From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Single mutations of specific amino acids within
the membrane-bound region of the sarco-endoplasmic reticulum
Ca2+ (SERCA)-1 ATPase interfere with Ca2+
inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two
Ca2+ that are known to bind to the enzyme. However, direct
measurements of Ca2+ binding in the absence of ATP have
been limited by the low quantities of available mutant protein. We have
improved the transfection efficiency by means of recombinant adenovirus
vectors, yielding sufficient expression of wild type and mutant SERCA-1
ATPase for measurements of Ca2+ binding to the microsomal
fraction of the transfected cells. We find that in the presence of 20 µM Ca2+ and in the absence of ATP, the
Glu771 Gln, Thr799
Ala,
Asp800
Asn, and Glu908
Ala mutants
exhibit negligible binding, indicating that the oxygen functions of
Glu771, Thr799, Asp800, and
Glu908 are involved in interactions whose single disruption
causes major changes in the highly cooperative "duplex" binding.
Total loss of Ca2+ binding is accompanied by loss of
Ca2+ inhibition of the Pi reaction. We also
find that, at pH 7.0, the Glu309
Gln and the
Asn796
Ala mutants bind approximately half as much
Ca2+ as the wild type ATPase and do not interfere with
Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309
Gln mutant does not bind any
Ca2+, and its phosphorylation by Pi
is not inhibited by Ca2+. On the contrary, the
Asn796
Ala mutant retains the behavior displayed at pH
7.0. This suggests that in the Glu309
Gln mutant,
ionization of acidic functions in other amino acids (e.g.
Glu771 and Asp800) occurs as the pH is shifted,
thereby rendering Ca2+ binding possible. In the
Asn796
Ala mutant, on the other hand, the
Glu309 carboxylic function allows binding of inhibitory
Ca2+ even at pH 6.2. In all cases mutational interference
with the inhibition of the Pi reaction by Ca2+
can be overcome by raising the Ca2+ concentration to the
mM range, consistent with a general effect of mutations on
the affinity of the ATPase for Ca2+.
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INTRODUCTION |
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Activation of the sarco-endoplasmic reticulum
Ca2+ (SERCA)1
ATPase requires binding of Ca2+, which is then
moved across the membrane upon utilization of ATP. Equilibrium binding
isotherms, obtained with sarcoplasmic reticulum (SR) vesicles as an
abundant source of enzyme, demonstrate that in the absence of ATP, two
Ca2+/ATPase bind cooperatively with Ka = 5 × 1012 M2 (2). As for the
topology of Ca2+ binding within the ATPase molecule,
involvement of six amino acid residues (Glu309,
Glu771, Asn796, Thr799,
Asp800, and Glu908) within four clustered
transmembrane helices (M4, M5, M6, and M8) was suggested by mutational
analysis (1). The low yield of recombinant enzymes, however, has
limited direct measurements of Ca2+ binding to the
pertinent mutants (with the exception of the Glu309
Gln
mutant; Skerjank et al. (3)). In fact, involvement of the
six amino acids in Ca2+ binding was suggested by their
mutations interfering with the inhibitory effect of Ca2+ on
enzyme phosphorylation by Pi. The shortcoming of such
experiments, however, was that they did not distinguish direct effects
of mutations on Ca2+ binding from mutational effects on
transmission of the Ca2+ binding signal within the ATPase
protein.
Alternative studies were performed to test whether the pertinent mutants retain the ability to occlude Ca2+ following addition of Cr-ATP (4). In this case, however, there is some degree of uncertainty as to whether Cr-ATP may affect Ca2+ binding by stabilizing the enzyme in a state analogous to that of the phosphorylated intermediate (i.e. "E2"). Other studies have utilized the effect of Ca2+ on the susceptibility of wild type and mutated enzyme to proteolytic digestion (5).
We have now improved the efficiency of ATPase gene transfer into COS-1 cells by using recombinant adenovirus vectors. The resulting expression yields sufficient amounts of protein for direct measurements of Ca2+ binding in the absence of nucleotide substrate, using native microsomal vesicles as the source of enzyme. We describe here our recombinant adenovirus constructs, the characteristics of ATPase overexpression in COS-1 cells under control of the SV40 promoter, the direct measurements of Ca2+ binding by wild type and mutated enzyme, and related tests of Ca2+ inhibition of the enzyme reaction with Pi.
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MATERIALS AND METHODS |
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DNA Constructs and Vectors-- The chicken fast muscle SR (SERCA-1) ATPase cDNA (6) was inserted into the pUC19 plasmid for amplification and then subcloned into the pSELECT-1 vector for site-directed mutagenesis by the Altered Sites In Vitro Mutagenesis System made available by Promega (Madison, WI) or by overlap extension using polymerase chain reaction (7). Eleven unique restriction sites were introduced into the SERCA-1 cDNA, retaining the original coding sequence. These sites are spaced at approximately equal intervals and facilitate further mutagenesis by generating cassettes of approximately three hundred bases that can be conveniently sequenced following mutagenesis and exchanged with the corresponding cassette in the wild type cDNA. Furthermore a c-myc tag was added to the 3' end to monitor ATPase expression using anti-c-myc antibodies, independently of mutations in the enzyme sequence.
Site-directed mutations were produced in the SERCA-1 cDNA by using the Altered Sites In Vitro Mutagenesis System (Promega) or by overlap extension using the polymerase chain reaction (7). Enhanced green fluorescence protein (EGFP) cDNA (containing the Phe64Cell Cultures and Transfections-- Cultures of HEK293 and COS-1 cells were maintained as described by Graham and Prevec (9) and Sumbilla et al. (10), respectively. The growth medium for COS-1 cells was Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and containing Penn-Strep (100 units/ml) and Fungizone (1 µg/ml).
Transfections of COS-1 cells with wild type or mutated SERCA-1 cDNA or EGFP cDNA subcloned into the shuttle vector pImmunostaining-- Lawns of cultured cells were first washed with PBS and then fixed with 4% formaldehyde for 10 min. After repeated washings with PBS, blocking of nonspecific sites was obtained by a 10-min incubation with 1% bovine serum albumin and 0.5% lysine in PBS. This was followed by a 45-min incubation with the primary antibody at a concentration of 5-10 µg/ml of PBS containing 1% albumin, 0.5% lysine, and 0.25% saponin (permeabilization medium). After washing with PBS, the cells were incubated for 45 min with biotinylated anti-mouse secondary antibody (Vector Laboratory, Burlingame, CA) at a concentration of 5 µg/ml of permeabilization medium. The cells were then washed with PBS and incubated for 20 min with Fluorescein Streptavidin (Amersham Pharmacia Biotech) at a concentration of 5 µg/ml of permeabilization medium. The sample was then washed again with PBS, 70% EtOH, and 90% EtOH, allowed to dry, and processed for fluorescence microscopy using a Zeiss Axioskop equipped with a mercury vapor lamp and fluorescence accessories.
Microsome Preparation and Immunodetection of Expressed
Protein--
The procedure for microsome preparation was as described
by Autry and Jones (11), and the final product was stored in small aliquots at 70 °C. The total microsomal protein was determined using bicinchoninic acid assay (Pierce). The expressed SERCA-1 ATPase
was detected by Western blotting. For this purpose, microsomal protein
were separated in 7.5% Laemmli (12) electrophoretic gels blotted onto
nitrocellulose paper. This was then incubated with a monoclonal
antibody (CaF3-5C3) to the chicken SERCA-1 (6) and in parallel with a
monoclonal antibody (9E10) to the c-myc epitope (13). After
incubation with secondary antibody (goat anti-mouse IgG-horseradish
peroxidase-conjugated), the bound proteins were probed using an
Enhanced Chemiluminescence-linked detection system (Amersham Pharmacia
Biotech). Quantitation of immunoreactivity was obtained by densitometry
and standardized with samples of the wild type ATPase used as controls
for the functional studies.
Functional Studies-- Studies of Ca2+ transport and phosphoenzyme formation with [32P]phosphate were carried out as described by Inesi et al. (14) and detected by autoradiography. ATPase hydrolytic activity was assessed by measuring Pi production (15).
Ca2+ binding to the expressed ATPase in the absence of ATP was measured by a filtration method. The equilibration mixture contained 20 mM MOPS, pH 6.8, 80 mM KCl, 3 mM MgCl2, 20 µM [45Ca]CaCl2 (including endogenous Ca2+), and 400 µg microsomal protein/ml. Endogenous Ca2+ in the equilibration medium was determined by titrating with EGTA in the presence of 50 µM Arsenazo and recording differential light absorption changes (660 and 687 nm wavelengths) with a DW-2000 SLM-Aminco spectrophotometer. Controls for nonspecific binding were conducted with microsomes preincubated with thapsigargin (6.75 µg/mg protein), and the binding observed with the inhibited microsomes was subtracted from the total binding obtained with noninhibited microsomes to yield "specific" Ca2+ binding. The Ca2+ binding assay was started by adding either 2.7 µg of thapsigargin in 2 µl of Me2SO or 2 µl of Me2SO alone to approximately 400 µg of microsomal protein in 50 µl of medium. After a brief incubation at room temperature, the samples were kept on ice for 10 min, and then 1.0 ml of binding medium was added. Following a 10-min incubation on ice, 0.75 ml was placed on a Millipore filter (HAWP 0.65 µm, 25-mm diameter) under suction for approximately 30 s. The vacuum was then turned off, and the filter was blotted on a paper towel to remove excess moisture, folded, and inserted into a 7-ml scintillation vial. The filters were dissolved with 1 ml of N,N-dimethylformamide, scintillation fluid was added, and the radioactivity was measured by scintillation counting. The measured Ca2+ binding levels were finally adjusted to compensate for slight variations of SERCA-1 expression in various preparations, with reference to a wild type preparation as indicated by Western blots. The thapsigargin-sensitive (i.e. specific) binding accounted for approximately 25% of the total Ca2+ binding by microsomes obtained from cells expressing wild type SERCA. It is known that thapsigargin is a global SERCA inhibitor (16), and we established that at the concentration used thapsigargin inhibited the Pi reaction in the wild type enzyme and in all mutants studied. ![]() |
RESULTS |
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Vector Efficiency and Transgenic Expression-- A preliminary assessment of adenovirus vector efficiency in COS-1 cells was obtained by microscopic visualization of the number of COS-1 cells expressing EGFP. Fig. 1 (A and B) shows phase contrast and fluorescent images of COS-1 cells following the DEAE-dextran transfection procedure. A comparison of the two images shows that only a few cells express EGFP. On the other hand, in Fig. 1C, nearly all cells express EGFP following infection by recombinant EGFP adenovirus (40 pfu/cell). A quantitative assessment of the percentage of cells expressing EGFP as a function of adenovirus concentration is given in Fig. 2. An asymptotic level of 97-100% transfection efficiency was obtained at 100 pfu/seeded cell, although the amount of expressed protein continued to rise as the number of gene copies introduced per cell was further increased.
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ATP-dependent Ca2+ Transport by Wild Type ATPase-- ATP-dependent Ca2+ transport is a highly specific functional parameter of SERCA that can be conveniently measured by following the uptake of radioactive calcium tracer by microsomal vesicles. Microsomes obtained from control COS-1 cells do not exhibit a significant rate of Ca2+ transport (Fig. 3). On the other hand, we found that under optimal conditions, the rate of Ca2+ transport by microsomes obtained from cells infected with the SERCA-1 adenovirus vector (100 pfu/cell) was approximately 10-fold higher than that of microsomes obtained from cells transfected by the DEAE-dextran method (Fig. 3). Conversely, their transport activity was approximately 10-fold lower than the rate of Ca2+ transport by native SR vesicles obtained from rabbit skeletal muscle (data not shown). Because SERCA-1 accounts for approximately 50% of the total protein in SR vesicles, it is apparent that transgenic SERCA-1 accounts for approximately 5.0% of the total microsomal protein obtained from COS-1 cells infected with recombinant adenovirus under our conditions. Similar conclusions were reached by comparative measurements of Ca2+-dependent ATPase activity (not shown). They are also consistent with Western blots showing no SERCA in control samples (Fig. 3, inset, lane 1) and much greater amounts of recombinant SERCA in microsomes obtained from cells infected with adenovirus (Fig. 3, inset, lanes 4 and 5) as compared with microsomes obtained from cells transfected by the DEAE-dextran method (Fig. 3, inset, lanes 2 and 3).
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ATP-independent Ca2+ Binding by Wild Type ATPase-- Ca2+ binding in the absence of ATP may be considered as the initial step of a single catalytic and transport cycle. In fact it is well known that Ca2+ binding to the ATPase is required to activate the enzyme before ATP can be utilized. The bound calcium is then displaced vectorially and released against a concentration gradient upon enzyme phosphorylation by ATP. The cycle is finally completed by hydrolytic cleavage of the phosphorylated enzyme intermediate.
We have previously used chromatography equilibration columns to measure Ca2+ binding to SR ATPase under equilibrium conditions, in the absence of ATP. The resulting binding isotherms demonstrate that two Ca2+ bind cooperatively to each ATPase with Ka = 5 × 1012 M
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Expression and Characterization of Mutants--
We observed in our
Western blots only slight variations in the levels of SERCA-1 protein
recovered in the microsomal fraction of COS-1 cells expressing the
various SERCA mutants under the same conditions (Fig.
5). We obtained densitometric evaluations of the electrophoretic bands to relate the functional parameters measured for each mutant to a corresponding quantity of wild type SERCA-1. We found, however, that the Glu309 Gln,
Asp771
Asn, Asn796
Ala,
Thr799
Ala, Asp800
Asn, and
Glu908
Ala mutants do not sustain significant rates of
either ATP hydrolysis or coupled Ca2+ transport (see also
Clarke et al. (1)). Furthermore, formation of phosphorylated
intermediate through Ca2+-dependent ATP
utilization is totally inhibited in these mutants. On the other hand,
phosphoenzyme formation by utilization of Pi still occurs
at normal levels (see below).
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Ca2+ Binding and Phosphorylation with Pi in
ATPase Mutants--
The main aim of our studies was to measure
Ca2+ binding by the ATPase mutants, taking advantage of
their overexpression in COS-1 cells infected with the adenovirus
vectors. In fact, we were able to unambiguously demonstrate specific
defects of Ca2+ binding in these mutants. It is shown in
Table I that at neutral pH and in the
presence of 20 µM Ca2+, some of the mutants
(i.e. Glu771 Gln, Thr799
Ala, Asp800
Asn, and Glu908
Ala)
displayed no significant binding, whereas others (i.e. Glu309
Gln and Asn796
Ala) bind
approximately half as much Ca2+ as the wild type enzyme
(Fig. 6). A 50% reduction of
Ca2+ binding by the Glu309
Gln mutation was
also noted by Skerjanc et al. (3). Interestingly, we found
that if the pH is reduced to 6.2, Ca2+ binding by the
Glu309
Gln mutant becomes negligible (0.02 ± 0.24 nmol/mg protein), whereas the Asn796
Ala mutant binds
approximately the same level of Ca2+ (0.44 ± 0.39 nmol/mg protein) as at neutral pH.
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DISCUSSION |
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Equilibrium and kinetic experiments have demonstrated that in the absence of ATP, SERCA binds Ca2+ with a stoichiometry of two Ca2+/enzyme. Binding occurs with a sequential and cooperative mechanism (2, 19, 20) whereby the enzyme is stabilized in the "E1" state. An important discovery for the understanding of the Ca2+ binding topology within the SERCA molecule was achieved when single mutations of specific residues in the transmembrane domain were found to interfere with Ca2+ inhibition of enzyme phosphorylation by Pi (1). Based on this interference, it was proposed that the six residues participate in Ca2+ complexation within the transmembrane domain (1). In fact it was shown by molecular modeling (21) that within the constraints imposed by the positions of these residues on four transmembrane helices, it is possible to arrange their oxygen functions to form a duplex calcium binding site with distribution and distances compatible with other duplex sites of known crystal structure, such as that of thermolysin (22).
Several attempts have been made to assign the oxygen functions of the
six amino acids implicated in binding to either calcium ion of the
duplex complex (21, 23, 24). Additional amino acids within a
neighboring cytosolic segment have been also implicated (5). However, a
shortcoming of this work has been the lack of direct Ca2+
binding measurements with the mutants, because of the low quantities of
recombinant protein. The only mutant tested for binding was Glu309 Gln expressed in insect cells infected with
recombinant baculovirus (3). This measurement required solubilization
of the membrane-bound enzyme, purification by affinity chromatography,
and reconstitution in liposomes, leaving some uncertainty on the
detergent effect on binding and the orientation of the reconstituted
enzyme in the liposomal membrane. Alternatively, "Ca2+
occlusion" was measured in the presence of Cr-ATP (4), which stabilizes the enzyme in a state similar to that obtained by enzyme phosphorylation with ATP. An advantage of our present experiments is
that Ca2+ binding was measured in the absence of ATP under
equilibrium conditions yielding strictly the E1 state. Another
advantage is the use of wild type and mutant proteins assembled in
native microsomal membrane with no need for detergent
solubilization.
Our measurements demonstrate that the Glu771 Gln,
Asp800
Asn, Thr799
Ala, and
Glu908
Ala mutations result in total loss of
Ca2+ binding in the presence of 20 µM
Ca2+ (Table I). The strong inhibition of Ca2+
binding in the first two mutants may be explained by considering that
Glu771 and Asp800 could contribute
electronegativity to both calcium ions, each donating its carboxyl
oxygen to one calcium and its carbonyl oxygen to the other. Yet, it is
remarkable that mutation of a single one of the acidic amino acids to
its corresponding amide (thereby leaving the carbonyl oxygen in place)
results in interference with binding of both calcium ions and with
inhibition of the Pi reaction by Ca2+ (Table
I).
The strong inhibition of Ca2+ binding by the
Thr799 Ala and Glu908
Ala mutations is
also remarkable, because Thr can contribute only one hydroxyl group to
the coordination complex. Furthermore, Glu908 appears to
contribute only a carbonyl group because the Glu908
Gln
mutation does not have functional consequences (18, 25).
It is then apparent that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 provide important stabilization to the cooperative Ca2+ complex, either by direct interaction with Ca2+ or by participation in hydrogen bonding with water or peptide amide functions, so much so that mutational interference with a single one of these functions results in major disruption of the duplex binding site. These effects, however, can be overcome by increasing the Ca2+ concentration (18), indicating that mutational disruption affects the affinity of the enzyme for Ca2+.
It is noteworthy that our finding of strong binding inhibition in the
Glu908 Ala mutant in the absence of ATP
(i.e. E1 state) is in apparent contrast with the
Ca2+ occlusion by the same mutant in the presence of Cr-ATP
(4, 23). This may be explained by the high concentration of
Ca2+ used in these experiments (23).
Although we observed strong inhibition of Ca2+ binding with
the mutants mentioned above we found that in the presence of 20 µM Ca2+ and pH 7.0, the Glu309
Gln and Asn796
Ala mutations result in reduction of
Ca2+ binding to approximately half the level observed with
wild type enzyme (Fig. 6). At pH 6.2, on the other hand, the
Glu309
Gln mutant exhibits no significant
Ca2+ binding, whereas the Asn796
Ala mutant
retains the same binding as at neutral pH. This suggests that
ionization of acidic functions of other amino acids (e.g.
Glu771 or Asp800) occurs as the pH is shifted
from 6.2 to 7.0, thereby facilitating Ca2+ binding in the
Glu309
Gln mutant. On the other hand, in the
Asn796
Ala mutant, the presence of the
Glu309 acidic function allows binding of inhibitory
Ca2+ even at pH 6.2.
The lack of Ca2+ inhibition of the Pi reaction
in the mutants allowing no Ca2+ binding is well
understandable. On the other hand, the strong inhibition of the
Pi reaction in mutants permitting binding of half the
normal Ca2+ level suggests that (a) these mutant
molecules bind only one of two Ca2+ known to bind to the
wild type enzyme and (b) the single bound Ca2+
is sufficient to inhibit the Pi reaction. Experimental
demonstration of the inhibition of the Pi reaction by
single Ca2+ binding is quite satisfactory, because it is
observed in both Asn796 Ala and Glu309
Gln mutants at pH 7.0 and only in the former mutant at pH 6.2.
That a single Ca2+ may be sufficient to inhibit the
Pi reaction was previously suggested based upon the effects
of sequential (and negatively cooperative) binding of strontium to
sarcoplasmic reticulum ATPase (26) and on different effects of various
mutations on the inhibition of the Pi reaction by
Ca2+ (23). Our direct measurements of Ca2+
binding at pH 7.0 and 6.2 in parallel with phosphorylation experiments confirm that in the Asn796 Ala and Glu309
Gln mutants, at suitable Ca2+ and H+
concentrations, a single Ca2+ is sufficient to inhibit the
Pi reaction. This would be unlikely to occur in the wild
type enzyme, because of the highly cooperative character of
Ca2+ binding.
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FOOTNOTES |
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* This work was supported by Grant P01HL-27867 and Training Grant 5-T32-AR07592 from the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, UMB, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-3220; Fax: 410-706-8297; E-mail: ginesi{at}umaryland.edu.
1 The abbreviations used are: SERCA, sarco-endoplasmic reticulum Ca2+; SR, sarcoplasmic reticulum; Cr-ATP, chromium ATP; EGFP, enhanced green fluorescence protein; pfu, plaque-forming unit; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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