Transient association of the sarcoplasmic reticulum Ca2+ ATPase with the Na+/K+-ATPase and H+/K+-ATPase ß-subunits during its biogenesis in Xenopus oocytes

Shunsuke Noguchi1, Nobuhito Sone1 and Masaru Kawamura2,*

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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We examined the effect of the ß-subunits of the Na+/K+ and H+/K+ ATPases on the biogenesis of the sarcoplasmic reticulum (SR) Ca2+ ATPase in Xenopus oocytes. Oocytes were simultaneously injected with cRNAs for both the SR Ca2+ ATPase and the ß-subunit of the Na+/K+ or the H+/K+ ATPase. Immunoprecipitation with antiserum specific for the ß-subunit of the Na+/K+ or the H+/K+ ATPase yielded not only the respective ß-subunit but also the SR Ca2+ ATPase, indicating that the SR Ca2+ ATPase was associated with the ß-subunits of the Na+/K+ and the H+/K+ ATPases. Pulse-chase experiments revealed that the complex between the SR Ca2+ ATPase and the ß-subunit of the Na+/K+ ATPase was formed transiently and dissociated during the course of maturation. This is the first report that demonstrates the association of the SR Ca2+ ATPase with the ß-subunit of the Na+/K+ and H+/K+ ATPases.

Key words: Ca2+ ATPase, Na+/K+ ATPase, ATPase ß-subunit, Chaperone


    Introduction
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The mammalian Na+/K+, H+/K+ and Ca2+ ATPases belong to the P2-type ATPase family, which transports cations across biological membranes (Lutsenko and Kaplan, 1995Go). The plasma membrane Na+/K+ and the gastric H+/K+ ATPases consist of a catalytic {alpha} and a glycosylated ß-subunit, whereas the sarcoplasmic reticulum (SR) Ca2+ ATPase is composed of an {alpha}-subunit alone. All the known functions of these ATPases have been attributed to the {alpha}-subunit (Lutsenko and Kaplan, 1995Go; Møller et al., 1996Go). The ß-subunit is needed for the correct folding of the {alpha}-subunit of Na+/K+ and H+/K+ ATPases in the membrane (Noguchi et al., 1987Go; Horowitz et al., 1990Go; Ackermann and Geering, 1990Go; Klaassen et al., 1993Go). The ß-subunit acts like a chaperone that influences the packing of the {alpha}-subunit by forcing the translocation of the transmembrane segments M7/M8 through translocons to the lumenal side of endoplasmic reticulum (ER) membranes, where the strong interactions between the two subunits lead to the maturation of the Na+/K+ and H+/K+ ATPases (Colonna et al., 1997Go; Béguin et al., 1998Go; Melle-Milovanovic et al., 1998Go; Beggah et al., 1999Go).

The SR Ca2+ ATPase shares a characteristic membrane topology with the {alpha}-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 {alpha}-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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Short-term labeling of cRNA expression products in Xenopus oocytes
Xenopus laevis oocytes (stage V-VI) were injected with cRNA for the rabbit SR Ca2+ ATPase or for the ß-subunit of Torpedo Na+/K+ ATPase or of the pig gastric H+/K+ ATPase separately or in combination (each 10 ng cRNA per 23 nl per oocyte). The oocytes were incubated at 19°C for 8-12 hours in modified Barth's medium containing 100 µg ml–1 each of ampicillin, streptomycin and cefmenoxime. Then, the oocytes were subjected to a second injection with [35S] methionine and [35S] cysteine (EXPRE35S35S; NEN, 1175 Ci mol–1) (11 mCi ml–1, 23 nl per oocyte) to label proteins without lag. At appropriate time intervals, the oocytes were frozen quickly in dry ice-ethanol.

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 ml–1) 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., 1979Go) 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., 1987Go; Kawamura and Noguchi, 1991Go). 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., 1987Go). 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., 1994Go) 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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Association of the SR Ca2+ ATPase with the ß-subunit of the Na+/K+ ATPase
Xenopus oocytes were injected with cRNA for either the SR Ca2+ ATPase or the ß-subunit of Na+/K+ ATPase, or with both cRNAs simultaneously. The left panel in Fig. 1 shows immunoprecipitations with the specific antisera followed by autoradiography. When cRNA for the SR Ca2+ ATPase was injected together with cRNA for the ß-subunit, the antiserum specific for the ß-subunit of Na+/K+ ATPase precipitated not only the ß-subunit but also a 100 kDa product (lane 4). When cRNA for the Na+/K+ ATPase ß-subunit was injected alone, no band of 100 kDa product was observed by immunoprecipitating with the antiserum specific for the ß-subunit (lane 6), suggesting that the band of 100 kDa found in the immunoprecipitates with the anti-ß-subunit antiserum was due to the translation product from the cRNA for the SR Ca2+ ATPase. We designated the anti-ß-subunit-antiserum-precipitatable 100 kDa product as Ca*. In order to confirm the identity of the Ca* and the SR Ca2+ ATPase, Ca* was characterized by western blotting (Fig. 1, right panel). After immunoprecipitation with the specific antisera and subsequent SDS-PAGE, the immunoprecipitates of lanes 1 to 4 in the left panel of Fig. 1 were blotted onto PVDF membrane. The membrane was washed as described in Materials and Methods. After three washes, the membrane was probed with goat anti-SR Ca2+ ATPase serum followed by detection with anti-goat second antibody. Bright bands can be seen corresponding to the SR Ca2+ ATPase in the autoradiogram shown in the left panel (lanes 7 and 9). The Ca* precipitated with anti-ß-subunit antiserum (raised in rabbit) could also be detected, although less brightly (lane 10). The weaker chemiluminescence intensity of the Ca* band was consistent with the lower band intensity level in the autoradiogram (lane 4). The second antibody was specific for the goat IgG and did not cross-react with the rabbit IgG. This excluded the possibility that the chemiluminescence of the Ca* on the blot was due to a cross-reaction between the primary antibody to the ß-subunit and the secondary antibody. These observations confirmed that Ca* was SR Ca2+ ATPase.



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Fig. 1. Immunoprecipitation and western blot of the SR Ca2+ ATPase. (Left) Oocytes injected with both cRNAs for the SR Ca2+ ATPase and the ß-subunit of the Na+/K+ ATPase (each 10 ng oocyte–1) were incubated in modified Barth's medium at 19°C for 10 hours. The oocytes were subsequently transferred to Barth's medium containing [35S]methionine and [35S]cysteine, and labeled for 60 minutes. The extracts of the labeled oocytes were immunoprecipitated and the precipitates were separated by SDS-PAGE followed by autoradiography. (Right) The separated proteins in lanes 1-4 of the left panel were transferred to PVDF membrane. After washing the blotted PVDF membrane three times with 2% SDS containing 0.7% 2-mercaptoethanol at 70°C for 30 minutes, the proteins were stained with antiserum specific for the SR Ca2+ ATPase raised in goat. The primary antibody was detected with anti-goat secondary antibody (20,000x dilution) that was labeled with peroxidase.

 

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, 1991Go). 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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Fig. 2. Short-term labeling of the SR Ca2+ ATPase. The oocytes that had been directly injected with radiolabeled amino acids as described in Materials and Methods, were incubated in modified Barth's medium for 3-30 minutes. The microsomes from the labeled oocytes were immunoprecipitated with antisera specific for the SR Ca2+ ATPase (lanes 1-4) and for the ß-subunit of the Na+/K+ ATPase (lanes 5-8).

 

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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Fig. 3. Pulse-chase of the SR Ca2+ ATPase. The oocytes that had been labeled for 1 hour were chased for 1-48 hours as described in Materials and Methods. Lanes 1-8 contain immunoprecipitates with antiserum to the SR Ca2+ ATPase; lanes 9-16 contain immunoprecipitates with antiserum to the ß-subunit of the Na+/K+ ATPase.

 

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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Fig. 4. Association of the H+/K+ ATPase ß-subunit and the SR Ca2+ ATPase. The oocytes were injected with cRNAs and radiolabeled as described in Fig. 1, except that antisera to the ß-subunit of the H+/K+ ATPase was used instead of that of the Na+/K+ ATPase.

 

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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Fig. 5. Prevention of the synthesis of the SR Ca2+ATPase by the anti-ß-subunit antibody. Oocytes were injected with cRNA for the SR Ca2+ATPase (10 ng oocyte–1) and incubated in modified Barth's medium at 19°C. After incubation for 10 hours, the oocytes were injected with varying volumes of antibody, adjusted to a total volume of injection at 23 nl with water and allowed to stand for 60 minutes. The volumes of antibody injected were 0 nl, 0.08 nl, 0.23 nl, 0.77 nl, 2.3 nl, 7.7 nl and 23 nl (from left to right). The oocytes were then labeled with [35S]methionine plus [35S]cysteine for 30 minutes, followed by immunoprecipitation with anti-Ca2+-ATPase antiserum.

 

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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Fig. 6. Tryptic digestion of the SR Ca2+ATPase. The oocytes were injected with cRNAs and radiolabeled as in Fig. 1. The microsomes from the oocytes were digested with trypsin (Worthington: L-1-tosylamide-2-phenylethy-chloromethyl-ketone-treated) at ratios of trypsin to protein of 0.01 and 0.1 on ice for 60 minutes. The digests were immunoprecipitated with antiserum to the SR Ca2+ ATPase (lanes 1-6) or to the ß-subunit of the Na+/K+ ATPase (lanes 7-9).

 

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) mg–1 h–1 and 3.32 (µmoles inorganic phosphate) mg–1 h–1, respectively (averages of two independent experiments).


    Discussion
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 Materials and Methods
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In this paper, we show that the SR Ca2+ ATPase associated transiently with the ß-subunits of the Na+/K+ and the H+/K+ ATPase at an early stage of its biogenesis. Although this association occurred in oocytes under the artificial experimental condition we used, the association seems to suggest some physiological role for the ß-subunit in the course of the biogenesis of the SR Ca2+ ATPase. One of the roles of the ß-subunit in the Na+/K+ ATPase is to assist the correct packing of the nascent {alpha}-subunit in membranes (Noguchi et al., 1987Go; Horowitz et al., 1990Go; Ackermann and Geering, 1990Go; Klaassen et al., 1993Go) through the association with the conserved amino acids (SYGQ) in the extracellular loop between M7 and M8 of the {alpha}-subunit (Colonna et al., 1997Go). Because the SR Ca2+ ATPase and the Na+/K+ ATPase {alpha}-subunits have similar membrane topologies, it is plausible that the {alpha}-subunits of not only the Na+/K+ ATPase but also the SR Ca2+ ATPase are assisted by association with the ß-subunit for correct packing into membranes. The extracellular loop between M7 and M8 might also be involved in the association of the SR Ca2+ ATPase. Because the M7/M8 loop of the SR Ca2+ ATPase does not contain the conserved four-amino-acid sequence SYGQ, the association might not be tight enough to associate permanently like the Na+/K+ ATPase.

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., 2002Go). 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 {alpha}-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 {alpha}-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.


    Acknowledgments
 
This work was supported by Grants-in-Aid for Scientific Research (C; 12670041) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Ackermann, U. and Geering, K. (1990). Mutual dependence of Na,K-ATPase {alpha}-subunits and ß-subunits for correct posttranslational processing and intracellular transport. FEBS Lett. 269,105 -108.[CrossRef][Medline]

Beggah, A. T., Béguin, P., Bamberg, K., Sachs, G. and Geering, K. (1999). b-Subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase {alpha}-subunit. J. Biol. Chem. 274,8217 -8223.[Abstract/Free Full Text]

Béguin, P., Hasler, U., Beggah, A., Horisberger, J.-D. and Geering, K. (1998). Membrane integration of Na,K-ATPase {alpha}-subunit assembly. J. Biol. Chem. 273,24921 -24931.[Abstract/Free Full Text]

Colonna, T. E., Huynh, L. and Fambrough, D. M. (1997). Subunit interactions in the Na,K-ATPase explored with the yeast two-hybrid system. J. Biol. Chem. 272,12366 -12372.[Abstract/Free Full Text]

Crambert, G., Béguin, P., Pestov, N. B., Modyanov, N. N. and Geering, K. (2002). ßm, a structural member of the X,K-ATPase ß subunit family, resides in the ER and does not associate with any known X,K-ATPase {alpha} subunit. Biochemistry 41,6723 -6733.[CrossRef][Medline]

Horowitz, B., Eakle, K. A., Scheiner-Bobis, G., Randolph, G. R., Chen, C. Y., Hitzeman, R. A. and Farley, R. A. (1990). Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. J. Biol. Chem. 265,4189 -4192.[Abstract/Free Full Text]

Kawamura, M. and Noguchi, S. (1991). Possible role of the ß-subunit in the sodium pump. In: The sodium pump: structure, mechanism, and regulation (ed. J. H. Kaplan and P. De Weer), pp. 45-61. New York: The Rockefeller University Press.

Klaassen, C. H. W., van Uem, T. J. F., de Moel, M. P., de Caluwe, G. L. J., Swarts, H. G. P. and de Pont, J. J. H. H. M. (1993). Functional expression of gastric H,K-ATPase using the baculovirus expression system. FEBS Lett. 329,277 -282.[CrossRef][Medline]

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Melle-Milovanovic, D., Milovanovic, M., Nagpal, S. and Sachs, G. (1998). Regions of association between the {alpha} and the ß subunit of the gastric H,K-ATPase. J. Biol. Chem. 273,11075 -11081.[Abstract/Free Full Text]

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