
10
cells/well. The medium was replaced by S-MEM
medium (Life Technologies, Inc.), a modification of MEM with lower
calcium content. After two washes with S-MEM, the cells were incubated
in the same medium for 2-4 h at 37 °C in the presence of 1.4
10
cpm
Ca
/well (7
10
cpm/ml). The 12-well plates were transferred to
room temperature, and the cells were quickly washed with S-MEM
containing 1 mM CaCl
. After the addition of S-MEM,
1 mM CaCl
, 1 mM ATP, and 10% FCS,
aliquots were withdrawn from the supernatant and counted. The
radioactivity remaining in the pellet was measured after solubilizing
the cells in 10 mM Tris-HCl, pH 8.0, and 1% SDS. The
percentage of Ca
released per unit of time was
calculated from the total radioactivity in the cells before the
induction of Ca
efflux.
Cell Growth Assay
Cells were plated in a 24-well
plate at a density of 2.4-2.6 10
cells/well.
They were trypsinized and counted at regular intervals. Their integrity
was checked by incubating them with trypan blue (Sigma). The cells were
plated in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 35 mg/liter proline in the
presence of different concentrations of FCS. The growth curves obtained
with other media were qualitatively identical.
Generation and Selection of CHO Stable Cell Lines
Expressing the PMCA Pump
The primary screening yielded about 30
MSX-resistant clones, 20 of which were analyzed for the presence of the
PMCA4CI cDNA by Southern blotting. Four clones giving strong positive
signals were subjected to additional rounds of selection at a higher
concentration of MSX. Fig. 1shows a Southern blot with three
clones: two isolated at 150 µM MSX (CHO-9-150 and
CHO-10-150, lanes 2 and 4, respectively) and one
(CHO-10-25, lane 3), at lower MSX concentration. Lanes
2-4 hybridized strongly with a EcoRI-EcoRI
1200-bp human PMCA4CI cDNA probe; the radioactive band was absent from
nontransfected CHO cell (Fig. 1, lane 1). A calibration
with known amounts of DNA (Fig. 1, lanes 5 and 6) indicated that the cells of the CHO-9-150 clone contained
10 copies of PMCA4CI-cDNA in their genome, that of clones CHO-10-150
and CHO-10-25, four to six copies. Additional clones (CHO-1 and CHO-3,
not shown) were positive in dot-blot hybridization experiments, but
showed a complex banding pattern on Southern blotting, indicating
rearrangement in the coding sequence of PMCA4CI during the
transfection.
Figure 1:
Southern blot of
genomic DNA. Genomic DNA was prepared from control CHO cells and the
stable transfected cells, digested with EcoRI, and transferred
to Nytran filters. The filters were hybridized with a random primed
labeled 1200-bp EcoRI-EcoRI PMCA4CI-specific DNA
fragment under high stringency conditions (Sambrook et al.,
1989). 5 µg of genomic DNA from CHO cells (lane 1),
CHO-9-150 (lane 2), CHO-10-25 (lane 3), and
CHO-10-150 cells (lane 4). To estimate the copy number of
PMCA4CI cDNA integrated in the cells, the amount corresponding to four (lane 5) or eight (lane 6) copies of PMCA4CI/cell was
separated, transferred, and hybridized in parallel. The autoradiogram
was exposed for 40 h at -20 °C. The densitographic scanning
of the 1200-bp radioactive band yielded the following values. Lane
2, 530,000 ± 30,000 scanning units; lane 3,
290,000 ± 20,000 scanning units; lane 4, 300,000
± 30,000 scanning units; lane 5, 220,000 ±
10,000 scanning units; and lane 6, 480,000 ± 20,000
scanning units.
Western Blotting Analysis of the Positive
Clones
Membrane proteins from the three Southern blot-positive
clones were prepared and analyzed with antibodies specific for the
PMCA4CI pump. A C-terminal PMCA4CI-specific antibody yielded a strongly
positive band at the expected molecular mass range (Fig. 2A,
lanes 3-5), which migrated as the PMCA protein from
erythrocytes (Fig. 2A, lane 1). The band at 60 kDa
resulted from an unspecific reaction of the serum, since it was also
present in control CHO cells (Fig. 2A, lane 2). The
overexpressed pump was also recognized by the 5F-10 monoclonal antibody (Fig. 2A, lanes 6-7, CHO-9-150 and CHO-10-25 gave
similar signals, not shown). It is important to emphasize that the
amount of PMCA4CI pump present in the crude membrane protein
preparations of the stable transfected cells was at least eight times
higher than in the membrane of erythrocytes. The level of expression
was highest in CHO-10-150 cells (Fig. 2A, compare lanes 3 and 4 with lane 5).
Figure 2:
Detection of the PMCA4CI protein by
Western blotting and stability of the overexpressing cell lines. A, crude membrane proteins were separated by SDS-PAGE
according to Laemmli (1979) and transferred to nitrocellulose
membranes. 10 µg of membrane proteins from normal CHO cells (lanes 2 and 6), CHO-9-150 (lane 3),
CHO-10-25 (lane 4), and CHO-10-150 cells (lanes 5 and 7) were loaded on the gels. As a control 60-70 µg of
erythrocyte membrane proteins were used (lane 1). The
nitrocellulose sheets were incubated with the polyclonal antibody 94.2
(specific for the C-terminal portion of PMCA4CI, D. Guerini unpublished
observation) (lanes 1-5) or with monoclonal antibody
5F-10 (Adamo et al., 1992b) (lanes 6 and 7). B, the stability of the overexpression was tested on 30 µg
of total cell protein. The cells were washed in TBS before denaturing
them in SDS-loading buffer. The PMCA4CI protein was detected with the
polyclonal antibody 94.2. The CHO-10-150 cells were passed 40-50
times in the presence of MSX (the selection drug) (lane 1), 40
times with MSX followed by four passages without MSX (lane 2),
or 40-50 times without MSX (lane 3). Nontransfected CHO
cells are shown in lane 4. 50-60 µg of erythrocytes
ghosts were used as a control (lane
5).
To test the
stability of the expression level, the stable transfected cells were
maintained for 50 passages (up to 6 months) in tissue culture with and
without addition of the selection drug MSX. The amount of overexpressed
pump was assessed by Western blotting. Culturing the cells for a short
time without selection didn't significantly affect the amount of
expressed pump (Fig. 2B, lanes 1 and 2), but
after 30-40 passages a substantial decrease became evident (Fig. 2B, lane 3). The expression level of the pump in
cells maintained in the selection media for as long as 6 months
remained constant.
Localization of the Overexpressed Protein
The
overexpressed pump was found to be targeted to the plasma membrane of
COS cells (Zvaritch et al., 1992) but other experiments
indicated that a significant fraction of it was apparently retained in
the endoplasmic reticulum (Adamo et al., 1992a). The latter
result may have been due to overexpression, causing a saturation of the
sorting machinery and accumulation of the pump in the endoplasmic
reticulum. Immunocytochemistry was therefore performed with the stable
transfected CHO cells. Strong signals were detected for the CHO-9-150
cells (Fig. 3a, D), the staining being concentrated on
the cell surface. No reticular staining was ever observed, indicating
that most of the expressed pump was correctly delivered to the plasma
membrane. This was also true for the stable cell lines CHO-10-25 and
CHO-10-150 (not shown). Not all the cells showed the same intensity of
staining, but a reasonable number (more than 50-60%) had a
similarly strong signal. The differences with the remainder of the
cells could have been due to the incomplete permeabilization of the
cells, which was necessary due to the cytoplasmic localization of the
epitope. Inhomogeneity of staining was also observed by others with
stable transfected CHO cells (Hussain et al., 1992).
Figure 3:
Localization of the overexpressed pump
protein. a, immunocytochemistry was performed as outlined
under ``Experimental Procedures.`` The experiments shown in
the figure have been carried out with non transfected CHO cells (A, B) and with CHO-9-150 cells (C, D). (Identical results were obtained with the CHO-10-25 and
CHO-10-150 cells). A and C, phase contrast
photograph; B and D, immunofluorescence after
incubating the permeabilized cells with the polyclonal antibody 94.2. b, surface labeling of the CHO cells. Cells were labeled with
[S]Met, treated with NHS-SS-biotin as described
under ''Experimental Procedures`` prior to
immunoprecipitation with the 5F-10 monoclonal antibody. Half of the
immunoprecipitated sample was separated on the gel (lanes 1 and 3). The other half was precipitated with avidin beads
before loading into the gel (lanes 2 and 4). The
experiment was performed with CHO-10-150 cells (lanes 1 and 2) and nontransfected CHO cells (lanes 3 and 4). The dried gel was exposed at -70 °C for
2-3 days. Exposure of up to 7 days failed to reveal radioactive
bands in lane 4.
Biotinylation experiments (Fig. 3b) confirmed that
the PMCA4CI protein was localized in the plasma membrane. After
immunoprecipitation a large amount of the pump protein was bound by the
streptavidin beads, indicating that the protein had become labeled in
the intact cells (Fig. 3b, lane 2). The
monoclonal antibody 5F10 (recognizing PMCA from different species,
Borke et al., 1989) was used for these experiments to detect
also the endogenous PMCA protein in nontransfected CHO cells (Fig. 3b, lane 3). In spite of the much longer exposure
time of the autoradiogram, the amount of biotinylated endogenous PMCA
was evidently too low to be detected with the protocol used (Fig. 3b, lane 4). The experiments thus supported the
conclusion (Fig. 3a) that the largest fraction of the
overexpressed PMCA4CI pump was correctly targeted to the plasma
membrane.
Formation of the Phosphorylated Intermediate by the
Overexpressed Pump
The formation of the phosphorylated
intermediate of the reaction cycle was studied on microsomes or crude
membrane preparations from CHO and CHO-10-150 cells. The relative
content of plasma membrane was estimated by measuring the endogenous
Na/K
-ATPase activity (see
``Experimental Procedures''). The membranes used for the
phosphoenzyme assay (preparations similar to the ones used for the
experiment in Fig. 4A) didn't show any significant
difference between membranes from CHO cells (380 ± 45 nmol
P
/mg protein
h
)
and CHO-10-150 cell line (410 ± 40 nmol P
/mg
protein
h
). The experiment
was performed in the presence of Ca
/La
to optimize the detection of the PMCA pump intermediate (Fig. 4A, lanes 1 and 4). The intermediate
formed by membranes from CHO-10-150 was 10-15 more abundant than
that of membranes from nontransfected cells (Fig. 4A,
compare lanes 1 and 4). Densitometric scanning of
gels similar to that shown in Fig. 4A resulted in a
value of 17,000 scan units for the 135-kDa band in lane 1 and
of 370,000 scan units for the corresponding band in lane 4. As
expected, the intermediate was sensitive to NH
OH (Fig. 4A, lanes 3 and 6) and was not seen in
the presence of EGTA (Fig. 4A, lanes 2 and 5).
The radioactive band comigrated with the corresponding pump band of
erythrocytes (Fig. 4, lane 7), i.e. it indeed
corresponded to the phosphoenzyme intermediate of the PMCA pump.
Interestingly, the amount of intermediate formed by the endogenous
SERCA pump (the radioactive band at 100 kDa) was reproducibly lower in
membrane preparations of CHO-10-150 cells than in control cells (Fig. 4A, compares lanes 1 and 4). Experiments
with different membrane preparations were performed to test if the
``artifactual'' enrichment of a given membrane fractions was
responsible for the observation: however, in all cases the amount of
SERCA intermediate in cells overexpressing the PMCA4CI pump was lower
than in control cells. This was found to be true for all three stable
transfected cell lines (not shown). To rule out possible artifacts
related to the ATP concentration used, experiments were performed with
different concentrations of the latter (Fig. 4B). A
total membrane fraction was used in this case (see ``Experimental
Procedures''), since the difference in the amounts of the SERCA
phosphoenzyme between transfected and nontransfected cells was
particularly evident in it. The results of Fig. 4B (lanes 1-6) demonstrate that the difference in the
amount of the SERCA intermediate (the radioactive band at 100 kDa)
between transfected and nontransfected cells was unrelated to the ATP
concentration. Since La
inhibits slightly the
formation of the SERCA intermediate (Carafoli and Guerini, 1993), the
phosphorylation reaction was also performed in the absence of
La
. Under these conditions the plasma membrane
intermediate was not visible (Fig. 4B, lanes 7 and 8), but the amount of the SERCA intermediate in CHO-10-150
cells (Fig. 4B, lane 8) was still lower than in control
cells (Fig. 4B, lane 7). It was important to demonstrate
that the 100-kDa band was indeed the SERCA pump. Addition of
La
in the assay buffer resulted in the appearance of
the PMCA-specific phosphoenzyme (Fig. 4B, lane 10). A
weaker band at the same molecular mass was also present in the lane
corresponding to the CHO cells (Fig. 4B, lane 9, compare
also Fig. 4A, lane 1 and Fig. 4B, lane
1). Under the same conditions (+La
), the
100-kDa band became weaker (Fig. 4B, lanes 9 and 10), which is a property of the phosphoenzyme intermediate of
the SERCA pump. The 100-kDa band was sensitive to thapsigargin, a well
known inhibitor of the SERCA pump, whereas the 135-kDa (PMCA) band was
not (Fig. 4C, lanes 1 and 2). Additionally
proofs that the 100-kDa band corresponded to the phosphoenzyme
intermediate of the SERCA were obtained by immunoprecipitation
experiments (Fig. 4C, lane 3-5).
Immunoprecipitation with a SERCA 2b-specific antibody resulted in the
almost quantitative recovery of the 100-kDa radioactive protein (Fig. 4C, lane 5). The PMCA-specific monoclonal antibody
precipitated only the 135-kDa protein (Fig. 4C, lane 4).
No bands around 100 kDa were ever observed. Since the 5F-10 monoclonal
antibody recognized an epitope present in the C-terminal end of the big
intracellular loop (amino acids 724-758) of all the PMCA isoforms
(Adamo et al., 1992b), proteolytic fragments bigger than 90
kDa should have been recognized by it. The lower amount of the PMCA
phosphoenzyme intermediate present before immunoprecipitation (Fig. 4C, lane 3) was due its low solubility in MEN
buffer after the trichloroacetic acid precipitation. Addition of
dithiothreitol and urea improved the solubilization, but no
immunocomplexes could be formed under these conditions. Similar
experiments were performed with nontransfected CHO cells, giving
essentially the same results (not shown).
Figure 4:
Activity measurements on the pump
overexpressed in the CHO cells. Formation of the phosphoenzyme
intermediate. A, 20-25 µg of microsomal proteins
from control CHO cells (lanes 1-3) and from CHO-10-150
cells (lanes 4-6), and 70 µg of erythrocyte membrane
proteins (lane 7) were phosphorylated with 0.4 µM [-
P]ATP (200-300 Ci/mmol) as
described under ''Experimental Procedures.`` The
phosphorylation reaction was performed in the presence of 100
µM Ca
and 100 µM La
(lanes 1, 4, and 7) or in
the presence of 1 mM EGTA (lanes 2 and 5).
The samples in lanes 3 and 6 were phosphorylated like
those of lanes 1 and 4 before incubating them for 30
min at room temperature with 200 mM NH
OH. The
proteins were separated by acidic gels after trichloroacetic acid
precipitation (Sarkadi et al., 1987). The dried gels were
exposed for 48 h at -70 °C. B, 25-30 µg of
total crude membrane proteins from control CHO cells (lanes
1-3, 7, and 9) and from CHO-10-150 cells (lanes
4-6, 8, and 10) were phosphorylated under different
conditions. Phosphorylation in lanes 1-6 was performed
at different concentrations of ATP in the presence of 100 µM Ca
and 100 µM La
.
The following ATP concentrations were used: lanes 1 and 4, 0.2 µM [
-
P]ATP
(500 Ci/mmol); lanes 2 and 5, 0.5 µM
[
-
P]ATP (200 Ci/mmol); lanes 3 and 6, 2 µM [
-
P]ATP (50
Ci/mmol). Phosphorylation in lanes 7 and 8 were done
in the presence of 0.3 µM
[
-
P]ATP (300 Ci/mmol) and 100 µM Ca
. Under these conditions (absence of
La
) the phosphoenzyme of the PMCA pump was not
visible. The scanning of the autoradiogram yielded the following
results: lane 7, 130,000 ± 20,000 scanning units for
the 100-kDa band; lane 8, 32,000 ± 4000 scanning units.
Phosphorylation in lanes 9 and 10 was done in the
presence of 0.3 µM [
-
P]ATP
(300 Ci/mmol), 100 µM LaCl
, and 100 µM CaCl
. Thapsigargin sensitivity and immunoprecipitation
of the phosphorylated intermediates. C, 25-30 µg of
CHO-10-150 cells membranes were phosphorylated in the presence of 100
µM LaCl
, 100 µM CaCl
,
and 0.3 µM [
-
P]ATP (300
Ci/mmol) (lanes 1 and 2). Prior to the
phosphorylation reaction, the membranes were treated with (lane
1) or without (lane 2) 2 µM thapsigargin, 10
min at 37 °C in the presence of 100 µM ATP. The
membranes were sedimented, washed twice in 20 mM MOPS-KOH, pH
6.8, and 100 mM KCl before the phosphorylation reaction. The
immunoprecipitation experiment (lanes 3-5) was performed
as described under ''Experimental Procedures.`` Lane
3, membrane proteins after the phosphorylation reaction and
solubilization in MEN buffer but before immunoprecipitation
(corresponding to 20 µg of membrane protein); lane 4,
phosphorylated proteins immunoprecipitated by the 5F-10 (PMCA specific)
antibody (corresponding to 100-130 µg of membrane proteins); lane 5, phosphorylated bands immunoprecipitated with the
polyclonal antibody against the SERCA2b pump (corresponding to
100-130 µg of membrane proteins). D, Ca
uptake by microsomes. Ca
uptake was measured at
a free Ca
concentration of 2.2 µM as
described under ''Experimental Procedures.`` Microsomes
obtained from the CHO-9-150 (
) and control CHO cells (
)
were used. Each point is the average of at least three measurements. In
the case of the CHO-9-150 cells the uptake was studied in the presence
(
) and in the absence of ATP (
). The Ca
dependence of the Ca
uptake is shown in the inset. The free Ca
concentration was
determined according to the program of Fabiato and Fabiato (1979). The
uptake was measured 10 min after the addition of ATP. 100% of the
Ca
transport was the maximal rate obtained at 2.2
µM Ca
.
, CHO-9-150 cells;
&cjs2123;, CHO-10-150 cells.
Ca
Fig. 4D shows
the uptake of Ca Uptake by Microsomes Obtained from
the Stable Transfected CHO Cells
by microsomes obtained from
CHO-9-150 and control cells. The fractionation scheme described under
``Experimental Procedures'' produced a fraction that
contained, in addition to vesicles of the endoplasmic reticulum, a
significant amount of inside/out plasma membrane vesicles. The specific
activity of the Na
/K
-ATPase in these
membranes was 1-2.2 µmol of P
released per mg
protein
h
. No significant
difference between control and stable transfected cells was observed.
In both cases the uptake was linear for the first 20-25 min,
after which saturation (in the case of the CHO-9-150 cell at 200 nmol
of Ca
/mg of microsomal protein) was reached. The
uptake by CHO-9-150 cell microsomes, 8 nmol of
Ca
/mg
min
was 8-10 times higher than that of nontransfected CHO
cells. The uptake by microsomes from CHO-10-150 cells was even higher
and amounted to 14.8 nmol of Ca
/mg of microsomal
protein
min
. This result was
in good agreement with those of the phosphorylation experiments (see
above), were a 15-fold increase of ATPase activity was also seen.
dependence of the uptake process was also
determined. Fig. 4D (inset) shows that in all
cases the K
for Ca
was
about 0.3 µM, in good agreement with the value obtained
for the purified pump (Niggli et al., 1981)
Ca
The
efflux of Ca Release from the Cells
was studied in nontransfected CHO cells
and from CHO-10-25 and CHO-10-150 cells. Cells were passively loaded
with
Ca
by incubating them for some
hours at 37 °C, and the release was started by the addition of ATP
and serum; ATP has been found to induce a transient increase of
Ca
in CHO cells, which normally lasts for 30-50
s (Pijuan et al., 1993). Fig. 5shows that under these
conditions higher amounts of Ca
were released by the
CHO-10-25 and CHO-10-150 cells 10 and 40 s after the addition of ATP
(14-17% of the total) as compared to nontransfected CHO cells
(7-8%). The increased efflux lasted about 60 s, the difference
with control cells disappearing at later times. Experiments on passive
Ca
accumulation or on passive Ca
release, i.e. cells were treated as described above, but
after transferring them to the release medium (see ``Experimental
Procedures''), no ATP and no serum were added, showed that cells
overexpressing the PMCA pump behaved as control CHO cells (not shown).
Figure 5:
Ca release from the
cells. Cells were plated in 12-well plates and loaded with
Ca
(see ''Experimental
Procedures``). After washing the cells, the efflux from them was
started by the addition of ATP and FCS. Aliquots of the reaction medium
were withdrawn and counted. The values reported in the figure are the
average of eight measurements and represent the amount of
Ca
released after the time indicated. 100% refers to
the total amount of
Ca
in the cells at
the beginning of the efflux measurements.
, CHO;
,
CHO-10-25;
, CHO-10-150.
Growth of the Stable Transfected Cells
It was
noticed during routine manipulation of the cells that those expressing
high levels of PMCA were not growing as fast as control cells or
transfected cells which were overexpressing lower amounts of the PMCA
pump. Different growth conditions were thus analyzed. Fig. 6A shows that nontransfected and transfected cells were unable to
grow at a concentration of FCS below 2%. In the presence of 5 or 10%
FCS, nontransfected CHO cells grew reproducibly faster than the
CHO-9-150 and CHO-10-150 cells. The semilogarithmic plot of Fig. 6A shows that CHO-10-150 and CHO-9-150 cells had a
growth delay in the first 12-24 h. This behavior persisted if the
cells were grown in different media, provided that adequate amounts of
FCS were present. The growth delay was not seen in MSX-resistant clones
which were not expressing the plasma membrane ATPase. In the inset of Fig. 6B the duplication time was calculated from
the curves between 40 and 140 h after plating; the values were very
similar for all cell lines, indicating that once the initial lag was
over, the stable transfected cells behaved like nontransfected CHO
cells. Thus cells overexpressing the PMCA4CI pump apparently needed a
longer ``recovery'' time after trypsinization and diluting in
fresh medium.
Figure 6:
Growth curve of the cells. A, the
growth curve of control CHO cell (circles), CHO-10-150 cells (squares), and CHO-9-150 cells (triangles) was determined at
four different FCS concentrations. (filled circles, triangles,
and squares, 1%; diagonally slashed open circles, triangles, and squares, 2%: dotted circles,
triangles, and squares, 5%; and open circles,
triangles, and squares, 10%). The values reported are the
average of two to three independent measurements. B, some of
the data of A (left: curve with 10% FCS; right, curve with 5% FCS) are represented in a semilogarithmic
diagram. The duplication times of the cells are given in the insets and were calculated between 40 and 140
h.
Purification of the Pump from the CHO Cells
The
overexpressed plasma membrane Ca-ATPase was purified
to essential homogeneity from CHO-10-150 cells by affinity
chromatography on calmodulin-Sepharose 4B (Fig. 7, lane
2). The preparation showed a major band at 135 kDa and minor
contaminants at 100 and 60 kDa (Fig. 7, lane 2). Some of
the latter were also present in a similar preparation from
nontransfected CHO cells (Fig. 7, lane 1). In the latter
no detectable band at 135 kDa was detected, again showing that the
endogenous plasma membrane pump is exceedingly low in CHO cells.
Figure 7:
Purification of the PMCA4CI pump from
CHO-10-150 cells. Membrane proteins (control CHO and CHO-10-150 cells)
were solubilized and incubated with calmodulin-Sepharose 4B,
essentially as described previously (Niggli et al., 1979).
Aliquots were separated by SDS-PAGE and visualized by silver staining
(Merryl et al., 1981). Fractions were eluted from
calmodulin-Sepharose with EDTA. Lane 1, control CHO cells; lane 2, CHO-10-150 cells. Molecular weight markers are shown
in lane 3.
pumps. Stable transfected cells produced
more PMCA than transiently transfected ones (generally three to six
times PMCA activity over the background is observed in COS cells (Heim et al., 1992; Enyedi et al., 1993)). An advantage of
the stable transfection system is that all the cells express similarly
high amounts of protein, whereas after transient transfection only a
smaller proportion of the cells (5-10%, Zvaritch et al.,
1992) expressed it. In addition, cells stable transfected with the
SERCA pump showed homogenous distribution of the expressed protein in
the membranes, at variance with transiently transfected COS(1) cells,
where only a small fraction of the membranes contained high amounts of
the pump (Hussain et al., 1992); this resulted in a fast
rising concentration of luminal Ca
in the vesicles
and in the inhibition of the pump activity. Although the duplication
time of cells overexpressing the PMCA pump was not different from that
of the parental cells, all clones had slower growth recovery after
trypsinization and plating. This was not related to the presence of
glutamine synthetase in their genomes, since clones of MSX-resistant
CHO cells transfected with glutamine synthetase had normal growth
pattern.
plays an important
role (Stossel, 1993). It is also known that addition of FCS containing
a variety of hormones and growth factors activates different signaling
pathways which are responsible for the reshaping of the cells (Stossel,
1989, 1993). Cells overexpressing the PMCA protein evidently extruded
higher amounts of Ca
and thus lowered it in the
cytosol at a faster rate. Possibly, this disturbed the FCS mediated
reshaping of the cells.
itself. It would
be interesting to see whether a similar phenomenon occurs in cells
overexpressing the other system exporting Ca
from the
cytosol, i.e. Na
/Ca
exchanger.
ATPase; FCS, fetal calf serum; MES,
2-(N-morpholino)ethanesulfonic acid; MOPS,
3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; SERCA, sarcoplasmic/endoplasmic reticulum
Ca
ATPase; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; bp,
base pair(s); CHO, Chinese hamster ovary; MSX, methionine sulfoximide;
TBS, Tris-buffered saline; PBS, phosphate-buffered saline; MEM, minimal
essential medium; S-MEM, minimal essential medium modified for
suspension cultures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.