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
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ABSTRACT |
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The catalytic -subunit of oligomeric P-type ATPases such as
Na-K-ATPase and H-K-ATPase requires association with a
-subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the
-subunit of Xenopus gastric
H-K-ATPase (
HK) in Xenopus oocytes together with
-subunits of H-K-ATPase (
HK) or Na-K-ATPase (
NK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of
Xenopus
HK from metabolically
labeled oocytes show that it is well expressed and, when synthesized
without
-subunits, can leave the ER and become fully glycosylated.
Xenopus
HK can associate with both coexpressed
HK and
NK, but the
-
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
HK may contain a
tissue-specific signal that is important in the formation or correct
targeting of functional
-
complexes in the stomach but that
cannot be recognized in Xenopus
oocytes and in consequence leads to cellular degradation of the
-
complexes in this experimental system.
intracellular transport; oligomerization; pre-Golgi degradation; Xenopus oocyte expression
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INTRODUCTION |
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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 -
cation-transporting ATPases expressed in Xenopus
laevis oocytes (7, 11, 14). The multi-membrane-spanning
-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
-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
-subunits share
~65% amino acid sequence identity (17), whereas the
-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
- and
-subunits of the gastric H-K-ATPase (
HK and
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
-subunits (
NK) and gastric
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
HK or
HK in combination with
NK or Na-K-ATPase
-subunits (
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
HK in oocytes along with
Xenopus
NK (10) or
Xenopus gastric
HK (17). To have a
strictly homologous Xenopus system, we
isolated a cDNA encoding Xenopus
HK
and generated an antibody against this protein. Using these tools and
the Xenopus oocyte expression system,
we show that, like rabbit gastric
HK, the
Xenopus
HK assembles with
Xenopus
HK as well as
NK in the
ER. However,
-
heterodimers formed with
Xenopus
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
-
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
HK is capable of rapidly leaving the ER, whereas
-assembled
HK
is not transported to the cell surface.
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METHODS |
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Cloning of a cDNA encoding the Xenopus
laevis gastric HK.
A cDNA fragment of Xenopus
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
HK.
Xenopus HK and
HK antibodies.
A glutathione S-transferase (GST)
fusion protein containing the carboxy-terminal 100 amino acids of
Xenopus
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
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
HK following cRNA injection
into oocytes (see below). Preimmune serum and immune serum preabsorbed
on the GST-Xenopus
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
HK.
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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 -subunit cRNA alone or in combination with
-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
-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.
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RESULTS |
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Primary structure and tissue distribution of the
Xenopus gastric HK.
We characterized recently a Xenopus
gastric
HK after coexpression with rabbit gastric
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
HK (GenBank
accession no. BankIt165014 AF042812). Xenopus
HK shares 55% amino acid
sequence identity with rabbit
HK, with two striking differences
(Fig. 1A). One area of difference is at the carboxy terminus, where the
Xenopus
HK extends eight amino acid
beyond that of the rabbit
HK. A second difference is in the
cytoplasmic amino terminus, where a tyrosine-based endocytosis motif
has been identified in mammalian
HK (4). In
Xenopus and chicken
HK, however,
phenylalanine replaces this tyrosine.
Xenopus HK does not
support the functional expression of
HK or
NK.
To test the ability of Xenopus
HK
to produce functional H-K-ATPase
-
complexes in
Xenopus oocytes, we expressed
Xenopus
HK or rabbit
HK together
with Xenopus or rabbit
HK and
compared the expression of functional H-K pumps at the cell surface by 86Rb uptake measurements. As
previously shown, coexpression of rabbit
HK with rabbit (10) or
Xenopus (17)
HK led to a
significant, approximately fivefold increase in
86Rb uptake compared with that
measured in oocytes expressing
HK alone (Fig.
2A).
Surprisingly, coexpression of Xenopus
HK with Xenopus or rabbit
HK did
not result in a significant change in the H-K pump activity compared
with that in
HK-expressing oocytes. Similarly,
Xenopus
HK coexpressed with
Xenopus
NK did not increase Na-K
pump activity at the cell surface compared with that found in oocytes
expressing Xenopus
HK alone (Fig.
2B).
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Xenopus HK can associate
with but not stabilize
HK or
NK.
The lack of an increased pump expression after coexpression of
Xenopus
HK with either
HK or
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
HK with the
-subunits. To test the two latter possibilities, we compared the
biosynthesis and assembly of metabolically labeled Xenopus or rabbit
HK expressed
alone or together with
HK or
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
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
HK could
associate with endogenous oocyte
NK, as reflected by
coimmunoprecipitation with a
HK antibody (lanes
1-3),
it was synthesized in large excess over the endogenous
-subunit.
Therefore our data confirm that rabbit
HK is able to be transported
to the plasma membrane without association with
-subunits. Rabbit
HK associated efficiently with coexpressed rabbit
(lane
10) and
Xenopus
(lane
4)
HK or
NK
(lane
7) and typically stabilized the
HK (lanes
5, 6,
11, and
12) and to a somewhat lesser extent
the
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
HK for
HK than for
NK and explains
the previously observed difference in the cell surface expression of
functional
HK-
HK and
NK-
HK complexes (14).
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DISCUSSION |
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In this study, we document an unusual characteristic of
Xenopus 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
-
complexes in the stomach.
Our data show that Xenopus HK
expressed in Xenopus oocytes can
indeed associate with rabbit or
Xenopus
HK and
NK but does not
permit the formation of functional pumps at the cell surface due to
rapid degradation of the
-
complexes. The
Xenopus
HK behaves in this respect
differently from all other
-subunits so far tested in the
Xenopus oocyte system, including
Xenopus, rat, and human
NK and
Bufo nongastric
HK and rabbit
gastric
HK. Although heterologous assembly between
NK and
HK
or
HK and
NK was found to be less efficient than homologous
assembly,
-assembly was always accompanied by a complete or at least
partial stabilization of the coexpressed
-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 HK expressed in
Xenopus oocytes can be stabilized and
form functional H-K pumps with rabbit
HK but not with
Xenopus
HK could involve the
presence of a molecular signal in the
Xenopus
HK, which might be
important for a tissue-specific control of the stable formation or
targeting of H-K-ATPase
-
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
HK could be of interest in
this respect. Analysis of the carboxy termini of
NK and
HK has
revealed that the last 10 amino acids may form an
amphipathic
-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
HK differs from all other
known
HK as well as from
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 HK
that could be involved in the particular behavior of this protein when
expressed in Xenopus oocytes, is the
cytoplasmic amino terminus. All
HK so far identified, with the
exception of chicken and Xenopus
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
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 HK
have functions similar to those of the tyrosine-based motifs in
mammals. If this is the case, the signal present in
Xenopus gastric
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
-
complexes might be sorted to lysosomes or another
degradation compartment.
Finally, it is interesting to note that there is degradation only of
assembled -
complexes but not of
Xenopus
HK expressed alone in
Xenopus oocytes. This result indicates
that a putative targeting signal present on
Xenopus
HK is exposed only after the
-subunit associates with the
-subunit, due either to a
conformational change or to targeting to a particular ER subcompartment
that is involved in the recognition of the signal.
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ACKNOWLEDGEMENTS |
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We thank P. Mangeat for the monoclonal HK antibody. P. J. Good
thanks Igor Dawid for help and support.
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FOOTNOTES |
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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.
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