From the Department of Biochemistry and Molecular Biology,
University of Maryland School of Medicine,
Baltimore, Maryland 21201
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INTRODUCTION |
The sesquiterpene lactone thapsigargin
(TG),1 isolated from the
plant Thapsia garganica (3), is a highly specific inhibitor of sarco-endoplasmic reticulum Ca2+ ATPases (SERCAs) (4,
5). Kinetic studies indicate that a 1:1 TG-ATPase stoichiometric
complex is formed at extremely low concentration of TG (6). The
inhibition involves Ca2+ binding, formation of
phosphorylated intermediate, ATP hydrolysis, and Ca2+
transport (7-10). These effects suggest a global effect of TG on the
enzyme, as also indicated by its influence on formation of ordered
ATPase arrays (11, 12). Hence, clarification of the TG binding domain
would contribute to the understanding of structural and mechanistic
features in the enzyme. A useful approach, in this regard, is to study
the TG sensitivity of chimeric proteins consisting of defined parts of
SERCA and Na+,K+-ATPase, because TG interacts
specifically with the former and not with the latter. Previous studies
with large chimeric exchanges, however, produced strong inhibition of
catalytic turnover and transport. Nevertheless,
Ca2+-dependent formation of phosphorylated
intermediate was preserved, and a reduced sensitivity of this parameter
to TG was obtained upon chimerization of the entire S3-M3 (stalk and
membrane-bound) region (13). In contrast, the TG sensitivity was not
altered significantly if other large regions were exchanged (13-15).
We describe here the construction and functional characterization of
more discrete chimeric changes, involving stepwise mutations of one and
up to several amino acids in the S3 or S4 stalk segments of the SERCA,
to match the corresponding residues of the
Na+,K+-ATPase. A similar strategy was
previously used in studies of ouabain binding by the
Na+,K+-ATPase, taking advantage of
ouabain-sensitive and ouabain-insensitive isoforms of the enzyme
(16).
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EXPERIMENTAL PROCEDURES |
PCR Mutagenesis and Protein Expression--
The chicken fast
muscle SERCA-1 cDNA (17), containing 11 artificial and unique
restriction sites spaced at approximately 300-bp intervals to
facilitate cassette exchange (18), was subcloned into pUC19 vector for
site-directed mutagenesis. For this purpose, the cassette delimited by
BssHII and Bsu36I restriction sites (579 bp
including S3 and M3 coding sequences), and the cassette delimited by
BamHI and Bsu36I (318 bp including M4 and S4
coding sequences), were amplified by PCR using oligonucleotide
"flanking" primers. Furthermore, complementary mutagenic
oligonucleotides of 23-35 bp length were synthesized for each
individual mutation. These primers were utilized to hybridize DNA
sequences internal to the flanking primers and were used for PCR
mutagenesis by the overlap extension method as described by Ho et
al. (19). Briefly, two overlapping fragments containing the
mismatched base(s) of the targeted sequence were amplified in separate
PCR reactions. The PCR mixtures contained 1 µM each of
flanking and mutagenic primers, 800 µM dNTPs, 20 ng of
SERCA-1 cDNA, 2.5 units of Pfu (Pyrococcus
furiosus) DNA polymerase, and Pfu buffer (Stratagene, Menasha, WI) in a final volume of 100 µl. The reaction products were
separated by electrophoresis on a 3% low melting agarose gel (FMC,
Rockland, ME), and the appropriate Mr band was
excised and melted at 72 °C for 5 min. The eluted fragments were
fused, and the entire cassette was amplified using both flanking
primers. The mutant cassette was then exchanged with the corresponding cassette of wild-type cDNA in pUC19 vector, and sequenced by the dideoxy chain-termination method using Sequenase (U. S. Biochemical Corp.). Additive chimeric mutations were introduced by sequential PCR
mutagenesis using mutant DNA as template. Finally, the mutated cDNA
was subcloned into COS-1 expression vector pCDL-SR
296 (20) for
transfection and overexpression of protein under control of the SV40
promoter. COS-1 cell cultures and transfections were carried out as
described by Sumbilla et al. (15).
Microsomal Preparation and Immunodetection of Expressed
Protein--
The microsomal fraction of transfected COS-1 cells was
obtained by differential centrifugation of homogenized cells (15). Immunodetection of expressed ATPase in the microsomal fraction was
obtained by Western blotting, using the CaF-5C3 monoclonal antibody to
SERCA-1 (17), as described by Sumbilla et al. (15).
Functional Studies--
ATP-dependent
Ca2+ transport was measured by following the uptake of
radioactive calcium tracer by microsomal vesicles. The reaction mixture
contained 20 mM MOPS, pH 7, 80 mM KCl, 5 mM MgCl2, 0.2 mM CaCl2,
0.2 mM EGTA, variable concentrations of TG, 5 µg of
microsomal protein/ml, 5 mM potassium oxalate, and 3 mM ATP. The reaction was started (37 °C) by the addition
of oxalate and ATP, and was terminated at sequential times by vacuum
filtration (0.45-µm Millipore filters). The filters containing the
calcium loaded vesicles were washed with 2 mM
LaCl3 and 10 mM MOPS, pH 7.0, and were then
processed for determination of radioactivity by scintillation counting.
The observed rates of Ca2+ transport were corrected to
reflect the level of expressed ATPase in each microsomal preparation,
as revealed by immunoreactivity and with reference to microsomes
obtained from COS-1 cells transfected with wild-type SERCA-1
cDNA.
ATPase activity was assayed in a reaction mixture containing 20 mM MOPS, pH 7.0, 80 mM KCl, 3 mM
MgCl2, 0.2 mM CaCl2, 5 mM sodium azide, 20 µg of microsomal protein/ml, 3 µM ionophore A23187, 3 mM ATP, and 0-1000
nM TG. Ca2+-independent ATPase activity was
assayed in the presence of 2 mM EGTA and no added
Ca2+. The reaction was started (37 °C) by the addition
of ATP, and samples were taken at serial times for determination of
Pi by the method of Lanzetta et al. (21). The
Ca2+-dependent activity was calculated by
subtracting the Ca2+-independent ATPase from the total
ATPase and was corrected to account for the level of expressed protein
in each microsomal preparation as revealed by immunoreactivity, and
with reference to microsomes obtained from COS-1 cells transfected with
wild-type SERCA-1 cDNA.
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RESULTS |
Description of Mutants--
The chimerization scheme for analysis
of the S3 stalk segment is shown in Table
I. In part A (top and bottom lines), nine amino acids of the SERCA S3 segment and of the corresponding
Na+,K+-ATPase segment are aligned according to
Norregaard et al. (1). In the intervening lines, it is then
shown how eight amino acids of the Ca2+ ATPase sequence
were stepwise mutated to complement the conserved Phe256,
and yield a nine-amino acid chimeric sequence identical to that of the
Na+,K+-ATPase. Seven mutants were derived from
this stepwise procedure and processed for transgenic expression in
COS-1 cells.
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Table I
Mutation schemes for the S3 (A) and M3 (B) segments
The native sequences of the S3 (stalk) and M3 (transmembrane) segments
of SERCA-1 (17, 25) and of the corresponding segments of the rat kidney
Na+,K+-ATPase 1 isoform (26, 27) are aligned (top
and bottom, respectively) according to Norregaard et al.
(1). In the S3 segment (A), one out of nine amino acids is identical in
the two ATPases, and eight are subjected to stepwise mutation from the
Ca2+ to the Na+,K+-ATPase sequence to yield a
nine amino acid segment of homology (Mutant S3,8). In the M3 segment
(B), four out of nine amino acids are homologous, and five are mutated
from the Ca2+ to the Na+,K+-ATPase sequence to
yield a nine amino acid segment of homology (Mutant M3,5).
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Table 1, part B, shows a similar chimerization scheme for analysis of
the M3 transmembrane segment. Five mutations were produced to yield a
nine-amino acid chimeric sequence (including four residues of native
homology).
Another chimerization scheme was directed to the Ca2+
ATPase S4 segment, and involved mutation of five amino acids to
complement 16 homologous amino acids and four conservative replacements
in the native sequence, yielding a 25-residue chimeric sequence that is
homologous to the corresponding segment of the
Na+,K+-ATPase (Table
II).
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Table II
Mutation scheme for the S4 segment
The native sequence of the S4 (stalk) segments of SERCA-1 (17, 25) and
of the rat kidney Na+,K+-ATPase (26, 27) are aligned
(top to bottom, respectively) by matching the aspartyl residues
undergoing phosphorylation. Sixteen amino acids are homologous, and
four are conservative replacements in the native sequences. Five
residues were mutated from the Ca2+ to the
Na+,K+-ATPase to yield, therefore, a 25-amino acid
homologous segment (Mutant S4,5).
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In addition to the chimeric exchanges described above, more discrete
mutations were produced in the S3 segment (as explained in Table
III and in the text below) to test the
effects of limited perturbations in this region.
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Table III
Effects of single mutations on SERCA-1, and alternative mutations
on the S3,5 chimera
Single mutations of the WT SERCA-1 to the corresponding
Na+,K+-ATPase residues, as well as single or double
mutations in the S3,5 chimera (to more conservative replacements as
compared to the corresponding Na+,K+-ATPase residues)
were produced, and their effects on the sensitivity of the enzyme to TG
were studied as shown in Figs. 4 and 5. The KI
values are the numbers used to fit the inhibition curves (three
experiments per each mutant), and correspond to the TG producing 50%
inhibition.
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Levels of Expression--
Approximately 10% of the COS-1 cells
transfected under our conditions overexpress and target the
Ca2+ ATPase to the endoplasmic reticulum, as shown by
in situ microscopic visualization following
immunofluorescent staining (18). In the experiments reported here,
Western blot analysis of microsomal fractions obtained from the
harvested cells revealed similar levels of expression for the wild-type
ATPase and ATPase mutants (Fig. 1). Minor
variations of expression levels were generally related to the
efficiency of transfection rather than the presence of mutations. At
any rate, the expression levels were quantitated by densitometry of
Western blots, and the resulting values were used to correct the
functional parameters to be described below, with reference to the
wild-type enzyme.

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Fig. 1.
Quantitation of protein expression.
Wild-type and mutated constructs were expressed in COS-1 cells, and
identical protein aliquots of solubilized cell homogenates were
analyzed by Western blotting as described under "Experimental
Procedures." The bands were quantitated by densitometry, and the
so-derived values were utilized to estimate the content of transgenic
protein per unit of total protein.
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Ca2+ Uptake and ATP Hydrolysis--
As originally
reported by Maruyama and MacLennan (22), microsomal vesicles obtained
from transfected COS-1 cells sustain ATP-dependent
Ca2+ uptake and related ATPase activity. These are specific
and useful functional signals, which, as shown in Fig.
2, proceed at constant rates for several
minutes. Ca2+ uptake is a highly specific functional
parameter, which is totally inhibited by TG (Fig. 2A). On
the other hand, the observed ATPase activity includes
Ca2+-independent and TG-insensitive components that must be
subtracted from the total in order to obtain the specific
Ca2+-dependent and TG-sensitive ATPase activity
(Fig. 2B).

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Fig. 2.
Examples of ATP-dependent
Ca2+ uptake (A) and ATPase activity
(B). These steady state measurements were performed as
explained under "Experimental Procedures," using wild-type SERCA-1
enzyme as the catalyst. Note the total inhibition of Ca2+
uptake by TG, whereas the ATPase activity displays a component that is
Ca2+-independent and TG-insensitive. The symbols refer to:
no TG ( ), 0.1 nM TG ( ), 0.2 nM TG ( ),
0.5 nM TG ( ), 10 nM TG ( ), 20 nM TG ( ), and no Ca2+ ( ).
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When we compare the Ca2+ transport activities of wild-type
and mutant proteins (Fig. 3), we find
that the transport rates are unaffected by mutations of up to four
amino acids in the S3 segment (mutants S3,2 and S3,3; Fig. 3), but
undergo a progressive reduction as the number of mutated amino acids in
the S3 segment is increased (mutants S3,4 to S3,8; Fig. 3). On the
other hand, the nine-amino acid chimeric homology with the
Na+,K+-ATPase in M3 (M3,5) and the 25-amino
acid homology in S4 (S4,5), produce only 60% and 30% reduction of the
transport rates, respectively (see Fig. 3). Similar effects of
mutations were observed on the ATPase hydrolytic rates (data not
shown). We then tested the TG concentration sensitivity of wild-type
enzyme and of mutants retaining at least 50% activity.

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Fig. 3.
Effects of various mutations on the activity
of SERCA-1 enzyme expressed in COS-1 cells. cDNA mutations and
transfections were carried out as described under "Experimental
Procedures." The microsomal fraction of the transfected cells was
obtained by differential centrifugation of homogenized cells, and
was utilized for measurements of ATP-dependent
Ca2+ uptake (see "Experimental Procedures").
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It is shown in Fig. 4A that
the KI for Ca2+ transport inhibition by
TG is gradually shifted by 3 orders of magnitude to higher
concentrations (1.7 × 10
10 M to
1.25 × 10
7 M) as the number of mutated
amino acids in the M3 segment is increased to five or six, to yield a
six- or seven-amino acid homologous segment between
Ca2+-ATPase and Na+,K+-ATPase.
Similar results were observed for
Ca2+-dependent ATPase activity (Fig.
4B).

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Fig. 4.
Sensitivity of wild-type SERCA-1 and S3
mutants to TG. Ca2+ uptake (A) and ATPase
(B) measurements were performed as described under
"Experimental Procedures." The symbols refer to wild-type SERCA-1
enzyme ( ) and to chimeras S3,2 ( ), S3,3 ( ), S3,4 ( ), S3,5
( ), and S3,6 ( ). Each point is the average of three steady state
velocities obtained as in Fig. 2, using two or three different protein
preparations. The experimental points were computer-fitted, using a
single site binding equation with dissociation constants yielding the
best fit for each set of data. Note that the theoretical fits obtained
for the Ca2+ uptake data are better than those obtained for
the ATPase data, perhaps due to a greater specificity of the
Ca2+ uptake measurements, relative to the ATPase
measurements.
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Because in the work of Norregaard et al. (1), the ATPase
sensitivity to TG was lost following an extensive chimeric replacement involving the entire S3-M3 segment, we produced a nine-amino acid chimeric exchange within the M3 transmembrane segment, to be compared with the analogous chimeric exchange produced in the S3 stalk segment.
We found that the chimeric exchange in the M3 segment produced
approximately 60% transport inhibition (Fig. 3), but had no effect on
the ATPase sensitivity to TG (Fig.
5).

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Fig. 5.
Sensitivity of wild-type SERCA-1 and the
S3,5, M3,5, and S4,5 mutants to TG. Ca2+ uptake and
ATPase measurements, as well as data analysis, as for Fig. 4. Symbols
refer to wild-type enzyme ( ), S3,5 ( ), M3,5 ( ), and S4,5 ( )
mutants.
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To test the specificity of mutations in the S3 segment with regard to
TG sensitivity, we then studied a five-amino acid chimeric mutation in
the S4 segment of the ATPase (S4,5 in Table II). It should be pointed
out that the segment chosen for our studies contains already 16 homologous, and four conservatively replaced, amino acids, when
compared with the corresponding segment of the Na+,K+-ATPase (Table II). Therefore, mutation
of five heterologous amino acids results in a 21-amino acid chimeric
segment. It is of interest that this S4 mutant produces only 30%
inhibition of function (Fig. 4), and its TG sensitivity is identical to
that of the wild-type enzyme (Fig. 5).
Considering that a five-amino acid mutation in the S3 stalk segment
reduces the ATPase sensitivity to TG by 3 orders of magnitude, we then
produced more discrete mutations to test the effects of limited
perturbations in this region. We found that a single mutation of
Gly257 to the corresponding
Na+,K+-ATPase residue (Ile) is sufficient, by
itself, to reduce the ATPase sensitivity to TG by 1 order of magnitude
(Table III). The important role of Gly257 is also revealed
by the significant reversal of TG sensitivity reduction observed when
the Gly257
Ile mutation in the S3,5 chimera is changed
to the more conservative Gly257
Ala mutation.
Additional reversal to WT behavior is produced by a further change
involving the Gln259
Leu mutation to the more
conservative Gly259
Asn mutation (Table III). This
further reversal is not obtained if Glu259 is mutated to
Gly (Table III).
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DISCUSSION |
TG produces global inhibition (i.e. phosphoenzyme
formation, hydrolytic activity, and Ca2+ transport) of
ATPase, whereas no inhibition is produced by TG on the
Na+,K+-ATPase (5). This specificity has
motivated chimeric studies to obtain information on the TG binding
site, assuming that replacement of a critical SERCA sequence with the
corresponding Na+,K+-ATPase sequence would
interfere with inhibition. Large chimeric exchanges produce nearly
total inhibition of hydrolytic activity and Ca2+ transport.
Nevertheless, formation of Ca2+-dependent
phosphoenzyme is retained by such chimeric proteins and, based on
phosphoenzyme measurements, it was shown that large chimeric exchanges
in the cytosolic SERCA region do not interfere with inhibition by TG
(15). In contrast, chimeric exchange of a 30-amino acid
(Leu253-Val283)
S3-M3 segment (1) reduces the inhibitory effect of TG concentrations as
high as 2 µM TG (Fig. 6 and Table
IV).

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Fig. 6.
Diagram of chimeric exchanges of SERCA
segments with corresponding Na+,K+-ATPase
segments. Diagram A shows the large cytosolic region
replacement described by Sumbilla et al. (15). Diagram
B represents the S3,M3 exchange reported by Norregaard et
al. (1). Diagrams C-E are S3, S4, and M3 chimeric
proteins, respectively, obtained by site-directed mutagenesis in our
experiments. Diagram F represents the localization of
radioactive azido-TG photolabel reported by Hua and Inesi (2).
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Table IV
Characteristics of the enzyme and chimeric constructs shown in Fig. 6
Characterization of chimera A was obtained from Sumbilla et
al. (15), B from Norregaard et al. (1), and C, D, and E
from the studies reported here. Diagram F was derived from Hua and
Inesi (2).
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In our experiments, we produced small chimeric changes by stepwise
site-directed mutations in the S3 stalk segment and studied the
so-derived SERCA mutants with regard to their ability to sustain Ca2+ transport and hydrolytic activity, and their
sensitivity to TG. Mutations of up to six amino acids in the S3 stalk
segment yield an enzyme retaining ample Ca2+ transport and
ATPase activities for studies of inhibition by TG. We then found that
small chimeric mutations of the S3 segment (Fig. 6 and Table IV)
produce a marked reduction of the ATPase sensitivity to TG. Single
mutation of Gly257
Ile is, by itself, very effective.
It should be noted that, given higher TG concentrations, the full
inhibitory effect is obtained in all cases. This indicates that
chimeric mutations in the S3 segment reduce the ATPase affinity for TG,
but do not interfere with the inhibitory mechanism.
It is of interest that the SERCA S4 segment includes already, in its
native structure, a very high and unique degree of structural homology
with the Na+,K+-ATPase as well as other cation
ATPases. Such a localized homology suggests that this segment may serve
as a common structural device for long range functional linkage of the
catalytic site in the extramembranous region, and the cation binding
site in the membrane-bound region (18, 23). On the other hand, such a
high degree of homology between the TG-sensitive SERCA and other
TG-insensitive ATPases suggests that S4 segment is not involved in the
enzyme interaction with TG. In fact, we found that a five-amino acid mutation, which (due to additional native homology) yields a 25-amino acid segment identical to that of the
Na+,K+-ATPase, does not interfere at all with
the concentration dependence of TG inhibition (Figs. 5 and 6).
Therefore, the lack of effect of larger chimeric mutations in the S4
segment underlines the topological specificity of the rather limited S3
chimeric mutations that interfere with ATPase sensitivity to TG.
Mutations in the transmembrane M3 segment are also ineffective.
Our experiments demonstrate that the S3 stalk segment is specifically
involved in determining the ATPase sensitivity to TG and suggest that
the S3 segment is involved in TG binding. That the reduced sensitivity
may be due to long range effects of the S3 mutations is unlikely,
because extensive chimeric exchanges in other regions (Fig. 6 and Table
IV) do not affect the ATPase sensitivity to TG (1, 15). It is possible
that specific interaction of TG with the S3 segment, in addition to
stacking of the TG ring structure at the membrane interface (24),
determines the high affinity of the inhibitor for the ATPase.