Regions of Association between the alpha  and the beta  Subunit of the Gastric H,K-ATPase*

Dominique Melle-Milovanovic, Marko Milovanovic, Sunil NagpalDagger , George Sachs§, and Jai Moo Shin

From the Department of Medicine and Physiology, UCLA and Wadsworth Veterans Affairs Hospital, Los Angeles, California 90073 and Dagger  Allergan Pharmaceuticals, Irvine, California 92715

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  and beta  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 alpha  subunit was bound to the beta  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 beta  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 alpha  subunit has strong interaction with the extracytoplasmic domain of the beta  subunit. Again, using yeast two-hybrid analysis, two different sequences in the beta  subunit Gln64 to Asn130 and Ala156 to Arg188 were identified as association domains in the extracytoplasmic sequence of the beta  subunit. These data enable identification of major associative regions of the alpha -beta subunits of the H,K-ATPase.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  subunit of 1035 amino acids with 10 transmembrane segments and a beta  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 beta  subunit, and the two enzymes share about 60 and 40% amino acid homology in their alpha  and beta  subunits, respectively (4). All the catalytic functions of both enzymes are contained within the alpha  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 beta  subunit is required for correct assembly of the enzyme. It has been shown for the Na,K-ATPase that the beta  subunit stabilizes the nascent alpha  subunit in the endoplasmic reticulum and plays a role in targeting the alpha -beta complex to the plasma membrane (7-10). Prevention of glycosylation of the beta  subunit also results in inadequate targeting and loss of catalytic function (11). There are also data showing that modification of the beta  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 alpha  and the beta  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 alpha  subunit. The region of the beta  subunit of the Na,K-ATPase near the first S-S bridge in the extracytoplasmic domain is thought to interact with the alpha  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 beta  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 beta  subunit extracytoplasmic sequence may be a region of interaction with the alpha  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 alpha -beta interaction.

The beta  subunit of H,K-ATPase can act as a surrogate for the beta  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 beta  subunit have provided direct evidence for binding of a region of the H,K-ATPase alpha  subunit, the TM7/loop/TM8 sector, to the beta  subunit (20). Since a monoclonal antibody Abl46 raised against rat parietal cells recognized both an extracytoplasmic epitope on the beta  subunit, between Cys162 and Cys178, and an epitope at the putative extacytoplasmic surface of TM7 on the alpha  subunit, between Ala875 and Asp879, it was suggested that those two regions may also be involved in alpha -beta contact (21). More recently, a chimer of the Na,K-ATPase alpha  subunit containing Gln905 to Val930 of rat gastric H,K-ATPase has been shown to preferentially assemble with H,K-ATPase beta  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 alpha  subunit that interact with the beta  subunit. It was found that the region TM7 to Arg922 was retained by the beta  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 alpha  and the beta  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 alpha  and one beta  construct were assayed by the yeast two-hybrid system. Interaction with the ectodomain of the beta  subunit was detected only with the TM7/loop/TM8 domain of the alpha  subunit. Shorter constructs of both the alpha  and the beta  subunits allowed us to define one unique smaller region in alpha , Arg898 to Thr928, and two different regions in the beta  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 alpha  subunit as being the major region of association with the beta  subunit.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  and beta  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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Plasmids used in the yeast two-hybrid assay

Transformation of S. cerevisiae-- Transformation of yeast was carried out by using the lithium acetate method described by Gietz et al. (25).

beta -Galactosidase Assays-- For qualitative evaluation of regions of contact, beta -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 beta -mercaptoethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside. Filters were then incubated at 30 °C and checked periodically for the appearance of blue colonies.

For quantitative studies, yeast strains were grown to stationary phase in synthetic medium lacking leucine and tryptophan, diluted to 5 × 106 cells/ml, and then incubated at 30 °C for 3-4 h. beta -Galactosidase activity was determined using the chemiluminescent Galacton-Star detection kit according to the manufacturer's instructions (CLONTECH). Values reported are the average of assays of three independent transformants.

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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The TM7/Loop Domain from Leu855 to Arg922 of the alpha  Subunit Associates with the beta  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 alpha  subunit interacts with the beta  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.

When the trypsin digest of solubilized H,K-ATPase was applied to the WGA column, nonbinding fragments were very widely distributed below 60 kDa, including trypsin and trypsin inhibitor (Fig. 1). However, the WGA-retained portion provided four strong fluorescent bands at 20, 11, 8, and 5 kDa. The first three of these fragments had the same N-terminal sequence, LVNEPLAA. Based on molecular weight and N-terminal sequence, the 20-kDa peptide contains the TM7/TM8/TM9/TM10 domain and the 11-kDa peptide contains the TM7/loop/TM8 domain as described previously (20). The 8-kDa peptide fragment corresponds to the sequence beginning at Leu855 and ending at Arg922, representing the TM7 and part of the extracytoplasmic loop. The 5-kDa peptide fragment provided only one N-terminal sequence, NIPELTPY, corresponding to a fragment containing the TM5/loop/TM6 domain.


View larger version (108K):
[in this window]
[in a new window]
 
Fig. 1.   SDS-polyacrylamide gel electrophoresis of WGA column chromatography of trypsin digestion of Nonidet P-40-solubilized H,K-ATPase. Nonidet P-40-solubilized H,K-ATPase was digested with trypsin and applied to a WGA column chromatography as described under "Experimental Procedures." Panel A shows Coomassie-stained gel following SDS-polyacrylamide gel electrophoresis, and panel B shows the fluorescent bands corresponding to the gel of panel A. Lane 1 represents molecular mass standards (106, 80, 47, 31, 24, 17, 16, 14.5, 10.8, 8.2, 6.2, 3.5, and 2.5 kDa). Lane 2 of panel A and panel B shows tryptic fragments of the solubilized H,K-ATPase. Heavy amounts of trypsin and trypsin inhibitor, and auto-digested fragments are shown together with the H,K-ATPase fragments. However, fluorescent bands represent major H,K-ATPase fragments. Lane 3 represents the fragments not retained on the WGA column. Lane 4 represents WGA-retained fragments. Bands a, b, and c were shown at 20, 11, and 8 kDa respectively, having the same N-terminal sequence, LVNEPLAA. Band d shown at 5 kDa provided one N-terminal sequence NIPELTPY.

The three fragments of 20, 11, and 8 kDa are associated with the beta  subunit, which enabled them to be retained by the WGA column. The 5-kDa peptide fragment corresponding to TM5/loop/TM6 seems to be associated with the C terminus containing the 20-kDa fragment, since the 5-kDa fragment disappears when the 20-kDa fragment is eliminated after longer digestion (Fig. 1). These data suggest an association of the beta  subunit with the region extending from TM7 through Arg922.

The Luminal Domain of the Rabbit Gastric H,K-ATPase beta  Subunit Interacts Only with the TM7/loop/TM8 Region of the alpha  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 alpha  subunit were fused to the binding domain of the transcription factor GAL4 (pASalpha 124-161TM1-2, pASalpha 319-337TM3-4, pASalpha 803-833TM5-6, pASalpha 869-933TM7-8, and pASalpha 972-1001TM9-10). Fig. 2 shows the putative structure of the alpha  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 beta  construct corresponding to the whole extracytoplasmic region of the beta  subunit fused to the activation domain of GAL4 (Fig. 4B, pACbeta 64-291). The resulting activation of the beta -galactosidase reporter gene was first checked for each co-transformation using the beta -galactosidase filter lift assay. Then the more sensitive and quantitative beta -galactosidase luminescent assay was performed on those clones showing a blue coloration, i.e. beta -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 pACbeta 64-291 was slightly auto-activating, giving rise to a weak activation of the reporter gene in the absence of an alpha  insert. However, a strong beta -galactosidase activity was observed when the TM7/loop/TM8 domain of the alpha  subunit was fused to the binding domain (pASalpha 869-933TM7-8) and co-transformed with the ectodomain of the beta  subunit fused to the activation domain (pACbeta 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 beta  construct fused to the activation domain is expressed together with the alpha  construct fused to the binding domain than when expressed with pAS. The same combination of alpha  and beta  fragments in the opposite orientation was not informative. The same alpha  subunit region fused to the GAL4 activation domain (pACalpha 860-928TM7-8) gave rise to a very strong auto-activation of the beta -galactosidase reporter gene when transformed with the nonrecombinant pAS2-1 vector (Table III). The beta  ectodomain was not found to interact with any other extracellular loop of the alpha  subunit, namely those loops between TM1 and TM2, TM3 and TM4, TM5 and TM6, and TM9 and TM10 in the yeast two-hybrid analysis.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Schematic representation of the rabbit H,K-ATPase alpha  subunit shows the putative general structure as well as the amino acid numbering.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Schematic representation of the rabbit H,K-ATPase alpha  subunit shows the different extracytoplasmic regions analyzed by the yeast two-hybrid system. TM, transmembrane domain. Boundaries for each construct are indicated by the position of the first and last amino acid, as well as by short segments of sequence corresponding to these positions.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Schematic representation of (A) the TM7/loop/TM8 region of the alpha  subunit and (B) the beta  subunit of the rabbit gastric H,K-ATPase shows the different constructs used in the two-hybrid assay. TM, transmembrane domain. The different constructs were cloned either as a fusion cDNA with the binding domain (BD) or the activation (AD) of the GAL4 transcription factor. Boundaries for each construct are indicated by the position of the first and last amino acid, as well as by short segments of sequence corresponding to these positions. Arrowheads on the beta  subunit point out the seven N-linked glycosylation consensus sequences. The three disulfide bonds are indicated by the filled circles linked by pair.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Interaction of the ectodomain of the beta  subunit with the extracellular loops of the alpha  subunit checked by the yeast two-hybrid assay

                              
View this table:
[in this window]
[in a new window]
 
Table III
Interaction of the ectodomain of the beta  subunit with different parts of the TM7/loop/TM8 region of the alpha  subunit checked by the yeast two-hybrid assay

A Unique Region of the TM7/Loop/TM8 Domain of the alpha  Subunit Interacts with the beta  Subunit-- To define more specifically which region or regions of the alpha  subunit are involved in the alpha -beta interaction, shorter fragments of the region were fused to both the binding domain (Fig. 4A, pASalpha 860-903TM7-8 and pASalpha 898-928TM7-8) and the activation domain of the GAL4 transcription factor (Fig. 4A, pACalpha 898-928TM7-8 and pACalpha 869-903TM7-8). Each construct was co-transformed in the yeast with pASbeta 64-291, pACbeta 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 pASalpha 860-903TM7-8. However, when linked to the activation domain of GAL4 this region of the alpha  subunit was not auto-activating and showed no interaction with the construct pACbeta 64-291 (pACalpha 869-903TM7-8). On the other hand, the downstream region of the TM7/loop/TM8 domain (pASalpha 898-928TM7-8) showed interaction with the beta  subunit ectodomain when fused to the binding domain. This sequence was auto-activating when fused to activating domain (pACalpha 898-928TM7-8). These results showed that the region of the alpha  subunit closer to the eighth transmembrane segment of the H,K-ATPase, alpha 898-928, was responsible for alpha -beta association in the yeast two-hybrid system.

Two Independent Domains of the beta  Subunit of the Rabbit H,K-ATPase Interact with the Same Region of the alpha  Subunit-- Six cDNA fragments of the ectodomain of the beta  subunit of the rabbit H,K-ATPase were fused to the activating domain of GAL4 (pACbeta 64-81, pACbeta 64-130, pACbeta 126-155, pACbeta 156-188, pACbeta 186-250, and pACbeta 197-291). A diagram of the beta  subunit is presented on Fig. 4B together with the different beta  constructs used in the two-hybrid system. Each of the six clones was transformed with either the whole TM7/loop/TM8 domain (pASalpha 869-933TM7-8), the downstream region of the TM7/loop/TM8 domain (pASalpha 898-928TM7-8) or the vector pAC. Results are presented in Table IV. Two independent sequences of the beta  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 alpha  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 beta  spanning the first disulfide bond (pACbeta 126-155). No analyzable results were possible for the C-terminal region of the beta  subunit due to strong auto-activation by sequences from this region. Two of these are represented in Fig. 4B (pACbeta 186-250 and pACbeta 197-291) that gave rise to auto-activation of the beta -galactosidase reporter gene when co-transformed with pAS. From these results, it can be concluded that at least two regions of the beta  subunit interact with the same region of the alpha  subunit close to the eighth transmembrane domain.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Interaction of the extracellular TM7/loop/TM8 region of the alpha  subunit with different parts of the ectodomain of the beta  subunit checked by the yeast two-hybrid assay

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

It is now well established that strong interactions between the alpha  and the beta  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 alpha  and the beta  subunit of the Na,K-ATPase as outlined earlier. There is direct and indirect evidence for the involvement of the TM7/loop/TM8 extracellular alpha  subunit region in alpha -beta assembly of the H,K-ATPase. No specific region of the beta  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 alpha  which was able to associate with the beta  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 beta  subunit by this biochemical assay. Since the association is known to occur in the lumen, the region on the alpha  subunit between TM7 and Arg922 was deduced as being involved in alpha -beta 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 beta  subunit that are important for alpha -beta 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 alpha  and the beta  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 alpha  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 beta  subunit by using this yeast two-hybrid system. An interaction between the ectodomain of the beta  subunit and only the TM7/loop/TM8 region of alpha  was detected.

Since the yeast two-hybrid system detects interaction in the probable absence of glycosylation, these data show that glycosylation of the beta  subunit is not required for the association with the alpha  subunit. This confirms previous observations (11).

Furthermore, the absence of the cytoplasmic and transmembrane domains in the beta  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 beta  subunit allowed assembly with the alpha  subunit (26). On the other hand, analysis of chimeric proteins between beta  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 beta subunit plays an important role for efficient association with alpha  subunits (27). It has therefore been proposed that assembly between the alpha  and beta  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 beta  subunit with any of the other extra
<AR><R><C><UP>Q</UP></C></R><R><C><UP>-</UP></C></R><R><C><UP>N</UP></C></R></AR><AR><R><C><UP>D</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>D</UP></C></R></AR><AR><R><C><UP>LQ</UP></C></R><R><C><UP> -</UP></C></R><R><C><UP>VE</UP></C></R></AR><AR><R><C><UP>D</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>D</UP></C></R></AR><AR><R><C><UP>S</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>S</UP></C></R></AR><AR><R><C><UP>Y</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>Y</UP></C></R></AR><AR><R><C><UP>G</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>G</UP></C></R></AR><AR><R><C><UP>QE</UP></C></R><R><C><UP> ‖</UP></C></R><R><C><UP>QQ</UP></C></R></AR><AR><R><C><UP>W</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>W</UP></C></R></AR><AR><R><C><UP>T</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>T</UP></C></R></AR><AR><R><C><UP>FG</UP></C></R><R><C><UP> -</UP></C></R><R><C><UP>YE</UP></C></R></AR><AR><R><C><UP>Q</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>Q</UP></C></R></AR><AR><R><C><UP>R</UP></C></R><R><C><UP>‖</UP></C></R><R><C><UP>R</UP></C></R></AR><AR><R><C><UP> H,K   907–922</UP></C></R><R><C></C></R><R><C><UP> Na,K   894–909</UP></C></R></AR>
Scheme 1.
cellular loops of the alpha  subunit was detected other than that contained within the TM7/loop/TM8 region. Although negative results should be viewed with caution since problems may exist in the expression, targeting, and stability of the fusion protein, this result suggests that the two subunits have strong interaction only in the region of the TM7/loop/TM8 domain. This finding confirms our previous biochemical data (20).

Shorter fragments of the alpha  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 beta  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 alpha  subunit, from Arg898 to Arg922, is the likely region of strong interaction with beta . This region contains the four amino acids, SYGQ, shown to be essential for the interaction between the alpha  and the beta  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 alpha  subunit containing the region from Gln905 to Val930 of the rat gastric H,K-ATPase preferentially assembled with beta subunit of H,K-ATPase (22). Therefore, a homologous 17 amino acid sequence of the alpha  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 beta  subunit while differences in the sequence in this region can account for selective assembly of the beta  subunits with their alpha 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 beta  subunit of the H,K-ATPase has been reported. In the present study, we sought direct evidence for assembly of specific H,K-ATPase beta  fragments with alpha  fragments. Six adjacent fragments of beta  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 alpha  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 alpha  and beta  subunits of the gastric H,K-ATPase (21).

The location of these two fragments showed that the disulfide bonds in the beta  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 beta -subunit of Torpedo californica Na,K-ATPase has shown that disruption of either the second or the third disulfide bond prevented alpha -beta 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 beta  subunit of the Na,K-ATPase result in less efficient assembly with the alpha  subunit (29).

Some of the C-terminal hydrophobic amino acids have been shown to be important in correct assembly of the alpha  and beta  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 alpha  subunit and two regions of the beta  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 alpha  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 beta  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 alpha -beta association.

    ACKNOWLEDGEMENT

We thank Audree Fowler of the UCLA Microsequencing Facility for sequencing.

    FOOTNOTES

* 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Bamberg, K., Mercier, F., Reuben, M. A., Kobayashi, Y., Munson, K. B., and Sachs, G. (1992) Biochim. Biophys. Acta 1131, 69-77[Medline] [Order article via Infotrieve]
  2. Reuben, M. A., Lasater, L. S., and Sachs, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6767-6771[Abstract]
  3. Tyagarajan, K., Lipniunas, P. H., Townsend, A. A., and Forte, J. G. (1997) Biochemistry 36, 10200-10212[CrossRef][Medline] [Order article via Infotrieve]
  4. Lingrel, J. B., Orlowski, J., Shull, M. M., and Price, E. M. (1990) Prog. Nucleic Acid Res. Mol. Biol. 38, 37-89[Medline] [Order article via Infotrieve]
  5. Palasis, M., Kuntzweiler, T. A., Arguello, J. M., and Lingrel, J. B (1996) J. Biol. Chem. 271, 14176-14182[Abstract/Free Full Text]
  6. Besanáon, M., Shin, J. M., Mercier, F., Munson, K., Miller, M., Hersey, S., and Sachs, G. (1993) Biochemistry 32, 2345-2355[Medline] [Order article via Infotrieve]
  7. Geering, K., Kraehenbuhl, J. P., and Rossier, B. C. (1987) J. Cell Biol. 105, 2613-2619[Abstract]
  8. Noguchi, S., Higashi, K., and Kawamura, M. (1990) J. Biol. Chem. 265, 15991-15995[Abstract/Free Full Text]
  9. Geering, K. (1991) FEBS Lett. 285, 189-193[CrossRef][Medline] [Order article via Infotrieve]
  10. Jaunin, P., Horisberger, J. D., Richter, K., Good, P. J., Rossier, B. C., and Geering, K. (1992) J. Biol. Chem. 267, 577-585[Abstract/Free Full Text]
  11. Klassen, C. H. W., Fransen, J. A. M., Swarts, H. G. P., and De Pont, H. H. M. (1997) Biochem. J. 321, 419-424[Medline] [Order article via Infotrieve]
  12. Lutsenko, S., and Kaplan, J. H. (1993) Biochemistry 32, 6737-43[Medline] [Order article via Infotrieve]
  13. Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255-8259[Abstract/Free Full Text]
  14. Fambrough, D. M., Lemas, M. V., Hamrick, M., Emerick, M., Renaud, K. J., Inmam, E. M., Hwang, B., and Takeyasu, K. (1994) Am. J. Physiol. 266, C579-C589[Abstract/Free Full Text]
  15. Beggah, A. T., Beguin, P., Jaunin, P., Peitsch, M. C., and Geering, K. (1993) Biochemistry 32, 14117-14124[Medline] [Order article via Infotrieve]
  16. Geering, K., Jaunin, P., Jaisser, F., Merilatt, A. M., Horisberger, J. D., Mathews, P. M., Lemas, V., Fambrough, D. M., and Rossier, B. C. (1993) Am. J. Physiol. 265, C1169-C1174[Abstract/Free Full Text]
  17. Colonna, T. E., Huynh, L., and Fambrough, D. M. (1997) J. Biol. Chem. 272, 12366-12372[Abstract/Free Full Text]
  18. Horisberger, J. D., Jaunin, P., Reuben, M. A., Lasater, L. S., Chow, D. C., Forte, J. G., Sachs, G., Rossier, B. C., and Geering, K. (1991) J. Biol. Chem. 266, 19131-19134[Abstract/Free Full Text]
  19. Gottardi, C. J., and Caplan, M. J. (1993) J. Biol. Chem. 268, 14342-14347[Abstract/Free Full Text]
  20. Shin, J. M., and Sachs, G. (1994) J. Biol. Chem. 269, 8642-8646[Abstract/Free Full Text]
  21. Mercier, F., Bayle, D., Besançon, M., Joys, T., Shin, J. M., Lewin, M. J. M., Prinz, C., Reuben, M. A., Soumarmon, A., Wong, H., Walsh, J. H., and Sachs, G. (1993) Biochem. Biophys. Acta 1149, 151-165[Medline] [Order article via Infotrieve]
  22. Wang, S. G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997) Am. J. Physiol. 272, C923-C930[Abstract/Free Full Text]
  23. Rabon, E. C., Im, W. B., and Sachs, G. (1988) Methods Enzymol. 157, 649-654[Medline] [Order article via Infotrieve]
  24. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve]
  25. Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve]
  26. Renaud, K. J., Inman, E. M., and Fambrough, D. M. (1991) J. Biol. Chem. 266, 20491-20497[Abstract/Free Full Text]
  27. Jaunin, P., Jaisser, F., Beggah, A. T., Takeyasu, K., Mangeat, P., Rossier, B. C., Horisberger, J. D., and Geering, K. (1993) J. Cell Biol. 123, 1751-1759[Abstract]
  28. Noguchi, S., Mutoh, Y., and Kawamura, M. (1994) FEBS Lett. 341, 233-238[CrossRef][Medline] [Order article via Infotrieve]
  29. Hamrick, M., Renaud, K. J., and Fambrough, D. M. (1993) J. Biol. Chem. 268, 24367-24373[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.