From the Department of Medicine and Physiology, UCLA and Wadsworth
Veterans Affairs Hospital, Los Angeles, California 90073 and
Allergan Pharmaceuticals, Irvine, California 92715
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ABSTRACT |
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A binding and a yeast two-hybrid analysis were
carried out on the gastric H,K-ATPase to determine interactive regions
of the extracytoplasmic domains of the and
subunits of this P
type ATPase. Wheat germ agglutinin fractionation of fluorescein
5-maleimide-labeled tryptic fragments of detergent-solubilized
H,K-ATPase showed that a fragment Leu855 to
Arg922 of the
subunit was bound to the
subunit. The
yeast two-hybrid system showed that the region containing only a part
of the seventh transmembrane segment, the loop, and part of the eighth
transmembrane segment was capable of giving positive interaction
signals with the ectodomain of the
subunit. The sequence in the
extracytoplasmic loop close to the eighth transmembrane segment, namely
Arg898 to Thr928, was identified as being the
site of interaction using this method. We deduced that the sequence
Arg898 to Arg922 in the
subunit has strong
interaction with the extracytoplasmic domain of the
subunit. Again,
using yeast two-hybrid analysis, two different sequences in the
subunit Gln64 to Asn130 and Ala156
to Arg188 were identified as association domains in the
extracytoplasmic sequence of the
subunit. These data enable
identification of major associative regions of the
-
subunits of
the H,K-ATPase.
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INTRODUCTION |
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The gastric H,K-ATPase is a polytopic integral membrane protein
belonging to the P type ATPase ion transport family that exchanges H3O+ for K+ to generate gastric
acid secretion. This ATPase consists of two noncovalently associated
subunits. There is, in the rabbit, a large subunit of 1035 amino
acids with 10 transmembrane segments and a
subunit of 290 amino
acids with a single transmembrane segment and with seven
N-linked glycosylation consensus sequences (1-3). The
Na,K-ATPase, or sodium pump, is the only other P type ATPase known to
have a
subunit, and the two enzymes share about 60 and 40% amino
acid homology in their
and
subunits, respectively (4). All the
catalytic functions of both enzymes are contained within the
subunit, such as the binding sites for ATP, cations, and the
phosphorylation consensus sequence, as well as the sites for ouabain
binding in the Na,K-ATPase, benzimidazole, and
K+-competitive inhibitors in the case of the H,K-ATPase (5,
6).
There is considerable evidence that the subunit is required for
correct assembly of the enzyme. It has been shown for the Na,K-ATPase
that the
subunit stabilizes the nascent
subunit in the
endoplasmic reticulum and plays a role in targeting the
-
complex
to the plasma membrane (7-10). Prevention of glycosylation of the
subunit also results in inadequate targeting and loss of catalytic
function (11). There are also data showing that modification of the
subunit of either enzyme has an effect on the catalytic function. For
example, reduction of the disulfide bonds appears to alter
K+ affinity in the case of the Na,K-ATPase (12). The
subunits can be separated using SDS but not with nonionic detergents.
Hence there are regions of strong interaction between the two subunits that have both assembly and structural effects on the holoenzyme.
The specific regions of interaction between the and the
subunits of the Na,K-ATPase have been investigated by the expression of
chimeras of the Na,K-ATPase and the sarcoplasmic Ca-ATPase (13).
Twenty-six residues in the extracellular loop between the seventh and
eighth transmembrane domains have been implicated as providing the
interactive region in the
subunit. The region of the
subunit of
the Na,K-ATPase near the first S-S bridge in the extracytoplasmic
domain is thought to interact with the
subunit (14). Some
hydrophobic C-terminal amino acids as well as a conserved proline
residue in the loop between the third disulfide bond of the
subunit
ectodomain have also been implicated in subunit assembly (15, 16).
Recently, the use of the yeast two-hybrid system has indicated that 63 amino acids of the
subunit extracytoplasmic sequence may be a
region of interaction with the
subunit (17). A specific sequence
containing SYGQ was identified as important using alanine scanning
mutagenesis. These data suggest that several regions of one or both
subunits of the Na,K-ATPase might be involved in
-
interaction.
The subunit of H,K-ATPase can act as a surrogate for the
subunit of Na,K-ATPase in the formation of functional Na,K-pumps in
Xenopus oocytes (18). This observation suggests some common regions of association in the two enzymes. However, the efficiency of
assembly is much lower and surrogate activity has not been found in a
mammalian cell expression system (19). Trypsinization of cytoplasmic
side out hog gastric vesicles and wheat germ agglutinin (WGA)1 column retention of
fragments associated with the bound
subunit have provided direct
evidence for binding of a region of the H,K-ATPase
subunit, the
TM7/loop/TM8 sector, to the
subunit (20). Since a monoclonal
antibody Abl46 raised against rat parietal cells recognized both an
extracytoplasmic epitope on the
subunit, between Cys162
and Cys178, and an epitope at the putative extacytoplasmic
surface of TM7 on the
subunit, between Ala875 and
Asp879, it was suggested that those two regions may also be
involved in
-
contact (21). More recently, a chimer of the
Na,K-ATPase
subunit containing Gln905 to
Val930 of rat gastric H,K-ATPase has been shown to
preferentially assemble with H,K-ATPase
subunit. This region also
contains the sequence, SYGQ, identified as important for the
Na,K-ATPase interaction (17, 22). Additional amino acids in this region
must determine selectivity.
Here, we have extended our biochemical approach using WGA fractionation
of trypsin digested, but now detergent solubilized H,K-ATPase, to
define more precisely regions of the subunit that interact with the
subunit. It was found that the region TM7 to Arg922 was
retained by the
subunit on the WGA column. A second independent technique, the yeast two-hybrid system, was also used in this study.
The putative extracellular regions of the
and the
subunits of
the rabbit H,K-ATPase were cloned as fusion cDNAs with either the
DNA binding or the activation domain of the GAL4 transcription factor
and all the possible combinations associating one
and one
construct were assayed by the yeast two-hybrid system. Interaction with
the ectodomain of the
subunit was detected only with the TM7/loop/TM8 domain of the
subunit. Shorter constructs of both the
and the
subunits allowed us to define one unique smaller region
in
, Arg898 to Thr928, and two different
regions in the
subunit, Gln64 to Asn30 and
Ala156 to Arg188, as interacting. Together with
the biochemical data, these results define the sequence
Arg898 to Arg922 on the
subunit as being
the major region of association with the
subunit.
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EXPERIMENTAL PROCEDURES |
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Trypsin Digestion of Solubilized H,K-ATPase-- The H,K-ATPase was derived from hog gastric mucosa by previously published methods (23). All manipulations were carried out at 4 °C.
Gastric vesicles (0.5 mg) enriched in gastric H,K-ATPase were solubilized at 4 °C in a buffer composed of 1% Nonidet P-40, 50 mM Tris/HCl, pH 8.0, at a protein concentration of 2 mg/ml. The mixture was spun at 100,000 × g for 10 min. The supernatant containing the solubilized protein (about 1.4 mg/ml) was digested for 20 min at room temperature with tosylphenylalanyl chloromethyl ketone-treated trypsin at a 1/4 ratio of trypsin/protein. The digestion was stopped by adding 10-fold excess of soybean trypsin inhibitor against trypsin, and the mixture was kept on ice before WGA column chromatography.WGA Fractionation of Solubilized H,K-ATPase Digest-- WGA fractionation was carried out as described previously (20). Tryptic digest was loaded on a WGA-Sepharose 6MB column (1 cm3 of column volume). After equilibration for 20 min at 4 °C, a fraction not retained on the column was eluted using 1 ml of a buffer composed of 1% Nonidet P-40, 50 mM Tris/HCl, pH 7.0, and collected for analysis. This fraction was concentrated in vacuo and precipitated with 0.7 ml of cold acetone for removing Nonidet P-40. Again, the column was extensively washed with a buffer (20 ml) composed of 50 mM Tris/HCl, pH 7.0, and 1% Nonidet P-40. This washing removed all peptide fragments not bound to WGA, including soybean trypsin inhibitor and trypsin. Elution of the WGA-retained components was carried out using 0.5 N acetic acid. The acetic acid eluate was collected and dried in vacuo. The WGA-binding fraction was resuspended in 100 µl of 50 mM Tris/HCl, pH 7.8, 0.3% SDS, and 0.1 mM fluorescein 5-maleimide. This allowed fluorescent labeling of peptide fragments enabling localization on SDS-polyacrylamide gel electrophoresis.
The labeled, SDS-solubilized peptide fragments were combined with a 20% volume of sample buffer (0.3 M Tris, 10% SDS, 50% sucrose, and 0.025% bromphenol blue), and the solution was placed on top of a 10% (34:1 acrylamide/methylene bisacrylamide) to 21% (17:1 acrylamide/methylene bisacrylamide) gradient slab gel of 1.5-mm thickness, using the Tricine buffer method of Schagger and von Jagow (24). The gel was run for 20-24 h at 48-mA constant current, along with a lane for prestained molecular mass (Bio-Rad, 16-106 kDa) standards and CNBr fragments of horse myoglobin (Sigma; 2.5-17 kDa). The gel was transferred to polyvinylidene difluoride membranes as described previously (20).Protein Sequencing-- Peptide bands were sequenced with a gas phase sequencer at the UCLA Protein Microsequencing facility using the Applied Biosystems 475 A system composed of a 470 A Sequencer, a 120 A phenylthiohydantoin analyzer, and a 900 A data module. For each peptide it was possible to follow the sequence for 10 amino acids or more, allowing unambiguous assignment of sequence.
Yeast Strains and Media-- The Saccharomyces cerevisiae yeast strain Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4D, met, gal80D, URA3::GAL1UAS-GAL1TATA-lacZ) (CLONTECH Matchmaker System II) was grown at 30 °C in either YPD medium or synthetic defined dropout yeast medium lacking the appropriate amino acids, i.e. tryptophan and leucine (CLONTECH). All the media contain 2% glucose as a source of carbon.
Construction of Fusion cDNAs--
Fusion proteins of
different fragments of the rabbit H,K-ATPase and
subunits with
either the DNA binding domain or the activation domain of the
transcription factor GAL4 were generated by cloning the corresponding
rabbit cDNA fragments into the multiple cloning site of pAS2-1 and
pACT2 vectors respectively (Matchmaker yeast two-hybrid system 2, CLONTECH). For clarity, these two vectors will be
referred to as pAS and pAC, respectively. The cDNA fragments were
generated by amplification by polymerase chain reaction (PCR) with
useful restriction sites incorporated into the primers. The plasmids
and the corresponding primers are listed in Table
I. The PCR reactions were carried out for
30 cycles using 1 unit AmpliTaq DNA polymerase (Perkin-Elmer) and as
followed: 30 s at 94 °C, 30 s at a temperature ranging
from 52 and 60 °C depending on the pair of primers used, and
72 °C for 1 min. An additional 5- min cycle at 72 °C ended each
program. The PCR product was then cleaved according to the conditions
recommended by the commercial supplier of the restriction enzymes
(Promega). It was purified using PCR Clean-up Promega columns and then
ligated for 1 h at room temperature into the pAS and pAC vectors.
The nucleotide sequence and the reading frame of each construct were
checked by automated DNA sequencing. The plasmids were then amplified in Escherichia coli and purified using Qiagen purification
columns.
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Transformation of S. cerevisiae-- Transformation of yeast was carried out by using the lithium acetate method described by Gietz et al. (25).
-Galactosidase Assays--
For qualitative evaluation of
regions of contact,
-galactosidase filter lift assays were carried
out. After 2-4 days of growth at 30 °C, yeast transformants were
transferred onto sterile VWR Scientific grade 410 paper filters, which
were then submerged in liquid nitrogen for permeabilizing the cells.
Filters were then placed onto filter paper presoaked in Z buffer (100 mM sodium phosphate (pH 7.0) 10 mM KCl, 1 mM MgSO4) supplemented with 50 mM
-mercaptoethanol and 0.07 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside. Filters
were then incubated at 30 °C and checked periodically for the
appearance of blue colonies.
Materials-- The materials were of the highest grade purity available. Trypsin type XIII was obtained from Sigma, polyvinylidene difluoride membranes were from Millipore, and Nonidet P-40 was from Sigma. Fluorescein 5-maleimide was obtained from Molecular Probes. WGA-Sepharose 6MB was obtained from Pharmacia.
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RESULTS |
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The TM7/Loop Domain from Leu855 to Arg922
of the Subunit Associates with the
Subunit as Shown by WGA
Fractionation of Trypsin-digested Solubilized H,K-ATPase--
In a
previous study, by using WGA fractionation of trypsin-digested
H,K-ATPase, we have demonstrated that the region TM7/loop/TM8 domain of
the
subunit interacts with the
subunit (20). In this study, we
applied the same technique, but on previously solubilized enzyme. When
solubilized H,K-ATPase was digested with trypsin, several peptide
fragments were observed in the range from 3 to 60 kDa, including
trypsin, auto-digested trypsin, and trypsin inhibitor. Among these, six
peptide fragments found at 20, 11, 8, 7.5, 6.2, and 5 kDa from the
SDS-gel were identified. The 20-kDa peptide fragment as well as the
other peptide fragments found at 11 and 8 kDa had an N-terminal
sequence LVNEPLAA corresponding to the N-terminal region of TM7. A
peptide of 7.5 kDa, with an N-terminal sequence TPIAIEI, defined the
domain TM3/loop/TM4, and another peptide of 6.2 kDa with the N-terminal
sequence, QLAGGLQ, contained the TM1/loop/TM2 domain as previously
shown (20). A peptide of 5 kDa with an N-terminal sequence NIPELTPY
represented the TM5/loop/TM6 domain.
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The Luminal Domain of the Rabbit Gastric H,K-ATPase Subunit
Interacts Only with the TM7/loop/TM8 Region of the
Subunit When
Assayed with the Two-hybrid System--
Five cDNA fragments
corresponding to the five putative extracellular loops of the rabbit
gastric H,K-ATPase
subunit were fused to the binding domain of the
transcription factor GAL4 (pAS
124-161TM1-2,
pAS
319-337TM3-4, pAS
803-833TM5-6,
pAS
869-933TM7-8, and
pAS
972-1001TM9-10). Fig.
2 shows the putative structure of the
subunit of the rabbit H,K-ATPase and Fig.
3 a diagram of the same subunit along with the different regions used in yeast two-hybrid assays. Each construct was co-transformed in yeast together with a
construct corresponding to the whole extracytoplasmic region of the
subunit fused to the activation domain of GAL4 (Fig.
4B, pAC
64-291). The resulting activation of the
-galactosidase reporter gene was
first checked for each co-transformation using the
-galactosidase filter lift assay. Then the more sensitive and quantitative
-galactosidase luminescent assay was performed on those clones
showing a blue coloration, i.e.
-galactosidase
expression, in the first 8 h. Several negative white clones, some
of them reported in this study (cf. Tables II-IV) have been
checked by the luminescent assay, and each always showed an activity
not significantly different from background. Results are presented in
Table II, together with the control
experiments corresponding to the transformation of each plasmid with
the plasmids pAS and pAC. We found that pAC
64-291 was slightly
auto-activating, giving rise to a weak activation of the reporter gene
in the absence of an
insert. However, a strong
-galactosidase
activity was observed when the TM7/loop/TM8 domain of the
subunit
was fused to the binding domain (pAS
869-933TM7-8) and
co-transformed with the ectodomain of the
subunit fused to the
activation domain (pAC
64-291). The time of appearance of the blue
color was 7 and 3 h for the former and the latter transformation,
respectively. Consistent with this blue lift assay, the luminescent
assay showed that the activation of the reporter gene is about 5 times
higher when the
construct fused to the activation domain is
expressed together with the
construct fused to the binding domain
than when expressed with pAS. The same combination of
and
fragments in the opposite orientation was not informative. The same
subunit region fused to the GAL4 activation domain (pAC
860-928TM7-8) gave rise to a very strong
auto-activation of the
-galactosidase reporter gene when transformed
with the nonrecombinant pAS2-1 vector (Table
III). The
ectodomain was not found to
interact with any other extracellular loop of the
subunit, namely
those loops between TM1 and TM2, TM3 and TM4, TM5 and TM6, and TM9 and TM10 in the yeast two-hybrid analysis.
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A Unique Region of the TM7/Loop/TM8 Domain of the Subunit
Interacts with the
Subunit--
To define more specifically which
region or regions of the
subunit are involved in the
-
interaction, shorter fragments of the region were fused to both the
binding domain (Fig. 4A, pAS
860-903TM7-8 and
pAS
898-928TM7-8) and the activation domain of the GAL4
transcription factor (Fig. 4A,
pAC
898-928TM7-8 and pAC
869-903TM7-8).
Each construct was co-transformed in the yeast with pAS
64-291, pAC
64-291, or the corresponding control plasmid. The results are
presented in Table III. The region close to the seventh transmembrane segment could not be analyzed when linked to the binding domain of GAL4
because of auto-activation found in the construct
pAS
860-903TM7-8. However, when linked to the activation
domain of GAL4 this region of the
subunit was not auto-activating
and showed no interaction with the construct pAC
64-291
(pAC
869-903TM7-8). On the other hand, the downstream
region of the TM7/loop/TM8 domain (pAS
898-928TM7-8)
showed interaction with the
subunit ectodomain when fused to the
binding domain. This sequence was auto-activating when fused to
activating domain (pAC
898-928TM7-8). These results
showed that the region of the
subunit closer to the eighth
transmembrane segment of the H,K-ATPase,
898-928, was responsible
for
-
association in the yeast two-hybrid system.
Two Independent Domains of the Subunit of the Rabbit H,K-ATPase
Interact with the Same Region of the
Subunit--
Six cDNA
fragments of the ectodomain of the
subunit of the rabbit H,K-ATPase
were fused to the activating domain of GAL4 (pAC
64-81,
pAC
64-130, pAC
126-155, pAC
156-188, pAC
186-250, and
pAC
197-291). A diagram of the
subunit is presented on Fig. 4B together with the different
constructs used in the
two-hybrid system. Each of the six clones was transformed with either
the whole TM7/loop/TM8 domain (pAS
869-933TM7-8), the
downstream region of the TM7/loop/TM8 domain
(pAS
898-928TM7-8) or the vector pAC. Results are
presented in Table IV. Two independent sequences of the
subunit ectodomain, one containing 66 amino acids
and positioned between the putative transmembrane domain and the first
disulfide bond (Glu64 to Asn130), and the
second consisting of 32 amino acids and spanning the second disulfide
bond (Ala156 to Arg188) were found to interact
with both
constructs. No interaction was detected with either a
small fragment of 17 amino acids directly adjacent to the transmembrane
domain (pAC64-81) or the fragment of
spanning the first disulfide
bond (pAC
126-155). No analyzable results were possible for the
C-terminal region of the
subunit due to strong auto-activation by
sequences from this region. Two of these are represented in Fig.
4B (pAC
186-250 and pAC
197-291) that gave rise to
auto-activation of the
-galactosidase reporter gene when
co-transformed with pAS. From these results, it can be concluded that
at least two regions of the
subunit interact with the same region
of the
subunit close to the eighth transmembrane domain.
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DISCUSSION |
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It is now well established that strong interactions between the
and the
subunits of the H,K-ATPase are necessary for the stabilization and targeting of a functional pump to the plasma membrane
of transfected cells or Xenopus oocytes and hence presumably to the tubulovesicles or secretory canaliculus of the parietal cell.
Identification of domains involved in assembly of this membrane protein
can therefore lead to a better understanding of not only the general
structure of the pump but also the mechanisms controlling its
biosynthesis and trafficking in the parietal cell.
Several assembly domains have been partially characterized in both the
and the
subunit of the Na,K-ATPase as outlined earlier. There
is direct and indirect evidence for the involvement of the TM7/loop/TM8
extracellular
subunit region in
-
assembly of the H,K-ATPase.
No specific region of the
subunit of this enzyme has been
identified heretofore.
In the present study, we extended our previous work using WGA
fractionation of tryptic digested solubilized H,K-ATPase. The minimal
fragment of which was able to associate with the
subunit was 8 kDa in length from Leu855 to Arg922. The
sequence from Arg922 to Arg948 which includes
the TM8 region did not interact significantly with the
subunit by
this biochemical assay. Since the association is known to occur in the
lumen, the region on the
subunit between TM7 and Arg922
was deduced as being involved in
-
interaction. Although direct, this technique does not allow much flexibility in the size of the
fragments that can be analyzed given the few tryptic cleavage sites in
the loop between TM7 and TM8. Moreover, it can not provide any
information on the regions of the
subunit that are important for
-
association, since there is little quantitative cleavage by
trypsin of this region. Further, it relies on stability of the
interaction in the nonionic detergents used to solubilize the
heterodimer.
Therefore, we took advantage of the sensitivity of the yeast two-hybrid
system for looking for possible interactions between any extracellular
fragments of the and the
subunit of the H,K-ATPase. Several
regions analyzed by this method gave auto-activation, but usually it
was possible to eliminate or minimize this effect by interchanging the
insertion between the activating or the binding domain of the yeast
expression vector.
The five extracellular domains of the subunit, as defined with
little variation in most structural models proposed today for this
subunit, were assayed for their interaction with the entire luminal
domain of the
subunit by using this yeast two-hybrid system. An
interaction between the ectodomain of the
subunit and only the
TM7/loop/TM8 region of
was detected.
Since the yeast two-hybrid system detects interaction in the probable
absence of glycosylation, these data show that glycosylation of the subunit is not required for the association with the
subunit. This
confirms previous observations (11).
Furthermore, the absence of the cytoplasmic and transmembrane domains
in the subunit construct showed that neither of those two domains
is critical for interaction of the two subunits as assayed here. It has
been shown in the case of the Na,K-ATPase, that deletion of the
cytoplasmic and most of the transmembrane domain of the chicken
subunit allowed assembly with the
subunit (26). On the other hand,
analysis of chimeric proteins between
subunits of
Xenopus Na,K-ATPase and rabbit H,K-ATPase that were constructed by exchanging their N-terminal plus transmembrane domain
and their extracytoplasmic COOH-terminal domain have been interpreted
as showing that the transmembrane domain of the
subunit plays an
important role for efficient association with
subunits (27). It has
therefore been proposed that assembly between the
and
ectodomains occurs first and then there is stabilization of the complex
by other interactions. Because of its high sensitivity, the yeast
two-hybrid system is able to detect weak interactions.
No interaction of the subunit with any of the other extra
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Shorter fragments of the subunit used in the yeast two-hybrid
system allowed us to define one unique region from Arg898
to Thr928, as being involved in the interaction with the
subunit. A combination of these results with the biochemical data
reported earlier in this study, shows that a region of only 25 amino
acids on the
subunit, from Arg898 to
Arg922, is the likely region of strong interaction with
. This region contains the four amino acids, SYGQ, shown to be
essential for the interaction between the
and the
subunits of
the Na,K-ATPase by yeast two-hybrid system using alanine scanning
mutagenesis and assumed to be similarly important for the H,K-ATPase
based on sequence similarity (17). On the other hand, previous work, using resistance to tryptic digestion and ouabain binding, showed that
a chimera of the Na,-K-ATPase
subunit containing the region from
Gln905 to Val930 of the rat gastric H,K-ATPase
preferentially assembled with
subunit of H,K-ATPase (22).
Therefore, a homologous 17 amino acid sequence of the
subunit of
the two enzymes, from position 907 to position 924 in the H,K-ATPase
and position 894 to 911 in the Na,K-ATPase, might be a point of stable
contact with the
subunit while differences in the sequence in this
region can account for selective assembly of the
subunits with
their
counterparts. The alignment of this region of the H,K- and
Na,K-ATPases as shown in Scheme 1 suggests that differences in this region, such as the smaller number of
charged amino acids in the H,K-ATPase sequence may account for the
selective association of the two subunits.
No experiment on the interactive regions of the subunit of the
H,K-ATPase has been reported. In the present study, we sought direct
evidence for assembly of specific H,K-ATPase
fragments with
fragments. Six adjacent fragments of
were analyzed with the yeast
two-hybrid system and two domains were found to interact with both the
whole TM7/loop/TM8 domain and the shorter Arg898 to
Thr928 fragment defined above on the
subunit. The first
fragment Gln64 to Asn130 is the extracellular
region directly adjacent to the transmembrane domain ending before the
first disulfide bond. The second fragment is a 32-amino acid fragment
spanning the entire sequence between the second disulfide bond
Ala156 to Arg188. This region of interaction
was deduced from reactivity of a monoclonal antibody, mAb 146 with both
the
and
subunits of the gastric H,K-ATPase (21).
The location of these two fragments showed that the disulfide bonds in
the subunit are nonessential for the assembly of the
heterodimer complex. This was also found to be true in the case of the
Na,K-ATPase when using the yeast two-hybrid system (17). However, the
expression in Xenopus oocytes of different mutants with a
substitution of one or both cysteine residues involved in the three
disulfide bonds of the
-subunit of Torpedo californica Na,K-ATPase has shown that disruption of either the second or the third
disulfide bond prevented
-
association (28). It might be that the
interactions detected by the yeast two-hybrid system are weak
interactions, otherwise not detected by other techniques because of a
lower sensitivity. This is consistent with the finding that deletions
of up to 146 extracellular amino acids from the carboxyl terminus of
the
subunit of the Na,K-ATPase result in less efficient assembly
with the
subunit (29).
Some of the C-terminal hydrophobic amino acids have been shown to be
important in correct assembly of the and
subunits of the
Na,K-ATPase in Xenopus oocytes (18). Unfortunately, the yeast two-hybrid system used here was unable to confirm these data
since all the variations tried in the C-terminal region resulted in
auto-activation.
In summary, both biochemical and yeast two-hybrid analysis
have shown that a single region of the subunit and two
regions of the
subunit of the H,K-ATPase are able to associate in
the absence of other regions. From the data above, the deduced
associative region of the
subunit is contained within the 22 amino
acids between Arg898 and Arg922. The selective
assembly of chimeras narrows this region down to and the two regions of
the extracytoplasmic surface of the
subunit are found between
Gln64 and Asn130 and between Ala156
and Arg188. The latter sequence is between the second
disulfide pair hence disulfide formation is not important for this
interaction. Since there is little or no glycosylation of the nuclear
transcription factors, the yeast two hybrid system shows that
glycosylation is not required for the initial phase of
-
association.
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ACKNOWLEDGEMENT |
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We thank Audree Fowler of the UCLA Microsequencing Facility for sequencing.
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FOOTNOTES |
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* This work was supported in part by United States Veterans Administration Senior Medical Investigator funds and National Institutes of Health Grants DK40615, DK41301, and DK17294.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. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Bldg. 113, Rm. 326, Wadsworth VA Hospital, Los Angeles, CA 90073. Tel.: 310-268-4672; Fax: 310-312-9478.
1 The abbreviations used are: WGA, wheat germ agglutinin; PCR, polymerase chain reaction; TM, transmembrane; Tricine, N-[2- hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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