1 Department of Biochemical Engineering and Science, Kyushu Institute of
Technology Iizuka, Fukuoka 820-8502, Japan
2 Department of Molecular Cell Biology, School of Medicine, University of
Occupational and Environmental Health, Kitakyushu 807-8555, Japan
* Author for correspondence (e-mail: xenopus{at}med.uoeh-u.ac.jp)
Accepted 27 January 2003
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Summary |
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Key words: Ca2+ ATPase, Na+/K+ ATPase, ATPase ß-subunit, Chaperone
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Introduction |
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The SR Ca2+ ATPase shares a characteristic membrane topology
with the -subunit of Na+/K+ and
H+/K+ ATPases. The overall hydropathy of the SR
Ca2+ ATPase is essentially identical to that of
Na+/K+ and H+/K+ ATPase
-subunits but it lacks a ß-subunit. How does the SR
Ca2+ ATPase fold correctly in the membrane without the help of the
ß-subunit?
We believed that a ß-subunit-like protein might be present in cells to assist with the correct folding of the SR Ca2+ ATPase. In the present work, we investigated whether the ß-subunit of the Na+/K+ and/or H+/K+ ATPase assists the folding of the SR Ca2+ ATPase during its biogenesis, and we showed that these ß-subunits associate transiently with the nascent SR Ca2+ ATPase.
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Materials and Methods |
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Pulse-chase analysis
After 8-12 hours incubation in modified Barth's medium, the cRNA-injected
oocytes were radiolabeled by the addition of radioactive amino acids to the
medium (EXPRE35S35S; NEN, 0.3 mCi ml1)
at 19°C for 1 hour. The labeled oocytes were transferred to the chase
medium containing methionine and cysteine at 100-fold that of the radiolabeled
amino acids in the medium and were incubated at 19°C for various time
intervals.
Ca2+ ATPase activity assay
About 150 oocytes were homogenized in 1.5 ml medium containing 0.25 M
sucrose, 50 mM Tris/malate buffer (pH 7.0) and 1 mM phenylmethylsulfonyl
fluoride. The homogenate was centrifuged in a swinging bucket rotor at 7000
g for 10 minutes on a 50% sucrose cushion (0.3 ml) to remove
the yolk granule, and the resulting supernatant was further centrifuged at
160,000 g for 30 minutes. The pellet was resuspended in 0.25 M
sucrose, 50 mM Tris/malate buffer (pH 7.0) and assayed for Ca2+
ATPase. The Ca2+ ATPase activity was measured colorimetrically
using the malachite green method (Lanzetta
et al., 1979) in a reaction mixture (45 µl) comprising 50 mM
Tris/malate buffer (pH 7.0), 0.1 M KCl, 5 mM MgCl2, 1%
C12E8, 4 mM ATP and either 0.1 mM CaCl2 or 1
mM EGTA at 37°C for 6 minutes. The reaction was started by adding the
microsomal fraction (12 µg protein).
Western blotting
After immunoprecipitation and subsequent SDS-PAGE, the separated proteins
were transferred onto PVDF membranes (Bio-Rad). The PVDF membranes were then
washed three times in 62.5 mM Tris/HCl buffer (pH 6.7) containing 2% SDS and
0.7% 2-mercaptoethanol at 70°C for 30 minutes with shaking to reduce the
immunoglobulin G (IgG) from the antiserum used for immunoprecipitation on the
PVDF membrane. The SR Ca2+ ATPase was detected with antiserum to
rabbit SR Ca2+ ATPase raised in goat. The primary antibody was
detected using anti-goat secondary antibody raised in donkey. The second
antibody was conjugated with horseradish peroxidase (Santa Cruz
Biotechnology). Chemiluminescence was detected using Lumi-LightPLUS
(Roche Diagnostics).
cRNA
The plasmids for cRNA synthesis of the Na+/K+ ATPase
and the SR Ca2+ ATPase were described previously
(Noguchi et al., 1987;
Kawamura and Noguchi, 1991
).
The cDNA for the pig gastric H+/K+ ATPase ß-subunit
was a gift of M. Futai (Osaka University, Osaka, Japan). cRNAs were
synthesized using MEGAscriptTM (Ambion) following the manufacturer's
instructions.
Immunoprecipitation
The antisera to the rabbit SR Ca2+ ATPase and the pig gastric
H+/K+ ATPase ß-subunit were gifts of H. Suzuki
(Asahikawa Medical College, Asahikawa, Japan) and of M. Futai (Osaka
University), respectively. Protein-G/agarose (Calbiochem) was used to
precipitate the anti-SR Ca2+ ATPase antibody. The antiserum to the
ß-subunit of the Torpedo Na+/K+ ATPase was
raised in rabbit as described previously
(Noguchi et al., 1987). The
monoclonal antibodies against the Na+/K+ ATPase and
H+/K+ ATPase ß-subunit were purchased from Upstate
Biotechnology (Lake Placid, NY) and Affinity BioReagents (Golden, CO),
respectively. Immunoprecipitation was carried out as described
(Noguchi et al., 1994
) and
autoradiographic images were detected and quantified with a BAS3000 Bioimage
Analyzer (FJIX). All experiments were performed at least twice and the results
were reproducible.
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Results |
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The Ca* and hence the SR Ca2+ ATPase was co-precipitated with the Na+/K+ ATPase ß-subunit by the antiserum specific for the Na+/K+ ATPase ß-subunit, strongly indicating that the ß-subunit of the Na+/K+ ATPase could associate with the SR Ca2+ ATPase.
Association of the SR Ca2+ ATPase and the ß-subunit
at the early stage of synthesis
When we previously examined the effect of the Na+/K+
ATPase ß-subunit on the expression of the SR Ca2+ ATPase in
Xenopus oocytes, the association between the two proteins was barely
observed (Kawamura and Noguchi,
1991). The sole difference in the experimental procedure between
the previous and this study was the labeling period of the injected oocytes, 3
days for the former and 1 hour for the latter
(Fig. 1), respectively.
Therefore, we examined the early events in biogenesis of the SR
Ca2+ ATPase in Xenopus oocytes.
Xenopus oocytes were injected with cRNA for the SR Ca2+ ATPase together with cRNA for the Na+/K+ ATPase ß-subunit and incubated for 10 hours in modified Barth's medium. Then the oocytes were subjected to a second injection with radiolabeled amino acids and incubated in modified Barth's medium for 3-30 minutes at 19°C. The Ca* precipitated with the antiserum specific for the Na+/K+ ATPase ß-subunit was detected as early as 3 minutes after initiation of labeling and increased with time up to 30 minutes (Fig. 2). The SR Ca2+ ATPase precipitated with the antiserum specific for the SR Ca2+ ATPase increased sharply with time of labeling, and the amount of the product was much more abundant than that of the Ca*. At 30 minutes of labeling, the SR Ca2+ ATPase precipitated with anti-SR Ca2+ ATPase was more than 30 times that of Ca*. Long-term labeling could result in accumulation of the SR Ca2+ ATPase in the ER in large quantities relative to the Ca*. Under the conditions of the previous experiment, the Ca* band was probably below the detection limit of the method that was used (SDS-PAGE followed by fluorography).
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The low level of the Ca* meant that only a small proportion of the SR Ca2+ ATPase synthesized in oocytes associated with the ß-subunit of the Na+/K+ ATPase and that most of the SR Ca2+ ATPase was free from the ß-subunit. One possible explanation for the low level of Ca* was that the Ca* was a precursor or an intermediate leading to the mature SR Ca2+ ATPase. If so, the association between the Ca* and the ß-subunit of the Na+/K+ ATPase should be transient. To demonstrate this, we performed pulse-chase experiments and followed the Ca* during the course of biogenesis of the SR Ca2+ ATPase.
Pulse-chase of the Ca*
After labeling for an hour by adding radioactive amino acids to the medium,
the oocytes injected with cRNA for the SR Ca2+ ATPase together with
cRNA for the ß-subunit of the Na+/K+ ATPase were
transferred to the chase medium and were incubated for 1-48 hours. Then, the
oocytes were immunoprecipitated with antiserum to the Ca2+ ATPase
or with antiserum to the ß-subunit of the Na+/K+
ATPase (Fig. 3). The
ß-subunit precipitated with the antiserum to the ß-subunit of the
Na+/K+ ATPase turned over from the core-glycosylated 43
kDa form (ßc) to the fully glycosylated 60 kDa form (ßm) during the
course of chase, indicating that the ß-subunit moved from the endoplasmic
reticulum to the Golgi. The Ca* precipitated with the antiserum to the
ß-subunit of the Na+/K+ ATPase decreased at
approximately the same rate as ßc
(Fig. 3, right panel), whereas
the SR Ca2+ ATPase precipitated with antiserum to the
Ca2+ ATPase was apparently constant through 48 hours of chase
(Fig. 3, left panel). Because
the Ca2+ ATPase precipitated with antiserum to the Ca2+
ATPase was much more abundant than the Ca*, it was uncertain whether the Ca*
was converted to the Ca2+ ATPase. From these results, we proposed
that the early complex of the Ca* and the ß-subunit was transient and
dissociated during the course of biogenesis.
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Association of the SR Ca2+ ATPase with the ß-subunit
of H+/K+ ATPase
It was interesting to see whether the ß-subunit of the gastric
H+/K+ ATPase could also associate with the SR
Ca2+ ATPase. When the microsomes of oocytes injected with cRNAs for
the SR Ca2+ ATPase and the ß-subunit of the
H+/K+ ATPase were immunoprecipitated with antiserum
specific for the ß-subunit of the H+/K+ ATPase, the
100 kDa band corresponding to the Ca* in
Fig. 1 was identified
(Fig. 4, lane 6), indicating
the association of the SR Ca2+ ATPase and the ß-subunit of the
H+/K+ ATPase.
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An additional important point shown in Fig. 4 was that the Ca* was precipitated with antiserum specific for the ß-subunit of the H+/K+ ATPase, although faintly, in oocytes injected with cRNA for the SR Ca2+ ATPase alone (Fig. 4, lane 5).
Inhibition of Ca2+-ATPase synthesis by antisera specific
for the ß-subunit
Based on the above observation that the Ca* was immunoprecipitated with the
H+/K+ ATPase ß-subunit antiserum in oocytes
injected with cRNA for the SR Ca2+ ATPase alone, we postulated the
presence of an endogenous protein in oocytes that not only shared a common
epitopic structure with the H+/K+ ATPase ß-subunit
but could also associate with the Ca*. If such a protein was involved in the
biogenesis of the SR Ca2+ ATPase, we would expect that the antibody
to the ß-subunit could prevent the synthesis of the SR Ca2+
ATPase when injected into oocytes that had been injected with cRNA for the SR
Ca2+ ATPase (Fig.
5). The more monoclonal antibody specific for
H+/K+ ATPase ß-subunit that was injected, the less
SR Ca2+ ATPase was synthesized; the SR Ca2+ ATPase was
barely detected when 23 nl of monoclonal antibody was injected. The antiserum
to the Na+/K+ ATPase ß-subunit also prevented the
SR Ca2+ ATPase synthesis in oocytes. By contrast, neither the
normal rabbit IgG nor the monoclonal antibody to the
Na+/K+ ATPase ß-subunit had any effect even when
injected in the highest amount (23 nl per oocyte). The epitope for the
Na+/K+ ATPase ß-subunit monoclonal antibody is
located on the extracellular (lumenal) side of the
Na+/K+ ATPase ß-subunit, which should be the reason
why the monoclonal antibody to the Na+/K+ ATPase
ß-subunit could not prevent the synthesis of the SR Ca2+
ATPase.
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Tryptic digestion of the SR Ca2+ ATPase
We examined whether the SR Ca2+ ATPase synthesized in the
presence of the ß-subunit of the Na+/K+ ATPase had
different trypsin sensitivity to that synthesized in the absence of the
ß-subunit. The SR Ca2+ ATPase was digested in a similar
fashion irrespective of the presence or the absence of the
Na+/K+ ATPase ß-subunit
(Fig. 6). Furthermore, the Ca*
was digested in a similar pattern to the SR Ca2+ ATPase. These
results suggest that the overall structure of the SR Ca2+ ATPases
synthesized with or without the ß-subunit and that of the Ca* were
similar or identical. Therefore, it is plausible to conclude that the
structural maturation of the SR Ca2+ ATPase was derived from
Ca*.
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Both SR Ca2+ ATPases synthesized in the presence and absence of the ß-subunit hydrolysed ATP, and the specific activities of the former and the latter were 3.52 (µmoles inorganic phosphate) mg1 h1 and 3.32 (µmoles inorganic phosphate) mg1 h1, respectively (averages of two independent experiments).
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Discussion |
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If the help of the ß-subunit is essential for the structural maturation of the SR Ca2+ ATPase as mentioned above, how was the functional SR Ca2+ ATPase formed in oocytes injected with cRNA for the SR Ca2+ ATPase alone? As shown in Fig. 5, a protein with chaperone-like activity could be expected in oocytes. In the absence of the ß-subunit, an endogenous protein with chaperone-like activity should assist the correct packing of the nascent SR Ca2+ ATPase.
We could not rule out the possibility that the postulated endogenous
chaperone-like protein could be the endogenous Na+/K+
ATPase ß-subunit. However, the possibility should be low because the
Na+/K+ ATPase ß-subunit is not an ER-resident
protein and, under physiological conditions, it leaves the ER for the plasma
membrane soon after the synthesis. Recently, a novel protein termed ß
muscle, has been identified (Crambert et
al., 2002). Despite a similarity with the
Na+/K+ ATPase ß-subunit, ß muscle is
concentrated in the SR and cannot associate with the
Na+/K+ or H+/K+ ATPase
-subunits. This ß muscle is one of the most probable candidates
for the endogenous chaperone-like protein reported here, but Crambert et al.
claim that ß muscle does not act as a chaperone for the maturation of any
known ATPase
-subunits, including the SR Ca2+ ATPase.
In oocytes injected with cRNA for the ß-subunit, the SR Ca2+ ATPase is matured in two different ways: one by way of the ß-subunit and the other by way of the endogenous chaperone-like protein of oocytes. Both the ATP hydrolytic activity and the trypsin sensitivity of the SR Ca2+ ATPase synthesized in the presence of the ß-subunit are essentially identical to those of the SR Ca2+ ATPase synthesized in its absence, suggesting that the SR Ca2+ ATPase is correctly matured, irrespective of the path it follows.
The monoclonal antibody to the H+/K+ ATPase ß-subunit recognizes a sequence at the N-terminal region that is on the cytoplasmic side of the plasma membrane, whereas the epitope of the monoclonal antibody to the Na+/K+ ATPase ß-subunit is extracellular. The antibody injected into oocytes rarely reaches the lumenal side of ER, where the epitope of the monoclonal antibody to the Na+/K+ ATPase ß-subunit is exposed. This is one of the possible reasons why the synthesis of the SR Ca2+ ATPase is prevented by the monoclonal antibody to the H+/K+ ATPase ß-subunit but not by the monoclonal antibody to the Na+/K+ ATPase ß-subunit.
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Acknowledgments |
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