(Received for publication, August 17, 1995; and in revised form, October 2, 1995)
From the
Mutants of individual residues of the plasma membrane
Ca-pump were made in the highly conserved region that
(in related P-type ATPases) has been associated with nucleotide
binding. Alteration of the strictly conserved Asp
to Glu
nearly eliminated the ability of the pump to transport
Ca
, while alteration at Val
,
Arg
, and Lys
reduced the activity. High
levels of ATP (25 mM) did not overcome the reduced activity,
indicating that it could not be due to a reduction in the affinity for
ATP. Effects not directly related to ATP binding seemed to result from
mutations in this area. For instance, the amount of phosphorylated
intermediate in the most severely inhibited mutant,
Asp
Glu, was nearly as high as that in the wild
type, a much larger amount of phosphorylated intermediate than was
expected from its low activity. However, the rate of decomposition of
this intermediate was much slower than that of the wild type,
indicating that the inhibition of this mutant resulted from an
inhibition of the E
P
E step in the enzyme
cycle.
The P-type ATPases of animals are divided into three main
classes: 1) the Ca-pumps of sarco-/endoplasmic
reticulum; 2) the Na
,K
-pumps (and
their close relatives, the
H
,K
-pumps); and 3) the plasma
membrane Ca
-pumps. Certain regions of the pump show
high identity between the different classes. These conserved regions
appear to be essential for pump function.
One region that displays some conservation is the ``nucleotide-binding'' region. It received this name because of the reaction of a lysine in this region with the nucleotide analog, fluorescein isothiocyanate and because of a supposed similarity between its secondary structure and that of the nucleotide-binding region of adenylate kinase and phosphofructokinase (Taylor and Green, 1989).
We have studied mutants of five residues
in crucial parts of the nucleotide-binding region. The locations of the
mutants are shown in Fig. 1, which also shows the alignment of
hPMCA4b ()with several other P-type ATPases. Since most of
the amino acids altered were highly conserved among the different
P-type ATPases, their alteration caused a substantial reduction in the
enzyme's activity. We tested whether the reduction in activity
was due to a change in the affinity of the enzyme for ATP and found no
change in the apparent ATP affinity. Instead we found that the most
severely inhibited mutant had a lower rate of dephosphorylation, which
accounted for its low activity.
Figure 1:
Alignment of altered regions with other
P-type ATPases. The numbers are the sequence numbers of
hPMCA4b, with the mutated residues marked by asterisks. The
consensus shows those residues that agree in five of the six sequences.
The other P-type ATPases, in order, are the following: human
Na,K
-ATPase (Kawakami et
al., 1986); rabbit fast twitch skeletal muscle sarcoplasmic
reticulum Ca
-ATPase (Brandl et al., 1986);
H
-ATPase from yeast (Serrano et al., 1986);
H
-ATPase from Arabidopsis thaliana (Harper et al., 1989); and another yeast ATPase, PMR2 (Rudolph et
al., 1989).
The construction of the hPMCA4b full-length cDNA was
described in detail previously (Adamo et al., 1992b).
Mutations were performed using the Altered Site mutagenesis kit
(Promega Corp.). Specific oligonucleotides containing the desired
changes were hybridized with the single-strand hPMCA4b cDNA contained
in the p-Alter-1 vector. After transformation and ampicillin selection
the clones containing the mutations were identified by double-strand
sequencing. The 426-base pair AvaI-AvaI fragment
containing the mutations was sequenced and then cloned into the pMM2
vector containing hPMCA4b for expression in COS-1 cells (Gluzman,
1981). The pMM2 vector was previously called pMT2-m (Adamo et
al., 1992b). The construction of the C-terminally truncated mutant
hPMCA4b(ct120) was described previously (Enyedi et al., 1993).
To obtain the truncated AspGlu(ct120) the AvaI-AvaI fragment was excised from the full-length
Asp
Glu DNA and cloned into hPMCA4b(ct120). The
cells were transfected by the DEAE-dextran-chloroquine method (Adamo et al., 1992b) and harvested after 48 h. For the isolation of
the microsomal fraction (Enyedi et al., 1993) the cells were
swollen at 4 °C for 10 min in 10 mM Tris-HCl (pH 7.5 at 37
°C), 1 mM MgCl
, 0.1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, and 1
µg/ml leupeptin and then homogenized with 30 strokes in a glass
Dounce homogenizer. The homogenate was diluted with an equal volume of
a solution of 0.5 M sucrose, 4 mM dithiothreitol, 0.3 M KCl, 10 mM Tris-HCl (pH 7.5 at 37 °C) and the
suspension was rehomogenized with 20 strokes in the same homogenizer.
The suspension was centrifuged at 5,100
g for 15 min.
The pellet was discarded, and 1.4 ml of 2.5 M KCl and 30
µl of 0.4 M EDTA were added to the supernatant. The
suspension was centrifuged at 100,000
g for 40 min,
and the pellet was homogenized in 0.25 M sucrose, 0.15 M KCl, 10 mM Tris-HCl (pH 7.5 at 37 °C) and 20
µM CaCl
with 20 strokes in a Teflon pestle
glass homogenizer. The final pellet was aliquoted and kept in liquid
nitrogen.
Ca transport activity was measured as
described (Enyedi et al., 1993). The reaction mixture
contained 28 mM KCl, 25 mM Tris-HCl (pH 7.5 at 37
°C), 7 mM MgCl
, 20 mM phosphate, 5
mM NaN
, 0.5 mM ouabain, 4 µg/ml
oligomycin, 400 nM thapsigargin, 250 nM calmodulin,
0.1 mM CaCl
, and enough EGTA to give the free
Ca
wanted. Five micrograms of membranes were
preincubated in the reaction mixture for 5 min at 37 °C, and the
reaction was initiated by the addition of 6 mM ATP. Unless
stated otherwise, Ca
uptake proceeded in the presence
of ATP for 5 min, and the reaction was terminated by filtering the
samples through a 0.45-µm Millipore filter. The
Ca
taken up by the vesicles was then determined by counting in a
scintillation counter The calcium uptake was linear with time for more
than 20 min.
The ATP dependence of the Ca transport was measured in the same medium but with the addition
of 1 mM phosphocreatine, 5 units/ml creatine phosphokinase,
and 0.1 mg/ml of bovine serum albumin. Because the ATP concentration
was being varied, it was also necessary to vary the total
Mg
in order to assure a constant level of free
Mg
. Therefore, the concentration of Mg
in the absence of ATP was 4 mM, and ATP and
Mg
were added in equimolar amounts.
The
phosphorylation reaction was carried out at 4 °C in a medium
containing 28 mM KCl, 25 mM Tris-HCl (pH 7.35 at 4
°C), 20 mM phosphate, 5 mM NaN, 0.5
mM ouabain, 4 µg/ml oligomycin, 400 nM thapsigargin, 250 nM calmodulin in the presence of 0.05
mM EGTA or of 0.02 mM CaCl
plus 0.02
mM LaCl
. The reaction was initiated by the
addition of 15 µM (
-
P)ATP and stopped
after 60 s with 10% trichloroacetic acid, 10 mM P
,
and 1 mM cold ATP. The denatured protein was collected by
centrifugation at 20,000
g for 10 min, washed once,
and separated by using SDS-electrophoresis according to Sarkadi et
al.(1986).
In the dephosphorylation experiments the enzyme was
phosphorylated at 4 °C for 30 s in the presence of 100 mM KCl, 25 mM Tris-HCl (pH 7.35 at 4 °C), 400 nM thapsigargin, 5 mM MgCl, 0.02 mM
CaCl
, and 1 µM (
-
P)ATP.
Dephosphorylation was initiated by adding cold ATP to give a final
concentration of 0.8 mM, and the reaction was stopped at
different times with 10% trichloroacetic acid and 10 mM
P
+ 1 mM cold ATP.
To quantify the
expressed Ca-ATPase a sandwich enzyme-linked
immunosorbent assay was used (Enyedi et al., 1993). In this
assay, the enzyme was adsorbed to the plates by using the monoclonal
antibody 5F10 (Borke et al., 1989), and the amount of
Ca
-ATPase was quantitated using a polyclonal antibody
(Verma et al., 1984).
Protein concentration was estimated by the method of Bradford using bovine serum albumin as a standard. SDS-electrophoresis and immunoblotting were carried out as described previously (Magocsi and Penniston, 1991). For the Western blot shown in Fig. 2, 5 µg of protein from membranes were loaded in each well and separated by SDS-electrophoresis on a 12.5% gel. Proteins were transferred to Millipore Immobilon membranes, and nonspecific binding was blocked by phosphate-buffered saline containing 1% bovine serum albumin for 1 h at 37 °C. For staining, biotinylated anti-mouse immunoglobulin G and avidin-horseradish peroxidase conjugate were used.
Figure 2:
Immunoblot using antibody JA9 of hPMCA4b
and its mutants, expressed in COS-1 cells. Lane a, 40 µg
of purified erythrocyte Ca-pump; lane b,
control membranes (from nontransfected cells); lane c,
membranes from COS cells expressing hPMCA4b (wild type); lane
d, Asp
Glu; lane e, Val
Pro; lane f, Arg
Lys; lane g, Arg
Asp; lane h,
Arg
Leu; lane i, Lys
Ile; lane j, Lys
Ile.
The size of the expressed Ca-pump was
assessed by Western blot, and the level of expression was assessed by
means of an enzyme-linked immunosorbent assay. The Western blot, shown
in Fig. 2, was done utilizing monoclonal antibody JA9, which
reacts with the extreme amino terminus of hPMCA4 (Adamo et
al., 1992a) and does not react with the endogenous plasma membrane
Ca
-pump of COS cells. This portion of the pump was
not altered in any of the mutations reported in this paper. These data
show that all of the mutants caused the expression of full-size
Ca
-pump. The amount of expression was tested more
quantitatively by sandwich enzyme-linked immunosorbent assay, which
showed (Table 1) that all of the mutants were expressed in a
quantity nearly equal to that of hPMCA4b.
Figure 3: Activity of hPMCA4b and mutants versus ATP. The activity of a control (membranes from COS cells transfected with the empty plasmid pMM2) was subtracted from each point.
Figure 4:
Phosphorylation of the wild-type and
mutant Ca-pumps by ATP. Phosphoenzyme formation was
carried out as described under ``Materials and Methods.''
Twenty-five micrograms of protein were loaded per well. Ca
and La
were present during the incubation of
all samples except for that shown in lane a. Lane a,
membranes from COS cells transfected with the empty plasmid pMM2; lane b, same membranes as in lane a; lane c,
Val
Pro; lane d, Arg
Lys; lane e, Arg
Ile; lane f,
hPMCA4b.
Figure 5:
Dephosphorylation of hPMCA4b(ct120) and
Asp Glu(ct120). The acylphosphate was chased with
unlabeled ATP as described under ``Materials and Methods.''
Because lanthanum was omitted from this experiment, the amount of
acylphosphate visualized in the plasma membrane Ca
band is less than it was in Fig. 4. In order to compensate
for this, a longer exposure of the x-ray film was used, which made the
SERCA band look stronger, even though most of the SERCA was inhibited
by thapsigargin.
As is shown in Fig. 1, most of the residues that were altered in this study are highly conserved among the different P-type ATPases, even those that are phylogenetically quite distant from one another. Our results confirmed that the activity of the pump is quite sensitive to changes in these conserved residues.
Particularly
sensitive to change was Asp. Even the relatively
conservative change to Glu nearly inactivated the enzyme. The
corresponding Asp has also been altered in the
Ca
-pump of SERCA1 (Clarke et al., 1990) and
in the H
-pump of yeast (Portillo and Serrano, 1988).
Changing Asp
of SERCA1 to either Glu or Asn reduced the
activity to undetectable levels.
Arg is also conserved
in all eucaryotic P-type ion pumps, but it was more tolerant of
mutation than Asp
. Changing this residue to Lys, Asp, or
Leu caused only partial inactivation of the enzyme, as shown in Table 2. In SERCA1, mutation of the corresponding residue,
Arg
, to Met also produced an enzyme with substantial
remaining activity. Thus, this residue is of substantial importance to
the activity of the enzyme, but a misfitting substitute does not
destroy the activity.
The alteration of Val to Pro is
interesting because the corresponding residue in other P-type ion pumps
is Pro. Because we had restored the consensus residue, we expected this
change to produce a fully active enzyme, but the mutant had only about
24% of the activity of the wild type. This result shows that the
occurrence of Val in this position in the plasma membrane
Ca
-pump is probably due to a difference in function.
This may indicate the existence of differences in the structural
organization of this region in the plasma membrane
Ca
-pump or compensating differences in other regions
that may cooperate with this region to create a similar structural
arrangement (Harris et al., 1991).
Lys is not
highly conserved, since the corresponding residue is a Gln even in
hPMCA1. Nonetheless, altering Lys
to Ile caused a small
reduction in activity. In SERCA1, alteration of the corresponding
Arg
to Ile did not reduce the activity. This residue is
evidently not very sensitive to alterations.
Conversion of
Arg to Ile had no effect on the activity, consistent with
the fact that this position contains Ile, Lys, or His in other
eucaryotes. This mutant provided a positive control, showing that the
mutation process did not, in itself, inactivate the enzyme.
The
effects of high ATP on the Ca uptake were examined
for three mutants that had reduced activity (Asp
Glu, Val
Pro, and Arg
Lys).
For all of them, even 6 or 24 mM ATP was not capable of
overcoming the inhibition. Since the region mutated here has been
thought to be the nucleotide fold, it might be expected that mutations
in this region would act by reducing the affinity of the enzyme for
ATP. We found no evidence that this had happened. The ATP dependence of
Ca
uptake by the mutants was similar to that for
inside-out vesicles from erythrocytes and for wild type enzyme
expressed in COS cells.
We found a substantial amount of
acylphosphate formation in all of the mutants tested, even in the
mutant Asp
Glu, which showed very low
Ca
uptake. This differed from the results of Clarke et al.(1990) on SERCA1, which reported that certain of the
mutations in this region showed no phosphorylation. In particular,
their mutation of the corresponding Asp
to Glu showed no
phosphorylation from ATP.
When chased with unlabeled ATP, the
acylphosphate formed by the mutant Asp
Glu
decomposed at a slower rate than did the wild type. In the normal cycle
of partial reactions of the plasma membrane Ca
-pump
(Rega and Garrahan, 1986), this would occur if the alterations slowed
the conformational change from E
P to E
-P or if it slowed the hydrolysis of E
-P to E
and P
.
The latter step is probably the one that is slowed, although the
experiments described here do not allow a definitive choice between
them.
Previous studies on mutations to this region of P-type ion
pumps have found complex effects on various aspects of pump function.
The most extensive study was by Clarke et al.(1990) on SERCA1.
Their data suggested that alteration of some residues in this region
affected the rate of the EP to E
-P transition, while alteration of others totally
prevented formation of the phosphorylated intermediate. Another report
from the same laboratory (MacLennan et al., 1992) showed that
mutations in this region prevented cross-linking by glutaraldehyde.
Since ATP also prevented such cross-linking, they infer some connection
between this region and ATP binding. The residues whose mutation
prevented cross-linking were Asp
and Pro
of
SERCA1a, which correspond to Asp
and Val
of
hPMCA4b.
The results presented here do not support a central role in
ATP binding of residues Asp, Val
, and
Arg
of the plasma membrane Ca
-pump.
Rather, they indicate that the alteration of these residues changes the
rate of the catalytic events that occur after phosphorylation.