From the Section de Biophysique des Protéines
et des Membranes, DBCM, Commissariat à l'Energie Atomique et
CNRS URA 2096, CE Saclay, 91191 Gif sur Yvette Cedex, France and the
** Danish Biomembrane Research Centre, Department of Biophysics,
University of Aarhus, DK-8000 Aarhus C, Denmark
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sarcoplasmic reticulum (SR)1 Ca2+-ATPase belongs to the family of P-type cation-transporting ATPases that transport cations by an active mechanism involving the formation of a phosphorylated intermediate (1-4). Members of this large family have been classified as type I or type II ATPases, corresponding to proteins transporting, respectively, heavy metals or cations such as H+, Na+, K+, or Ca2+ (5). Ca2+-ATPase in mature skeletal muscle contains 994 residues, as deduced from cloning and sequencing of the SERCA1a gene by MacLennan and co-workers (6). According to a topological model based on both sequence-derived predictions and experimental protein-chemical data, 70% of the polypeptide consists of two cytosolic domains connected to the membrane-embedded part by a stalk of putative helices. The membranous part comprises about 20% of the residues, arranged in 10 putative transmembrane spans, M1-10, with 10% additional residues that could form a small luminal domain. Structural three-dimensional data, obtained to date only at low resolution, have confirmed this description of the general organization of the protein (7), but the detailed structure of the Ca2+-ATPase remains an open question (8, 9).
During Ca2+ transport, Ca2+-ATPase probably undergoes several conformational changes, although only two main states were initially considered (1), which denote forms with a high and low affinity for Ca2+, respectively (see also Refs. 9 and 10). Binding of cytosolic Ca2+ at the Ca2+ activating and transport sites of the ATPase was measured directly under equilibrium conditions and found to occur with a stoichiometry of 2:1 and a positive cooperativity, which was interpreted on the basis of a sequential mechanism (11). It has been proposed to occur in a multistep process accompanied by conformational changes. Evidence has been presented that binding of only one Ca2+ is sufficient to prevent phosphorylation from Pi, while the binding of the second Ca2+ is required to allow subsequent phosphorylation from ATP (12). Mainly based on the kinetics of Ca2+ dissociation, Ca2+-binding sites have been suggested to be arranged in a channel-like structure including two sites (13-15). After phosphorylation with ATP, the Ca2+ ions can initially no longer dissociate from the high energy phosphoenzyme formed and are found in what has been described as an "occluded" state (16, 17). The Ca2+ ions are subsequently released toward the SR lumen because of reorganization of both sites and loss of their affinity for Ca2+ (18-20). It has been shown that after this stage, protons (or hydronium ions) may combine with and be countertransported by the enzyme (21, 22). Chemical and/or genetic modification of the polypeptide chain has allowed us to examine the functional role of different domains (for reviews, see Refs. 5, 10, 23, and 24). Residues critical for Ca2+ binding and transport were not found in the stalk sector, despite the fact that this region has a large content of acid residues (25). Instead, such residues are clustered in the transmembrane domain, in M4 (Glu-309), M5 (Glu-771), M6 (Asn-796, Thr-799, and Asp-800) and M8 (Glu-908) (26). Further experiments showed that the five residues in M4, M5, and M6 were also critical for occlusion in the presence of Cr-ATP (27), but not Glu-908, which was therefore considered to be involved in the initial recognition of the Ca2+ ions but not in its final intramembranous binding (10, 28).
In addition to the molecular biology approach, limited proteolysis of the SR Ca2+-ATPase has also been developed for identification of regions critical for Ca2+ activation of the ATPase (29-31). Experiments showed that the rate of electrophoretic migration of a small C-terminal ATPase peptide, p20C, was sensitive to Ca2+, while a slightly shorter peptide, p19C, was not. We found that p20C starts at Gly-808, at the beginning of the loop connecting putative transmembrane spans 6 and 7, while p19C starts at Asp-818, in the middle of loop. This led us to suggest that the L6-7 loop might interact with Ca2+ (32). The N-terminal part of the loop contains three aspartic residues (Asp-813, -815, and -818), which were mutated in a cluster, D813A/D818A and D813A/D815A/D818A. After expression in yeast, we found that these mutants had a low Ca2+-ATPase activity (32). It is noteworthy that in gastric H+,K+-ATPase, residues Glu-837 and Asp-839, which correspond to Asp-813 and Asp-815 in Ca2+-ATPase, were found to render the ATPase unphosphorylatable by ATP when mutated to glutamine and asparagine residues, respectively (33).
In the present work, we have carried out more detailed experiments to investigate the role of the L6-7 loop in Ca2+-ATPase. The experiments were designed to characterize the functional consequences of mutations of aspartic residues, D813A/D818A and D813A/D815A/D818A, as well as those of mutations of the proline residues in the same loop, Pro-811, -812, -820, and -821. We found that cluster mutations of aspartate residues led to a clear reduction in the apparent affinity with which Ca2+ controlled ATPase phosphorylation or dephosphorylation. Although Asp to Ala mutations are often thought of as nonconservative, susceptibility to proteolytic digestion suggested that our mutation of the L6-7 loop did not cause any major conformational change. Thus, the present findings are consistent with an interaction of the aspartic residues of the L6-7 loop with the first Ca2+ ion that binds to Ca2+-ATPase during the transport process and suggest that the L6-7 loop contributes to the control of the activation of Ca2+-ATPase by Ca2+ during the initial steps of Ca2+ binding. A model is proposed describing one possible mechanism by which such a control might occur.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutation and Expression of Ca2+-ATPase in Yeast-- The single mutants E309Q and E771Q and the cluster mutants D813A/D818A (referred to later as ADA) and D813A/D815A/D818A (referred to later as AAA) were obtained as in Ref. 32. Using Ca2+-ATPase SERCA1a cDNA (34), single mutants P812A and P821A and cluster mutants P811A/P812A and P820A/P821A were obtained by site-directed mutagenesis performed with the pAlter kit (Promega). The presence of the desired mutations and the absence of unexpected ones resulting from the polymerase chain reaction were verified by DNA sequencing. Wild type and mutant DNA were inserted into a yeast expression vector and expressed as described previously (32). A light membrane fraction obtained by differential centrifugation was prepared for each mutated ATPase and stored at 10 mg of proteins/ml in 10 mM Hepes, pH 7.4 (Tris), 0.3 M sucrose, and 0.1 mM CaCl2, as described in Ref. 32. Protein concentrations were measured by the bicinchoninic assay (35) in the presence of 0.5% SDS. The amount of Ca2+-ATPase was quantified by Western blotting as in Ref. 34, using the polyclonal antibody 577-588 (31) and a GS-700 imaging densitometer with Molecular Analyst software (Bio-Rad); for reference, we used native SR membranes in which 75% of the protein content is assumed to be Ca2+-ATPase. Typically, light membranes expressed wild type or mutant Ca2+-ATPase at about 0.75 mg of Ca2+-ATPase/100 mg of membrane proteins.
ATPase Assay-- ATP hydrolysis was assayed spectrophotometrically at 30 °C as in Ref. 32, generally with 50 µg of yeast light membranes/ml in the assay and with a final Ca2+ concentration of 0.1 mM. The reaction was started by the addition of 1 mM Na2ATP. Thapsigargin (1 µg/ml; see Ref. 41) was added after 100 s, and the rate of hydrolysis was followed for an additional 100 s. The Ca2+-ATPase activity was calculated as the difference between the slopes obtained in the presence and in the absence of thapsigargin and was corrected for the (very small) background of thapsigargin-dependent activity obtained with light membranes of control yeasts.
Ca2+ Dependence of Ca2+-ATPase
Phosphorylation from [-32P]ATP, as Measured by a
Filtration Method--
Ca2+-ATPase phosphorylation from
[
-32P]ATP was carried out in an ice-cold buffer
containing 20 mM MOPS-Tris, pH 7, 100 mM KCl, 5 mM MgCl2, and various free Ca2+
concentrations, as indicated in Figs. 3 and 4. Final pCa
values (obtained by mixing Ca2+ with various amounts of
EGTA) were calculated using Maxchelator software, taking into account
the endogenous Ca2+. Five mM NaN3,
0.05 µg/ml bafilomycine A, and 0.1 mM ammonium molybdate
were also included in the assay medium to inhibit other ATPases. Fifty
µg of light yeast membrane proteins were added to a final volume of
50 µl. The reaction was started by adding 2 µM
[
-32P]ATP (1 Ci/mmol) and quenched after 15 s by
adding 2 ml of cold quenching solution containing 150 mM
perchloric acid and 15 mM NaH2PO4.
Acid-denatured proteins were retained on a glass fiber filter (Gelman,
A/E type) and washed five times with 4 ml of quenching solution (36).
The radioactivity in the filter was then counted in 4 ml of
scintillation liquid (Packard, Filter-Count). A filter containing 100 pmol of [
-32P]ATP was used as a standard, to convert
measured cpm to pmol of 32P.
Ca2+ Dependence of Ca2+-ATPase
Phosphorylation from [-32P]ATP, as Measured after
Electrophoretic Separation--
Ca2+-ATPase
phosphorylation was carried out as in the case of the filtration
experiments except that proteins were diluted 10 times more
(i.e. into a final volume of 500 µl with the same buffer), and the various free Ca2+ concentrations were established
more reliably, by mixing Ca2+ with EGTA and MgEDTA (37). In
this case, the reaction was stopped by adding 1 ml of a cold solution
containing 9% trichloroacetic acid and 27 mM
NaH2PO4. Acid-denatured proteins were treated
according to Ref. 38 and sedimented by centrifugation for 15 min at
18,000 rpm (25,000 × gav) at 4 °C. The
pellet was neutralized by the addition of 1 µl of 1M
Tris-base and then suspended in 50 µl of 150 mM Tris-Cl,
pH 6.8, 10 mM EDTA, 2% SDS, 16% glycerol (v/v), 0.04%
bromphenol blue (v/v), and 0.84 M
-mercaptoethanol.
After 10 min at room temperature, proteins corresponding to 150 ng of Ca2+-ATPase were loaded on 7% polyacrylamide gels and run
for 90 min at 120 V in 170 mM MOPS-Tris, pH 6.0, 0.1% SDS.
After electrophoresis, the gels were fixed in 45% methanol and 10%
acetic acid for 25 min and then dried overnight between two sheets of
cellophane paper. Radioactivity was revealed with a PhosphorImager
(Molecular Dynamics, Inc.) and a Biomax film (Amersham Pharmacia
Biotech) and quantified by comparison with known amounts of
[
-32P]ATP.
Ca2+ Dependence of Ca2+-ATPase
Phosphorylation from
[32P]Pi--
Phosphorylation from
Pi was assayed both at pH 6.0 and at pH 7.0. The reaction
was carried out at 20 °C in a total volume of 500 µl of 100 mM MES-Tris, pH 6.0, or 100 mM MOPS-Tris, pH 7.0, 5 mM MgCl2, 20% dimethyl sulfoxide, 5 mM NaN3, 0.05 µg/ml bafilomycine A, and 0.1 mM ammonium molybdate, also containing various mixtures of
Ca2+ and EGTA or MgEDTA to obtain the free Ca2+
concentrations indicated in Figs. 5 and 6. The reaction was started by
adding 50 µg of light membranes (corresponding to 375 ng of Ca2+-ATPase) and 0.1 mM
[32P]Pi and stopped after 15 s by the
addition of 2 ml of cold quenching medium (9% trichloroacetic acid, 27 mM NaH2PO4). Quantification of
phosphoenzyme formed was carried out by the electrophoretic method as
described above for phosphorylation from
[-32P]ATP.
Proteolysis, Electrophoresis, and Blotting-- Proteinase K digestion of wild type and mutated Ca2+-ATPases was carried out in 100 mM Tes-Tris, pH 7.0, 5 mM MgCl2, at various free Ca2+ concentrations. For each membrane sample, containing either wild type or mutated ATPase, 20 µg of proteins were rapidly thawed and diluted in 200 µl of proteolysis buffer. Proteolysis was started by the addition of 0.36 µg of proteinase K and was performed for 30 min at 20 °C. The reaction was stopped by the addition of 1 mM phenylmethylsulfonyl fluoride, and samples were left on ice for 10 min and then centrifuged at 75,000 rpm (200,000 × gav) for 60 min at 4 °C in a TLA100 rotor and a Beckman TL100 ultracentrifuge. The pellet was suspended in 42 µl of urea-SDS denaturing buffer (39) and heated for 1 min at 100 °C, and aliquots corresponding to 150 ng of Ca2+-ATPase were loaded on a 11.4% polyacrylamide gel containing 1 mM Ca2+. After electrophoresis, proteins were transferred to PVDF membranes for immunodetection (31). Peptides were first immunodetected with polyclonal 79B Ab and then revealed by an ECL kit (Amersham Pharmacia Biotech). Polyclonal 79B Ab is a kind gift of A-M. Lompré (40); it reacts with a main epitope located in the N-terminal portion of the Ca2+-ATPase, as previously discussed for 78(7) Ab in Ref. 31. The membrane was stripped, and peptides were then immunodetected with 577-588 Ab (31).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Choice of Mutations in the L6-7 Loop--
The primary structure
of the L6-7 loop is shown in Fig. 1. The
N-terminal region of the loop, which is highly conserved among SERCA
ATPases, presents two remarkable features; it contains three aspartic
residues, Asp-813, Asp-815, and Asp-818 (note that conservative replacements with Glu or Asn residues are found for Asp-818 among SERCA
ATPases) (triangles in Fig. 1), and these residues are
located between two couples of proline residues, Pro-811-Pro-812 and
Pro-820-Pro-821 (squares in Fig. 1). We initially wondered
whether the acidic residues could be part of an initial
Ca2+-binding site, located at the membrane-cytosol
interface and structured by the proline residues. Therefore, these
residues were mutated to alanine residues to give the following
mutants: D813A/D818A (referred to as ADA), D813A/D815A/D818A (referred
to as AAA), P811A/P812A, P812A, P820A/P821A, and P821A. Cluster
replacement of the negatively charged Asp residues by the small neutral
Ala residues was performed in preference to the a priori
more conservative Asp Asn or Asp
Glu substitutions with the aim
of suppressing all potential calcium-liganding oxygen atoms in the side
chain. A few single mutations (D813A and D815A) were also tested (see "Ca2+-ATPase Activities of Mutants").
|
Ca2+-ATPase Activities of Mutants-- Fig. 2 shows the overall ATPase activity of native, wild type, and mutant enzymes in the presence of Ca2+. Examples of typical traces are displayed in Fig. 2A, which shows ATP hydrolysis as measured by the rate of NADH oxidation recorded at 340 nm. In the presence of several ATPases inhibitors, even control membranes displayed measurable ATPase activity. However, this activity was virtually insensitive to thapsigargin, a specific inhibitor of SERCA ATPases (41), while the additional ATPase activity of expressed wild type Ca2+-ATPase (WT assay), or that of an equivalent amount of native SR Ca2+-ATPase added to control membranes (SR + control membrane assay) was specifically inhibited by thapsigargin.
|
Ca2+ Dependence of Phosphoenzyme
Formation--
Mutants were then tested for their ability to become
phosphorylated from [-32P]ATP, a reaction that
requires binding of two Ca2+ ions to the protein. These
experiments were first carried out by using a method based on
filtration of perchloric acid-precipitated protein (Fig.
3) and were subsequently confirmed using
a method based on electrophoretic separation of the phosphorylated
intermediate (Fig. 4).
|
|
Inhibition by Ca2+ of Phosphorylation from
[32P]Pi--
The filtration method used in
Fig. 3 could not be used to measure phosphorylation from
Pi, due to the fact that the Pi concentration that had to be used (100 µM) resulted in a much larger
background than in the experiments with [-32P]ATP (2 µM ATP). Thus, phosphorylation from Pi was
quantified by our second method only, i.e. after separation
on SDS-PAGE of the phosphorylated Ca2+-ATPase. The
experiments were carried out both at pH 6.0 and 7.0, under
conditions previously described (26-28, 49).
|
|
Ca2+-dependent Pattern of Proteolysis by Proteinase K of Wild Type and Mutated Ca2+-ATPases-- The above described phosphorylation experiments were supplemented with a completely different type of experiments, in which the ability of various mutants to bind Ca2+ was deduced from the Ca2+ dependence of their proteolysis pattern. Wild type Ca2+-ATPase as well as E309Q, E771Q, and ADA mutants were submitted to proteolytic attack by proteinase K in the presence of various Ca2+ concentrations at neutral pH. The fragments were separated by SDS-PAGE, followed by Western blot. Among the fragments produced, peptides p95 and p83C (see their locations in Fig. 7D) were recognized by immunodetection with 577-588 Ab (Fig. 7A), and peptide p28N was recognized with 79B Ab (Fig. 7B). As illustrated in Fig. 7D and found previously for native Ca2+-ATPase treated under similar conditions (31), p95 is produced by a proteolytic cleavage between residues Leu-119 and Lys-120, while p28N and p83C are both produced by a proteolytic cleavage between residues Thr-242 and Glu-243.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Functional Properties of the L6-7 Mutants-- The N-terminal side of the L6-7 loop in Ca2+-ATPase (see Fig. 1) is characterized by the presence of three aspartic residues, surrounded by two couples of proline residues. An important outcome of our investigation is that cluster mutations of the aspartic residues shifted the Ca2+-ATPase affinity for Ca2+ to much higher concentrations than those required to activate wild type ATPase; the K0.5 for Ca2+ activation of phosphorylation from ATP of the D813A/D818A mutated ATPase was shifted from the micromolar to the millimolar range, while the maximal level of phosphorylation remained the same as that of wild type ATPase (Figs. 3 and 4). It can therefore be concluded that the ADA mutant has an intact phosphorylation site and can still bind two Ca2+ ions, although at higher Ca2+ concentrations than WT ATPase. In agreement with this, the double and triple mutants D813A/D818A and D813A/D815A/D818A only displayed a very low ATPase activity when they were tested at pCa 4, (Fig. 2). Note that the single mutant D815A gave a fully active ATPase, while mutation of D813A alone resulted in only a moderate loss of activity of 56%.2
An unexpected feature of our phosphorylation experiments was that Ca2+ stimulation of phosphoenzyme formation from ATP by WT ATPase revealed two steps, separated by a plateau around pCa 5.5-3.5 (Figs. 3A and 4B). However, a comparable behavior has been observed previously for purified ATPase or leaky SR vesicles under similar conditions (see, for example, Fig. 7 in Ref. 45). Stimulation of phosphorylation by micromolar Ca2+ is caused by binding of Ca2+ to the high affinity transport sites, but saturation of these sites is not necessarily accompanied by complete phosphorylation, since the latter is dependent on the balance between the overall rates of phosphorylation and dephosphorylation. Increasing the Ca2+ concentration to millimolar values slows down the rate at which phosphoenzyme is hydrolyzed, presumably because of substitution of Ca2+ for Mg2+ at the catalytic site, and thereby increases the steady state level of phosphorylation (see Refs. 45-48). Conceivably, for the ADA mutant, the same substitution of Ca2+ for Mg2+ could also contribute to the rise of the steady state EP level at millimolar Ca2+ concentrations. Consequently, the "apparent" affinity for Ca2+ of the ADA mutant deduced from the phosphorylation data probably represents a "mixed" affinity, partly reflecting the usual exchange of Mg2+ for Ca2+ at the ATPase catalytic site and partly reflecting the binding of Ca2+ at the transport sites with reduced affinity. Mutation of both couples of proline residues to alanine affected the Ca2+-ATPase in different ways. For the P811A/P812A double mutant, the resulting Ca2+-ATPase activity (Fig. 2) and the ability to become phosphorylated from ATP (Fig. 3B) were not drastically affected, in good agreement with similar data previously reported for proline residue 812 (42). This suggests that the proline residues of the first doublet are not essential for Ca2+-ATPase function. On the other hand, proline residues 820-821 seem to play a more important role; we found that the Ca2+-ATPase activity at pCa 4 of the corresponding double alanine mutant was only 10% that of wild type ATPase (Fig. 2B), and maximal phosphorylation from ATP and Pi was low. Comparable effects were obtained after a single mutation of Pro-821 to alanine, suggesting that this residue is more critical than Pro-820. Note that Pro-821 is well conserved in all transport ATPases (5). Nevertheless, a clear difference from the effect of mutation of the aspartic residues in the L6-7 loop is that after mutations of proline residues the apparent affinity of the ATPase for Ca2+ was not modified, as judged from the Ca2+-dependence of ATPase phosphorylation from either ATP or Pi (Figs. 3-5). This shows that the proline residues of loop L6-7 do not participate in the formation of the Ca2+-binding site, even indirectly by structuring it. One possible explanation for the effects observed on Vmax and EPmax is that the mutation of Pro-821 stabilizes the nonphosphorylated E1 species of the ATPase and that the integrity of this residue may be required to overcome rate-limiting step(s) in the cycle, associated with the E2/E1 conformational conversion.The Relationship between Phosphorylation and Ca2+ Binding-- Because of the above mentioned biphasic Ca2+ dependence of ATPase phosphorylation from ATP, the complementary experiments in which we tested the inhibition by Ca2+ of Pi-derived phosphorylation are of critical importance. In agreement with the low affinity for Ca2+ of the ADA mutant suggested by the ATP-derived phosphorylation experiments, we observed that in this mutant Pi-derived phosphorylation was also less sensitive to Ca2+ inhibition than in WT ATPase, both under acidic and under neutral conditions (Figs. 5A and 6A). Some of the "classical" mutations have been reported to make the ATPase less sensitive to Ca2+ irrespective of pH in Pi-derived phosphorylation experiments (e.g. the well known E771Q, T799A, D800N, and E908A mutations), while for another subset of the "classical" mutants (e.g. E309Q and N796A) Ca2+ inhibits Pi-derived phosphorylation at pH 7 with an affinity that is not very different from that of wild type ATPase (10, 28, 50) (it should be mentioned that in acid media, the behavior of the N796A mutant becomes different from that of the E309Q mutant (26, 51). Our results in Fig. 6B for E771Q and E309Q mutants fully confirm the different behavior reported for these mutants at pH 7. In addition, it is clear from the results shown in Fig. 6A that, from a phenomenological point of view, the behavior of the ADA mutant is more closely related to the subclass of "classical" mutants that have a reduced affinity for Ca2+ irrespective of pH.
It has previously been emphasized (10) that the effect of mutations on sensitivity to Ca2+, as determined in phosphorylation assays, may be accounted for by any one of the following reasons: (a) one or both Ca2+ sites are disrupted; (b) the conformational equilibrium of ATPase is displaced in favor of an "E2-like" conformation that can be phosphorylated from Pi but does not bind Ca2+ efficiently; and (c) the signal transduction from the Ca2+-binding sites to the phosphorylation site is disrupted so that, irrespective of the ability to bind Ca2+, there is no concomitant change in the chemical specificity at the phosphorylation site. The last possibility can be excluded in the case of the ADA mutation, since the presence of Ca2+ was found to result in the expected phosphorylation or dephosphorylation events, although with a reduced affinity. It is worth recalling that binding of a single Sr2+ is believed to be sufficient to prevent Pi-derived phosphorylation, while occupation of the two Sr2+-binding sites is required to permit phosphorylation from ATP (12). On this basis, and assuming that the same is true for the binding of Ca2+, Andersen (10) originally proposed that the classical mutants in which Ca2+ is able to prevent Pi-derived phosphorylation at neutral pH presumably have the first binding site intact, while mutants in which Ca2+ is unable to prevent Pi-derived phosphorylation have the first binding site altered by the mutation. Applying the same rationale to the results obtained with the ADA mutant would suggest that in the ADA mutant the initial steps in Ca2+ binding are also severely altered, consistent with a role of the L6-7 aspartic residues in controlling at least the first of the two Ca2+-binding sites. Whether such control is exerted through the first or second mechanism described above is the next question to be addressed.Proteolytic Degradation of ATPase Mutants by Proteinase K--
In
the absence of a highly resolved three-dimensional structure, mild
proteolysis is a powerful tool with which to investigate the spatial
organization of transport ATPases (29-31, 52). Since several
conformational changes probably occur upon Ca2+ binding to
the Ca2+-ATPase, it was of interest to study by this method
these conformational changes in wild type and mutated
Ca2+-ATPase, and the dependence of these changes on
pCa. In the absence of bound Ca2+, proteinase K
predominantly cleaves Ca2+-ATPase in SR vesicles at two
previously identified sites (31). The most N-terminal one of these two
sites produces a short N-terminal peptide as well as peptide p95
(Lys-120-COOH terminus), while the other site gives rise to p28N
(Thr-242-NH2 terminus) and p83C (Glu-243-COOH terminus).
The differential accessibility of these sites in the absence of
Ca2+ can be assumed to reflect the conformational
equilibrium between the various forms of Ca2+-free ATPase.
On the other hand, binding of Ca2+ results in masking of
the most N-terminal proteolytic site (Lys-120) and a higher degree of
exposure of the second proteolytic site (Glu-243) in the -strand
region.
A Model for Control by the L6-7 Loop of Binding of
Ca2+ by Ca2+-ATPase--
As a starting point,
our results with the ADA mutant could be interpreted by assuming that
the double Asp Ala mutation within the L6-7 loop produces a
reduction in the overall affinity of the ATPase for Ca2+
because of an indirect structural perturbation exerted by these mutations on the position of the Ca2+ liganding residues in
the contiguous M6 segment. Since M6 is suspected to contribute 2 (51)
or 3 (26) residues participating in Ca2+ complexation (with
other residues in M4, M5, and M8), it is indeed likely that any
displacement of M6 in the Ca2+ binding cluster would reduce
the affinity of the site for Ca2+. The fact that long range
perturbations are possible is demonstrated by the effect of L6-7
proline mutations on the catalytic site.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Anne-Marie Lompré (Université Paris Sud, Orsay) for the gift of the 79B antibody; Drs. D. Pompon and P. Urban (CNRS, Gif sur Yvette) for the gift of the yeast strain and the vector; and Adrienne Gomez de Gracia for technical assistance. We are grateful to Stéphanie Soulié and Drs. Francisco Centeno, Béatrice de Foresta, and Birte Juul for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Commissariat à l'Energie Atomique (CEA) and CNRS, by "Ministère de la Recherche et de la Technologie" fellowships (to T. M.), and by "Association Française contre les Myopathies" grants (to M. le M., F. C., and L. B.).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.
§ Present address: Institut de Biotechnologie des Plantes, Université Paris XI, 91405 Orsay Cedex, France.
¶ Present address: Centre National de Séquençage Genoscope, 2 rue Gaston-Cremieux, BP 191, 91006 Evry, France.
Present address: Biologie Cellulaire et Reproduction,
UPRES-A6026 CNRS, Université de Rennes I, Campus de Beaulieu,
35042 Rennes Cedex, France.
To whom correspondence and reprint request should be addressed:
SBPM/DBCM/CEA, Bât 528, Centre d'Etudes de Saclay, 91191 Gif sur
Yvette, Cedex, France. Tel.: 33-169089882; Fax: 33-169088139; E-mail:
falson{at}dsvidf.cea.fr.
The abbreviations used are: SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPasep95, C-terminal proteolytic fragment of SR Ca2+-ATPase starting at residue Lys-120p83C, C-terminal proteolytic fragment of SR Ca2+-ATPase starting at residue Glu-243p28N, N-terminal proteolytic fragment of SR Ca2+-ATPase ending at residue Thr-242p20C or p20, C-terminal proteolytic fragment of SR Ca2+-ATPase starting at residue Gly-808p19C or p19, C-terminal proteolytic fragment of SR Ca2+-ATPase starting at residue Asp-818C12E8, octaethylene glycol monododecyl etherTes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidPAGE, polyacrylamide gel electrophoresisAb, AntibodyWT, wild typeADA, aspartate mutant D813A/D818AAAA aspartate mutant D813A/D815A/D818A, PVDF, polyvinylidine difluorideMOPS, 4-morpholinepropanesulfonic acidMES, 4-morpholineethanesulfonic acidE1, conformational state of Ca2+-ATPase of high affinity for calciumE2, conformational state of Ca2+-ATPase of low affinity for calcium.
2 T. Menguy, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|