Unusual degradation of alpha -beta complexes in Xenopus oocytes by beta -subunits of Xenopus gastric H-K-ATPase

Pei-Xian Chen1, Paul M. Mathews1, Peter J. Good2, Bernard C. Rossier1, and Käthi Geering1

1 Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland; and 2 Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The catalytic alpha -subunit of oligomeric P-type ATPases such as Na-K-ATPase and H-K-ATPase requires association with a beta -subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the beta -subunit of Xenopus gastric H-K-ATPase (beta HK) in Xenopus oocytes together with alpha -subunits of H-K-ATPase (alpha HK) or Na-K-ATPase (alpha NK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of Xenopus beta HK from metabolically labeled oocytes show that it is well expressed and, when synthesized without alpha -subunits, can leave the ER and become fully glycosylated. Xenopus beta HK can associate with both coexpressed alpha HK and alpha NK, but the alpha -beta complexes formed are degraded rapidly in or close to the ER and do not produce functional pumps at the cell surface as assessed by 86Rb uptake. A possible explanation of these results is that Xenopus beta HK may contain a tissue-specific signal that is important in the formation or correct targeting of functional alpha -beta complexes in the stomach but that cannot be recognized in Xenopus oocytes and in consequence leads to cellular degradation of the alpha -beta complexes in this experimental system.

intracellular transport; oligomerization; pre-Golgi degradation; Xenopus oocyte expression

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CELLS CONTROL THE FIDELITY of secretory protein biosynthesis before transport through the Golgi complex, assuring that proteins in distal compartments of the secretory pathway are conformationally intact and targeted to the correct cellular compartment. To a major extent, cells achieve this by synthesizing proteins that form multimeric complexes and by permitting only fully assembled multimers to exit the endoplasmic reticulum (ER). In many cases, unassembled and ER-retained subunits are degraded rapidly by a pre-Golgi degradation system (for review see Ref. 6).

Our laboratory has studied the subunit assembly and transport from the ER to the plasma membrane of the heterodimeric alpha -beta cation-transporting ATPases expressed in Xenopus laevis oocytes (7, 11, 14). The multi-membrane-spanning alpha -subunit (relative mol mass ~100 kDa) is the catalytic subunit that hydrolyzes ATP and undergoes the E1-to-E2 conformational transition during ion translocation. The glycosylated beta -subunit (relative mol mass ~45-80 kDa) is required for the plasma membrane expression of functional pumps (for review see Ref. 6), and it may influence the apparent K affinity (12, 14). Well characterized in vertebrates are the Na-K-ATPase, a ubiquitous component of the plasma membrane, and the gastric H-K-ATPase, expressed only in the parietal cell of the stomach mucosa. The alpha -subunits share ~65% amino acid sequence identity (17), whereas the beta -subunits are less well conserved (24).

In the parietal cell of the stomach, the H-K-ATPase resides in the apical membrane and subapical tubulovesicles (20), whereas the Na-K-ATPase is basolateral, as it is in most polarized epithelia (18). Gottardi and Caplan (9) have identified apical targeting domains in the alpha - and beta -subunits of the gastric H-K-ATPase (alpha HK and beta HK, respectively), suggesting a mechanism for the correct plasma membrane localization of the molecule. Arrival in the apical or basolateral membrane, however, must be preceded by appropriate subunit assembly in the ER. The available data indicate that assembly can be promiscuous, with Na-K-ATPase alpha -subunits (alpha NK) and gastric beta HK forming heterodimers and functional pumps in the plasma membrane (5, 10, 14, 16). Clearly, however, the parietal cell must extend greater control over the expression of mixed pumps.

To better understand these control mechanisms, we expressed gastric alpha HK or beta HK in combination with alpha NK or Na-K-ATPase beta -subunits (beta NK) in Xenopus oocytes and analyzed their ability to assemble and to support the expression of functional H-K or Na-K pumps at the cell surface. With this approach, we wanted to test whether Xenopus oocytes lack the parietal cell-specific control factors for correct assembly and transport of cation pumps. Our previous work has involved the functional expression of rabbit gastric beta HK in oocytes along with Xenopus alpha NK (10) or Xenopus gastric alpha HK (17). To have a strictly homologous Xenopus system, we isolated a cDNA encoding Xenopus beta HK and generated an antibody against this protein. Using these tools and the Xenopus oocyte expression system, we show that, like rabbit gastric beta HK, the Xenopus beta HK assembles with Xenopus alpha HK as well as alpha NK in the ER. However, alpha -beta heterodimers formed with Xenopus beta HK were not expressed at the cell surface due to early degradation. This may suggest a mechanism by which the parietal cell can assure that only appropriate alpha -beta heterodimers are routed to the trans-Golgi network for trafficking to either the apical or basolateral membranes. Additionally, this presents a novel paradigm for the ER exit of a multisubunit protein. Unassembled beta HK is capable of rapidly leaving the ER, whereas alpha -assembled beta HK is not transported to the cell surface.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cloning of a cDNA encoding the Xenopus laevis gastric beta HK. A cDNA fragment of Xenopus beta HK was generated by PCR, using as template cDNA prepared from Xenopus stomach mRNA with oligo(dT) priming. The degenerate oligonucleotide primers used in the PCR were a sense primer encoding the amino acid sequence Pro-Asp-Tyr-Gln-Asp-Gln with an added EcoR I site at the 5' end (CGGAATTCCNGAYTAYCARGAYCA) and an antisense primer encoding the amino acid sequence His-Tyr-Phe-Pro-Tyr-Tyr with an added BamH I site at the 5' end (CCGGATCCRTARTANGGRAARTARTG). A 480-bp fragment was isolated, subcloned, sequenced, and found to encode the appropriate region of Xenopus beta HK.

A Xenopus stomach mucosal cDNA library was constructed in the vector pBluescript (Stratagene) from size-fractionated poly(A)+ RNA enriched for Xenopus beta HK message (see Ref. 17 for details). Approximately 40,000 independent colonies were screened in pools of 2,000 colonies by PCR using 2 oligonucleotides (GGTGTGACATTGAGACC and TGAACAGTTCACAAGAGG), the sequences of which were based on the Xenopus beta HK cDNA fragment described above. Sequential pool size reduction and PCR screening yielded a clone containing a full-length Xenopus beta HK cDNA, which was sequenced on both strands. The full-length cDNA was subcloned into the expression vector pSD3 (8) for cRNA synthesis (19).

Xenopus beta HK and alpha HK antibodies. A glutathione S-transferase (GST) fusion protein containing the carboxy-terminal 100 amino acids of Xenopus beta HK (Fig. 1) was constructed in the vector pGEX-2T (Pharmacia). A BamH I site, in frame with the GST open reading frame, was introduced by PCR into the Xenopus beta HK cDNA before the codon for Pro-195. Bacterially produced fusion proteins were affinity-purified using glutathione-Sepharose 4B (Pharmacia), recovered by elution with glutathione, and used to immunize rabbits. The specificity of the antiserum was tested by immunoprecipitation of digitonin extracts of metabolically labeled Xenopus beta HK following cRNA injection into oocytes (see below). Preimmune serum and immune serum preabsorbed on the GST-Xenopus beta HK fusion protein used as antigen did not immunoprecipitate any labeled proteins (data not shown). On the other hand, both immune serum preabsorbed on GST and immune serum without treatment immunoprecipitated a core-glycosylated ER form (~50 kDa) and a fully glycosylated post-Golgi form (60-75 kDa) of the Xenopus beta HK.


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Fig. 1.   A: primary structure and tissue distribution of Xenopus gastric H-K-ATPase beta -subunit (beta HK). Isolated cDNA coding for Xenopus beta HK is ~1.1 kb long and contains a 295-codon open reading frame with start codon beginning at nucleotide 22. Shown is an alignment of amino acid sequences of Xenopus laevis (X) and rabbit (rab) gastric beta HK. Identical amino acids are indicated by dashes and conservative substitutions by dots. Single transmembrane domain is underlined in bold. The 7 putative glycosylation sites are boxed. Two additional, less conservative consensus glycosylation sequences NDT and NPT are also present. A glutathione S-transferase fusion protein that was used to generate antibodies against beta HK contained the carboxy-terminal 100 amino acids (underlined). B: Northern blot analysis of tissue distribution of Xenopus beta HK. Total RNA (7.5 µg) from different tissues of adult X. laevis was hybridized with cDNA probes. Top: RNA hybridized with a Xenopus gastric beta -subunit probe. Bottom: same filter reprobed with a Xenopus elongation factor 1alpha (EF1alpha ) probe. mRNA encoding EF1alpha is a major transcript at midblastula transition in Xenopus (15). Lanes are labeled with source of RNA; st 14/15 RNA is from stage 14-15 embryos. Migration of 18S RNA is indicated at right.

A synthetic peptide corresponding to a 15-amino acid sequence near the amino terminus of the Xenopus alpha HK (Ser-Val-Glu-Met-Glu-Arg-Glu-Gly-Asp-Gly-Ala-Met-ValLys) linked to a lysine core (multiple antigenic peptide; Ref. 21) was used to immunize rabbits to generate the alpha HK antiserum. This antiserum recognizes in vitro-translated alpha HK and does not recognize alpha NK either from in vitro translation or expressed in oocytes (data not shown). The anti-rabbit beta HK monoclonal antibody was a gift of P. Mangeat (20). Antibodies against alpha NK and beta NK have been described (1).

Protein expression in Xenopus oocytes and immunoprecipitations. Oocytes were obtained from X. laevis as previously described (7). Oocytes were injected with the indicated amounts of beta -subunit cRNA alone or in combination with alpha -subunit cRNA, metabolically labeled in modified Barth's solution (MBS) containing 0.6 µCi/ml [35S]methionine, and chased in MBS containing 10 mM unlabeled methionine. Digitonin extracts were prepared, and immunoprecipitations were performed under denaturing or nondenaturing conditions as described (7). In some instances, immunoprecipitated beta -subunits were digested with endoglycosidase H (Calbiochem-Novabiochem, La Jolla, CA) (13). SDS-PAGE, fluorography, and laser densitometry were performed as previously described (7).

86Rb uptake. Three days after cRNA injection of oocytes, 86Rb uptake was measured as previously described (13). The assay solution used throughout was (in mM) 90 NaCl, 1 MgCl2, 0.33 Ca(NO3)2, 0.41 CaCl2, 5 BaCl2, and 10 HEPES (pH 7.4). For H-K-ATPase transport measurements, oocytes were preincubated for 15 min in an assay solution containing 10 µM ouabain, which completely inhibits the endogenous oocyte Na-K-ATPase but has no effect on H-K-ATPase activity (17). After preincubation, oocytes were incubated for 12 min in an assay solution containing 5 µCi/ml 86RbCl (Amersham) and 0.5 mM KCl for H-K-ATPase transport measurements or 5 mM KCl for Na-K-ATPase transport measurements. Oocytes were washed, and the 86Rb uptake in single oocytes was determined by scintillation counting.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Primary structure and tissue distribution of the Xenopus gastric beta HK. We characterized recently a Xenopus gastric alpha HK after coexpression with rabbit gastric beta HK in Xenopus oocytes (17). To test whether functional H-K pumps could be formed in a strictly homologous system different from parietal cells, we isolated a cDNA encoding Xenopus gastric beta HK (GenBank accession no. BankIt165014 AF042812). Xenopus beta HK shares 55% amino acid sequence identity with rabbit beta HK, with two striking differences (Fig. 1A). One area of difference is at the carboxy terminus, where the Xenopus beta HK extends eight amino acid beyond that of the rabbit beta HK. A second difference is in the cytoplasmic amino terminus, where a tyrosine-based endocytosis motif has been identified in mammalian beta HK (4). In Xenopus and chicken beta HK, however, phenylalanine replaces this tyrosine.

Northern blot analysis of Xenopus beta HK performed in different tissues (Fig. 1B) revealed its exclusive expression in the stomach, confirming that the isolated cDNA encoded the gastric beta HK.

Xenopus beta HK does not support the functional expression of alpha HK or alpha NK. To test the ability of Xenopus beta HK to produce functional H-K-ATPase alpha -beta complexes in Xenopus oocytes, we expressed Xenopus beta HK or rabbit beta HK together with Xenopus or rabbit alpha HK and compared the expression of functional H-K pumps at the cell surface by 86Rb uptake measurements. As previously shown, coexpression of rabbit beta HK with rabbit (10) or Xenopus (17) alpha HK led to a significant, approximately fivefold increase in 86Rb uptake compared with that measured in oocytes expressing alpha HK alone (Fig. 2A). Surprisingly, coexpression of Xenopus beta HK with Xenopus or rabbit alpha HK did not result in a significant change in the H-K pump activity compared with that in alpha HK-expressing oocytes. Similarly, Xenopus beta HK coexpressed with Xenopus alpha NK did not increase Na-K pump activity at the cell surface compared with that found in oocytes expressing Xenopus beta HK alone (Fig. 2B).


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Fig. 2.   Expression of Xenopus gastric beta HK in Xenopus oocytes does not produce functional H-K or Na-K pumps at cell surface. Oocytes were injected with indicated combinations of cRNAs for different alpha -subunits of N-K-ATPase (alpha NK; 11 ng) or H-K-ATPase (alpha HK; 11 ng) and beta HK or beta -subunit of N-K-ATPase (beta NK) [2 ng rabbit (r) beta HK, 0.5 ng Xenopus beta HK, or Xenopus beta NK]. Three days after injection, 86Rb uptake into oocytes was measured as described in METHODS. A: 86Rb uptake mediated by expressed H-K pumps. Shown is mean of 2 experiments performed on 2 different batches of oocytes. In each experiment, 16-20 oocytes were measured for each condition. Values obtained in oocytes expressing rabbit alpha HK-rabbit beta HK complexes were set to 1 and amounted to 14.3 ± 0.5 and 12.8 ± 0.4 pmol · min-1 · oocyte-1, respectively, in 2 experiments. B: 86Rb uptake mediated by expressed Na-K pumps. Values obtained in oocytes expressing Xenopus alpha NK-Xenopus beta NK complexes were set to 1 and amounted to 129.2 ± 15.5 pmol · min-1 · oocyte-1. Data are means ± SE (n = 20).

Xenopus beta HK can associate with but not stabilize alpha HK or alpha NK. The lack of an increased pump expression after coexpression of Xenopus beta HK with either alpha HK or alpha NK could be an indication that Xenopus oocytes lack mechanisms for the sorting of H-K-ATPases vs. Na-K-ATPases that parietal cells need to express H-K pumps in the correct cellular compartment. However, it is also possible that the results obtained were only due to inefficient translation of the injected cRNA or to lack of assembly of the newly synthesized Xenopus beta HK with the alpha -subunits. To test the two latter possibilities, we compared the biosynthesis and assembly of metabolically labeled Xenopus or rabbit beta HK expressed alone or together with alpha HK or alpha NK in oocytes. After cRNA injection, oocytes were subjected to a 16-h pulse with [35S]methionine and various chase periods, digitonin extracts were prepared, and the expressed proteins were immunoprecipitated under nondenaturing conditions that preserve subunit interaction. As previously observed (10), rabbit beta HK was well expressed in oocytes and when expressed alone was immunoprecipitated mainly in its core glycosylated form after a pulse period (Fig. 3A, lane 1) and in its fully glycosylated form after various chase periods (lanes 2 and 3). Although rabbit beta HK could associate with endogenous oocyte alpha NK, as reflected by coimmunoprecipitation with a beta HK antibody (lanes 1-3), it was synthesized in large excess over the endogenous alpha -subunit. Therefore our data confirm that rabbit beta HK is able to be transported to the plasma membrane without association with alpha -subunits. Rabbit beta HK associated efficiently with coexpressed rabbit (lane 10) and Xenopus (lane 4) alpha HK or alpha NK (lane 7) and typically stabilized the alpha HK (lanes 5, 6, 11, and 12) and to a somewhat lesser extent the alpha NK (lanes 8 and 9), which in an unassembled form are degraded completely during the chase period (1). This result reflects the higher specificity of beta HK for alpha HK than for alpha NK and explains the previously observed difference in the cell surface expression of functional alpha HK-beta HK and alpha NK-beta HK complexes (14).


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Fig. 3.   Xenopus beta HK assembles with but does not stabilize alpha HK and alpha NK. Oocytes were injected with beta HK (0.25 ng Xenopus beta HK, 2.0 ng rabbit beta HK) cRNA alone or in combination with alpha HK (8 ng) or alpha NK (7 ng) as indicated. After metabolic labeling for 16 h and chase periods of 0, 24, or 48 h, digitonin extracts were prepared and subjected to immunoprecipitation under nondenaturing conditions with an anti-rabbit beta HK monoclonal antibody (A) or a Xenopus beta HK antiserum (B). Core-glycosylated (cg) and fully glycosylated (fg) beta HK and beta NK, coprecipitated exogenous alpha -subunit (alpha ), and endogenous alpha -subunit (alpha  endo) are indicated.

Inefficient synthesis of Xenopus beta HK is not responsible for the lack of formation of functional H-K pumps. Xenopus beta HK also was well expressed in oocytes when expressed either alone (Fig. 3B, lane 1) or together with alpha HK (lanes 4 and 7). When expressed alone, Xenopus beta HK was found mainly in its core glycosylated form after a pulse period (lane 1) and in its fully glycosylated form after chase periods (lanes 2 and 3), indicating that, like rabbit beta HK, Xenopus beta HK can leave the ER without alpha -association.

The association efficiency of Xenopus beta HK with alpha -subunits was tested by following the coimmunoprecipitation of coexpressed Xenopus alpha HK or alpha NK. The results revealed that Xenopus beta HK was indeed able to associate with both alpha -subunits during a pulse period (lanes 4 and 7) but that the association was lost during the chase periods (lanes 5, 6, 8, and 9). The results indeed suggest that association of Xenopus beta HK with Xenopus alpha HK or with alpha NK, which is stabilized completely by association with beta NK (lanes 10 and 11), provokes the degradation not only of the associated alpha -subunit but also of the Xenopus beta HK itself rapidly after synthesis. This event is responsible for the lack of formation of functional H-K or Na-K pumps at the cell surface.

To further document this finding, we expressed Xenopus beta HK alone or together with Xenopus alpha HK in oocytes and followed the degradation and the glycosylation processing of the Xenopus beta HK after a pulse and various chase periods. The glycosylation processing was followed via the sensitivity to endoglycosidase H digestion, which characteristically cleaves only high-mannose core sugars acquired during synthesis and not complex type sugars added to the protein in the trans-Golgi compartment after mannose trimming. Typically, after a 12-h pulse, the total population of Xenopus beta HK synthesized in Xenopus oocytes in the absence (Fig. 4, lanes 1 and 2) or presence (lanes 7 and 8) of Xenopus alpha HK was endoglycosidase H sensitive, indicating that the protein resides at the level of the ER. In the absence of coexpressed alpha HK, Xenopus beta HK became progressively fully glycosylated and thus was transported to the plasma membrane, as reflected by the decrease in endoglycosidase H-sensitive species and a parallel increase in higher molecular mass species that were partially but not completely endoglycosidase H resistant (lanes 3-6). Analysis of the N-linked sugars in beta HK revealed recently that oligomannose structures persist on some of the seven glycosylation sites even in fully glycosylated beta HK (23), which could explain the partial endoglycosidase H sensitivity of the high-molecular-mass species. Compared with individual Xenopus beta HK, alpha -assembled Xenopus beta HK are not processed to the same high-molecular-mass species during the chase periods but are slowly degraded, mainly in their core-glycosylated, endoglycosidase H-sensitive form (lanes 7-9) and to a lesser extent in an intermediate, poorly defined, endoglycosidase H-resistant form. This result suggests that association of Xenopus beta HK with alpha -subunits induces retention of the alpha -beta complex in or close to the ER compartment and its concomitant degradation.


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Fig. 4.   Carbohydrate processing of Xenopus beta HK expressed alone or with alpha HK. Oocytes were injected with Xenopus beta HK cRNA (0.25 ng) alone or in combination with alpha HK cRNA (8 ng), metabolically labeled for 16 h, and chased for indicated times. Digitonin extracts were denatured and immunoprecipitated with Xenopus beta HK antiserum. One-half of eluted proteins were digested with endoglycosidase H (Endo H) before SDS-PAGE. Nonglycosylated (ng), core-glycosylated, and fully glycosylated forms of Xenopus beta HK are indicated.

Xenopus oocytes contain an endogenous alpha NK pool that is not associated with beta -subunits and that is in a highly trypsin-sensitive form (13). Expression of exogenous beta NK is able to recruit this immature alpha NK pool and permits the formation of functional Na-K pumps at the cell surface. In contrast to exogenous alpha -subunits, which are degraded when expressed alone in oocytes, the endogenous, unassembled alpha -subunits are stable for unknown reasons. To further characterize the degradation event induced by Xenopus beta HK assembly, we tested in a final series of experiments whether association of Xenopus beta HK with the endogenous, stable alpha -subunit would promote its degradation or rather permit expression of functional Na-K pumps at the cell surface. Figure 5A (lanes 1 and 2) shows the metabolically labeled, endogenous oocyte alpha -subunit pool, which was stable during a 48-h chase period. Expression of either exogenous rabbit (lanes 5 and 6) or Xenopus (lanes 3 and 4) beta HK did not destabilize the endogenous alpha NK pool but indeed led to a small but significant increase in the number of functional Na-K pumps at the cell surface, as measured by 86Rb uptake (Fig. 5B). These data indicate that Xenopus beta HK can indeed associate with both alpha NK and alpha HK and induce their functional maturation but only if the alpha -subunit is in a stable form, possibly due to association with an unknown factor and/or a particular cellular localization that protects it from pre-Golgi degradation.


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Fig. 5.   Assembly and functional maturation of endogenous alpha -subunit with Xenopus beta HK. Oocytes were injected or not with Xenopus beta HK (0.25 ng) or rabbit beta HK (2 ng). A: immunoprecipitation of endogenous alpha -subunits. Oocytes were labeled for 16 h and chased for 40 h. Extracts were denatured and immunoprecipitated with alpha NK antiserum. B: 86Rb uptake. After cRNA injection, oocytes were incubated for 3 days before 86Rb uptake measurements. Data are means ± SE (n = 20). * P < 0.5 vs. noninjected control oocytes.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In this study, we document an unusual characteristic of Xenopus beta HK expressed in Xenopus oocytes that may be indicative of specific mechanisms governing the selective formation or targeting of H-K-ATPase and Na-K-ATPase alpha -beta complexes in the stomach.

Our data show that Xenopus beta HK expressed in Xenopus oocytes can indeed associate with rabbit or Xenopus alpha HK and alpha NK but does not permit the formation of functional pumps at the cell surface due to rapid degradation of the alpha -beta complexes. The Xenopus beta HK behaves in this respect differently from all other beta -subunits so far tested in the Xenopus oocyte system, including Xenopus, rat, and human beta NK and Bufo nongastric beta HK and rabbit gastric beta HK. Although heterologous assembly between alpha NK and beta HK or alpha HK and beta NK was found to be less efficient than homologous assembly, beta -assembly was always accompanied by a complete or at least partial stabilization of the coexpressed alpha -subunit and a corresponding increase in the number of functional pumps at the cell surface (for review see Ref. 6).

A possible explanation for the observation that rabbit as well as Xenopus alpha HK expressed in Xenopus oocytes can be stabilized and form functional H-K pumps with rabbit beta HK but not with Xenopus beta HK could involve the presence of a molecular signal in the Xenopus beta HK, which might be important for a tissue-specific control of the stable formation or targeting of H-K-ATPase alpha -beta complexes in the Xenopus stomach but which cannot be interpreted correctly in the Xenopus oocyte expression system. At first sight, two domains in the Xenopus beta HK could be of interest in this respect. Analysis of the carboxy termini of beta NK and beta HK has revealed that the last 10 amino acids may form an amphipathic beta -strand that exposes on one side a hydrophilic domain and on the other side a continuous hydrophobic domain that is important for subunit assembly (2). Xenopus beta HK differs from all other known beta HK as well as from beta NK by an extension of eight amino acids, which could be necessary for a tissue-specific control of subunit assembly.

A second domain in the Xenopus beta HK that could be involved in the particular behavior of this protein when expressed in Xenopus oocytes, is the cytoplasmic amino terminus. All beta HK so far identified, with the exception of chicken and Xenopus beta HK, contain a tyrosine-containing sequence in the amino terminus that is a reversed version of the motif that is responsible for transferrin receptor internalization (3). Recently, it was shown in transgenic mice that the Phe-Arg-His-Tyr motif in the mammalian beta HK is necessary for endocytosis of H-K pumps and termination of acid secretion in the stomach (4). In addition, tyrosine-based motifs have been shown to be responsible for targeting to various endosomal compartments or lysosomes (for review see Ref. 22).

It is not known whether the signals that are involved in these processes are similar in amphibia or birds and in mammals, but it could be that the corresponding Phe-Arg-Arg-Phe or the Phe-Gly-Arg-Phe sequences in chicken or Xenopus beta HK have functions similar to those of the tyrosine-based motifs in mammals. If this is the case, the signal present in Xenopus gastric beta HK might specifically mediate targeting to the tubulovesicular structure in the Xenopus stomach cells. Due to the lack of these structures in Xenopus oocytes, the newly synthesized Xenopus H-K-ATPase alpha -beta complexes might be sorted to lysosomes or another degradation compartment.

Finally, it is interesting to note that there is degradation only of assembled alpha -beta complexes but not of Xenopus beta HK expressed alone in Xenopus oocytes. This result indicates that a putative targeting signal present on Xenopus beta HK is exposed only after the beta -subunit associates with the alpha -subunit, due either to a conformational change or to targeting to a particular ER subcompartment that is involved in the recognition of the signal.

    ACKNOWLEDGEMENTS

We thank P. Mangeat for the monoclonal beta HK antibody. P. J. Good thanks Igor Dawid for help and support.

    FOOTNOTES

This work was supported by Swiss National Fund for Scientific Research Grants 31.33598.92 (to B. C. Rossier) and 31.42954.95 (to K. Geering) and by a Fogarty Foundation foreign-funded fellowship to P. M. Mathews.

Present addresses: P.-X. Chen, Cytogenetics Lab, Dept. of Reproductive Genetics, Northwestern Memorial Hospital, 333 E. Superior St., Chicago, IL 60611; P. J. Good, Dept. of Biochemistry and Molecular Biology (and Center for Excellence in Cancer Research), LSU Medical Center, Shreveport, LA 71130-3932; P. M. Mathews, Natham Kline Institute, 140 Old Orangeburg Rd., Orangeburg, NY 10962.

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. §1734 solely to indicate this fact.

Address for reprint requests: K. Geering, Institute of Pharmacology and Toxicology, University of Lausanne, du Bugnon 27, CH-1005 Lausanne, Switzerland.

Received 28 January 1998; accepted in final form 13 April 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(1):C139-C145