Interaction of alpha - and beta -subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta -subunit

Curtis T. Okamoto1,*, Dar C. Chow2,*, and And John G. Forte2

1 Department of Pharmaceutical Sciences, University of Southern California, Los Angeles 90089-9121; and 2 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The assembly of the beta -subunit of the gastric H-K-ATPase (HKbeta ) with the alpha -subunit of the H-K-ATPase or the Na-K-ATPase (NaKalpha ) was characterized with two anti-HKbeta monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKbeta , MAb 2/2E6 was observed to bind to HKbeta only when interactions between alpha - and beta -subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S226LHY229 of the extracellular domain of HKbeta . The epitope for MAb 2G11 was mapped to the eight NH2-terminal amino acids of the cytoplasmic domain of HKbeta . In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKbeta with alpha -subunits of the endogenous cell surface NaKalpha , as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKbeta . In HKbeta -transfected LLC-PK1 cells, significant immunofluorescent labeling of HKbeta at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKbeta could also be coimmunoprecipitated with NaKalpha . Thus assembly of HKbeta with NaKalpha does not appear to be a stringent requirement for cell surface delivery of HKbeta in LLC-PK1 cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid alpha -beta heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKbeta that is associated with alpha -beta interaction.

sodium-potassium-adenosinetriphosphatase; Madin-Darby canine kidney cells; LLC-PK1 cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE BIOSYNTHETIC ASSEMBLY of the Na-K-ATPase, the best-characterized member of the P-type ion transporters, has been extensively studied (see Refs. 8, 16, and 33 for review). The minimal functional unit of the Na-K-ATPase consists of a polytopic alpha -subunit and a glycosylated, single membrane-spanning beta -subunit. Additional tools for the investigation and characterization of interactions between alpha - and beta -subunits in ATPases have been provided by the identification and characterization of a beta -subunit from another member of the P-type ATPases, the gastric H-K-ATPase (5, 21, 36, 37, 41), and of homologs of the alpha -subunit of the gastric H-K-ATPase expressed in colon (10, 11) and kidney cells (4, 24, 28).

The alpha -subunit is typically cited as the catalytic subunit for the H-K-ATPase and Na-K-ATPase, but enzymatic function is dependent on the interaction between the respective alpha - and beta -subunits (7, 15, 23). Therefore, identification of subunit interactive sites is important to understand the functional cycle of these cation-exchange ATPases. The association between subunits is stable to mild detergents, such as Triton X-100 or Nonidet P-40, whereas the forces of interaction are clearly disrupted by denaturing detergents, such as dodecyl trimethyl ammonium bromide (DTAB) or SDS. These detergent-sensitive properties were first used to demonstrate the existence of a beta -subunit for the H-K-ATPase (HKbeta ) (36) and have also been exploited to identify specific segments of the alpha -subunit that interact with the beta -subunit (40). Recently, Wang et al. (43), using a heterologous yeast expression system to monitor assembly of transfected chimeric ATPase subunits, found a region in an extracellular domain of the alpha -subunit of the H-K-ATPase (HKalpha ) spanning Gln-905 to Val-930 that interacts with the extracellular domain of HKbeta . In addition, Melle-Milovanovic et al. (34) used the yeast two-hybrid system to probe for sites of interaction between alpha - and beta -subunits. They reported two regions in the ectodomain of the beta -subunit, Gln-64 to Asn-130 and Ala-156 to Arg-188, as containing sites of interaction with the alpha -subunit. Geering et al. (17) demonstrated that a sequence motif Y242Y(or F)PYY in the ectodomain of the Na-K-ATPase beta -subunit (NaKbeta ) is conserved among all beta -subunits, and the presence of P244 in this motif is essential for assembly of Na-K-ATPase alpha - and beta -subunits. Moreover, consistent with the results of Wang et al., Melle-Milovanovic et al. showed that the region Arg-898 to Arg-922 in the alpha -subunit interacts strongly with the extracytoplasmic domain of the beta -subunit. The ability to form hybrid alpha -beta heterodimers between the Na-K-ATPase and the H-K-ATPase has provided significant insight into not only the regulation of assembly of native alpha -beta heterodimers but also the role of the beta -subunit in the modulation of transport activities of the holoenzyme (15, 19, 22, 23, 30).

We previously reported on two monoclonal antibodies (MAbs) against HKbeta : MAb 2G11, which bound to the native enzyme at a site within the cytoplasmic NH2-terminal region, and MAb 2/2E6, which bound within the large extracytoplasmic segment, but only when the enzyme was denatured (7). An objective for the present work was to test the hypothesis that MAb 2/2E6 recognizes a specific site of interaction between alpha - and beta -subunits and to identify the epitope within HKbeta .

Another objective of this work was to use the MAbs against topologically distinct regions of HKbeta to study the regulation of differential assembly of HKbeta and its impact on plasma membrane delivery of P-type ATPases in epithelial cells, a topic that has received relatively little attention (6). Investigation of this phenomenon is critically important to understanding the physiology of transport across epithelia, inasmuch as the Na-K-ATPase is responsible for the maintenance of transepithelial ion gradients that, in turn, drive the transepithelial transport of other ions and nutrients. Moreover, in the gastric acid-secreting oxyntic cell, the mechanism by which the Na-K-ATPase and the H-K-ATPase are separately targeted to the basolateral or apical membranes, respectively, must be precise, notwithstanding the high degree of homology between the alpha - and beta -subunits of the pump enzymes (6). A similar process may exist in colon and kidney. When expressed in Xenopus, HKbeta has been shown to act as a surrogate for conveying Na+-pumping functions for the alpha -subunit of the Na-K-ATPase (NaKalpha ) (23). However, the situation appeared to be quite different when Gottardi and Caplan (18) transfected HKbeta in a mammalian epithelial cell line, LLC-PK1 cells. These authors reported that the expressed HKbeta was exclusively targeted to the apical plasma membrane and endogenous Na-K-ATPase was targeted to its standard basolateral location, with very poor efficiency of HKbeta /NaKalpha assembly or surrogation of activity. In the present work, HKbeta was transfected into two polarized kidney cell lines, MDCK cells and LLC-PK1 cells, and the biosynthesis, assembly, and cell surface expression of HKbeta were investigated.


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MATERIALS AND METHODS
RESULTS
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Materials. All chemicals were reagent grade. The anti-HKbeta MAbs 2/2E6 and 2G11 have been previously described (7). The MAb against HKalpha was the kind gift of Dr. Adam Smolka (Medical University of South Carolina, Charleston, SC). The polyclonal anti-Na-K-ATPase antibodies were a kind gift from Dr. W. James Nelson (Stanford University, Palo Alto, CA). The anti-NaKalpha MAb 6H (18) was obtained as a hybridoma supernatant from Dr. Alicia McDonough (University of Southern California, Los Angeles, CA). Prestained molecular weight and secondary antibodies coupled to horseradish peroxidase (HRP) were purchased from Bio-Rad (Hercules, CA). Pro-mix L-[35S]amino acids in vitro cell labeling mix, streptavidin coupled to HRP, enhanced chemiluminescence (ECL) kit, protein A-Sepharose, protein G-Sepharose, and Sepharose CL-2B were purchased from Amersham Pharmacia Biotech. Trans-35S-label in vitro labeling mix was purchased from ICN (Irvine, CA). Sulfosuccinimidobiotin (sulfo-NHS-biotin) was purchased from Pierce (Rockford, IL). MEM was purchased from Mediatech (Washington, DC). Cys- and Met-free DMEM, MEM with Hanks' salts, clostripain, and streptavidin-agarose were purchased from Sigma-Aldrich. Penicillin, streptomycin, amphotericin B (Fungizone) cocktail, and Hanks' balanced salt solution (HBSS) were purchased from the Cell Culture Facility at the University of California, San Francisco, Bio-Whittaker (Washington, DC), or Mediatech. FBS was purchased from Hyclone Laboratories (Logan, UT). G418 and 14C-labeled molecular weight markers were purchased from Life Technologies (Bethesda, MD). Endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) were purchased from Boehringer Mannheim. Lysyl endopeptidase C (Lys C) was obtained from Wako Chemical (Osaka, Japan). 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF) · HCl was purchased from Calbiochem-Novachem (La Jolla, CA). Pepstatin, leupeptin, chymostatin, antipain, and benzamidine were purchased from Chemicon (Temecula, CA). Wheat germ agglutinin (WGA) coupled to agarose was purchased from E-Y Laboratories (San Mateo, CA).

Immunohistochemistry of gastric glands and cell cultures. Gastric glands were isolated as previously described (1). The glands were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature, then they were permeabilized by 1% Triton X-100 in PBS for 15 min. The fixed, permeabilized glands were immunostained directly or subjected to further treatment with denaturants before they were immunostained. Treatment with urea or detergents was carried out at room temperature, and the treated glands were subsequently washed with PBS before incubation with antibodies. The treated cells were incubated for 1 h with MAbs against HKbeta (MAb 2/2E6 or MAb 2G11), washed three times in PBS, and incubated with fluorescent-labeled anti-mouse IgG antibody for 1 h. Previous work has shown that the epitope on HKbeta for MAb 2/2E6 is in the extracellular domain and that for MAb 2G11 is in the NH2-terminal cytoplasmic tail (7).

To localize the expressed HKbeta in transfected MDCK cells or LLC-PK1 cells, confluent monolayers of cells were fixed in 3.7% formaldehyde in PBS for 20 min. The cells were then treated with denaturants and/or permeabilizing agents, as described in individual experiments, to immunostain HKbeta in the plasma membrane pool or the intracellular pool. Treatment with primary and secondary antibodies was as described above.

Stained glands and cells were examined by conventional fluorescence microscopy or by confocal microscopy. Confocal images of cells were taken with a Bio-Rad MRC-600 equipped with a Nikon ×60 plan-Apo NA 1.4 oil-immersion objective. A series of confocal sections were taken in the horizontal (x-y) plane with 0.5-mm vertical spacing. Vertical (x-z) plane reconstructions were made from the confocal section series by use of the MRC-600 software.

Biochemical characterization of alpha - and beta -subunit interaction. H-K-ATPase-enriched gastric microsomes were prepared as previously described (29). As a general protocol, the microsomes were solubilized for 1 h at room temperature in 1% Triton X-100 in suspending medium (SM) consisting of 300 mM sucrose, Tris · HCl (pH 7.4), and 0.5 mM EDTA. The solubilized materials were applied to different affinity matrices before or after the H-K-ATPase was subjected to several modifying procedures.

The antibody-protein A and the antibody-protein G columns were made by mixing protein A-agarose or protein G-agarose, respectively, with a saturating amount of antibody in culture media. WGA-agarose beads were used to immobilize HKbeta .

FITC labeling of H-K-ATPase was carried out with 6 mg of microsomal protein in 2 ml of 100 mM MOPS (pH 8.0), 2% Triton X-100, and 2 mg of FITC at room temperature for 1 h. The labeled material was passed through a column containing 1.5 ml of WGA to immobilize the H-K-ATPase. The column was washed overnight with 100 ml of SM containing 1% Triton X-100. The column material was divided into seven smaller columns, each of which was washed with 2.5 ml of SM with a specified concentration of DTAB or SDS. The washes were collected, and optical density was measured at 494 nm to estimate the FITC in the washes. After the columns were washed, 10 ml of MAb 2/2E6 culture medium was passed through the columns. The columns were further washed with 15 ml of SM containing 1% Triton X-100 and then SM without Triton X-100. The beads were collected in Eppendorf tubes, 0.3 ml of nonreducing 4× sample buffer was added, and aliquots were taken for SDS-PAGE and Western blotting.

In some experiments, Triton X-100-solubilized H-K-ATPase was treated with urea. The solubilized material was split into two aliquots. In one sample, urea was added to 6 M, the other served as control. Both samples were incubated at room temperature for 1 h and then diluted threefold or more before they were passed through WGA columns. After the columns were washed, samples of the beads were aliquoted and 0.3 ml of nonreducing 4× sample buffer was added to release bound materials.

Phage display peptide library kit. The heptapeptide display library phage kit was purchased from New England Biolabs. Phage selection, amplification, and DNA extraction were carried out according to manufacturer's instructions. Using polystyrene petri dishes coated with 2/2E6 antibody as selection plates, we carried out four rounds of selection (biopanning) to choose a pool of phages. Twenty individual clones were isolated, amplified, and analyzed with Western blot for 2/2E6. Seven of them showed that their minor coat protein strongly reacted with 2/2E6. The corresponding DNA sequences were sequenced, and the amino acids were deduced. Synthetic peptides were synthesized by BioMed. Competitive Western blots were carried out with primary antibody preincubated with the synthetic peptide for 1 h.

Construction of HKbeta clones and transfection. cDNA encoding rat HKbeta (5) was excised from the pBluescript SK(-) vector (Stratagene) with Apa I and Avr II and subcloned into pCB6, a vector in which the expression of the insert cDNA is driven by a cytomegalovirus promoter (2). The Apa I-digested end was filled in with Klenow and blunt-end ligated into the unique Hind III site of pCB6; the Avr II-cut end was ligated into the Xba I site. MDCK strain II cells, which are of European Molecular Biology Lab parentage and are the clone that delivers newly synthesized Na-K-ATPase exclusively to the basolateral membrane, were transfected with this plasmid by the calcium phosphate method, as described previously (35). After selection in G418, four positive clones were identified, expanded, and maintained in MEM supplemented with 5% FBS and antibiotic-antimycotic. The polarity of these four clones was verified by assaying for polarized secretion of the endogenous MDCK glycoprotein gp80, as described previously (42). The apical-to-basal ratio of gp80 secretion in all the clones described here was >= 4:1. The LLC-PK cells used in this study, stably transfected with HKbeta (18), were the kind gift of Dr. Michael Caplan (Yale University).

Cell surface biotinylation. Cells grown on Costar 24-mm Transwell polycarbonate filters were cooled to 4°C with several washes of cold HBSS supplemented with 20 mM sodium HEPES, pH 7.4. The apical or basolateral surface of transfected MDCK cells was biotinylated with 0.5 mg/ml sulfo-NHS-biotin in HBSS with two 15-min incubations at 4°C. After biotinylation, cells were washed quickly three times with cold HBSS and incubated for 15 min at 4°C with cold MEM supplemented with 0.6% BSA, 20 mM sodium HEPES, and 50 mM glycine to quench any remaining unreacted biotin. The cells were lysed under denaturing conditions (0.5% SDS, 100 mM NaCl, 50 mM triethanolamine · HCl, 5 mM EDTA, 0.1 mM AEBSF, and 0.2% NaN3, pH 8.1) or nondenaturing conditions [2.5% (wt/vol) Triton X-100, 100 mM NaCl, 100 mM triethanolamine · HCl, 5 mM EDTA, 0.1 mM AEBSF, 0.2% NaN3, 5 µg/ml each of pepstatin and leupeptin, 10 µg/ml each of chymostatin and antipain, and 0.5 mM benzamidine]. After cell lysates were precleared with Sepharose CL-2B, HKbeta was immunoprecipitated with MAb 2/2E6-protein A-Sepharose (specific for the ectodomain of HKbeta ) or MAb 2G11-protein G-Sepharose (specific for the cytoplasmic domain of HKbeta ), and biotinylated proteins were isolated with streptavidin-agarose. Immunoprecipitates were run on SDS-PAGE, transferred to nitrocellulose, and probed with MAb 2/2E6 to detect HKbeta , anti-NaKalpha monoclonal or polyclonal antibodies to detect NaKalpha , or streptavidin-HRP to detect biotinylated proteins. After incubation with appropriate secondary antibodies coupled to HRP (if necessary), the reactive proteins were visualized by ECL. Signals from ECL blots were quantitated on a Bio-Rad GS-670 imaging densitometer.

Pulse-chase analysis of HKbeta . Transfected MDCK or LLC-PK1 cells grown on 24-mm Transwell plates were washed with PBS containing Ca2+ and Mg2+ and starved for 15 min at 37°C with Cys- and Met-free DMEM supplemented with 5% dialyzed FBS, 20 mM sodium HEPES (pH 7.4), and antibiotic-antimycotic. Cells were pulse labeled with 50 µCi of Pro-mix (New England Nuclear) or Trans-35S-label (ICN, Irvine, CA) L-amino acids from the basolateral surface and chased for various periods. Cells were lysed under denaturing or nondenaturing conditions, as described above. Lysates were processed for immunoprecipitation, as described above. Immunoprecipitates were run on SDS-PAGE and fluorographed. In some cases, immunoprecipitates of pulse-labeled HKbeta were digested with Endo H before SDS-PAGE and fluorography. The immunoprecipitated material was resuspended by boiling in 0.1 M sodium citrate (pH 6.0) and 1% SDS; Endo H was added to 3 mU, and the material was incubated overnight at 37°C.


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Immunostaining of parietal cells by 2/2E6 requires denaturation. When we first introduced MAb 2/2E6, we pointed out that immunostaining did not occur with conventional formaldehyde fixation and permeabilization by Triton X-100 but "required alcohol delipidation of the glands." Figure 1 shows that H-K-ATPase of Formalin-fixed parietal cells is immunostained by MAb 2/2E6 only when a denaturant is applied (e.g., alcohol, SDS, DTAB, alkali). A titration with urea as a denaturant is shown in Fig. 2. For gastric glands fixed by Formalin and permeabilized with Triton X-100, immunostaining by 2/2E6 was barely evident up to 4.5 M urea and became uniformly apparent at 5 M urea. These data demonstrate that the epitope for 2/2E6 is not simply related to delipidation but, rather, is buried and becomes exposed only after denaturation.


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Fig. 1.   H-K-ATPase of Formalin-fixed parietal cells is immunostained by monoclonal antibody (MAb) 2/2E6 only when a denaturant is applied. Gastric glands were fixed with Formalin and then subjected to a series of permeabilization procedures as follows: 1% Triton X-100 for 15 min (TX-100), 1% Triton X-100 for 15 min followed by a brief exposure to methanol (TX-100 + MeOH), 0.5% dodecyl trimethyl ammonium bromide for 15 min (DTAB), and 0.5% SDS for 15 min (SDS). Glands were then probed with MAb 2/2E6 followed by secondary FITC-labeled anti-mouse antibody and examined by fluorescence (top) and differential interference (bottom) microscopy. All fluorescent micrographs were captured with identical times of exposure. Scale bar, 20 µm.



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Fig. 2.   Immunostaining by MAb 2/2E6 occurs when H-K-ATPase is denatured by urea. Formalin-fixed gastric glands were permeabilized with 1% Triton X-100 and subjected to titration with 0-5.5 M urea before they were probed with MAb 2/2E6. All fluorescent micrographs were captured with identical times of exposure. Scale bar, 20 µm.

Dissociation of alpha - and beta -subunits exposes the 2/2E6 epitope. We previously reported that alpha -beta association was stable in nonionic detergents and disrupted by ionic detergents, such as DTAB or SDS (36). The unmasking of the 2/2E6 epitope by DTAB or SDS in fixed gastric glands further supported an epitopic site within the sphere of alpha -beta interaction. To investigate the action of SDS on the alpha -beta association and unmasking of 2/2E6 epitope, we treated purified H-K-ATPase-containing membranes with various amounts of SDS, then added Triton X-100 to mop up the excess SDS. The treated samples were passed through a 2/2E6-protein A affinity column. The bound materials were eluted with 2% SDS and analyzed by SDS-PAGE and Western blot, as shown Fig. 3. For samples where the membranes were treated with >= 0.3% SDS, HKbeta bound to the 2/2E6 column, demonstrating that these levels of SDS were sufficient to expose the binding site.


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Fig. 3.   SDS dissociates subunits of H-K-ATPase and exposes epitope for MAb 2/2E6 on beta -subunit. H-K-ATPase-rich vesicles were treated with 0-1% SDS. An excess of Triton X-100 (10-fold volume dilution in 2% Triton X-100) was added to all samples, which were then passed through a 2/2E6-protein A affinity column. Bound materials were eluted with 2% SDS, separated by SDS-PAGE, and developed by silver staining. A significant quantity of beta -subunit was adsorbed by 2/2E6 column only when original treatment with SDS was >= 0.3%. Right lane shows a sample of control, untreated microsomes run of same gel, where relative amounts of alpha - and beta -subunits can be seen in starting membrane material. mw, Molecular mass (in kDa).

In an alternative test, we immobilized Triton X-100-solubilized H-K-ATPase on a series of WGA columns and then washed the columns with various amounts of SDS. After the SDS washes were collected, 2/2E6 antibody was passed through the columns. The dissociated alpha -subunit that appeared in the SDS washes and the 2/2E6 that bound to the column were separately analyzed by SDS-PAGE and Western blot. The results in Fig. 4 show that increasing the amount of SDS progressively eluted the HKalpha from the columns and exposed the 2/2E6 epitope on the bound HKbeta . Quantitation of the blots indicated a strong correlation between the amount of HKalpha liberated and the amount of 2/2E6 bound, as a function of SDS concentration. In separate tests of Coomassie blue-stained gels, we were able to estimate that the amount of IgG heavy chain bound to the column was stoichiometrically equivalent to the HKalpha eluted, suggesting that the 2/2E6 antibody bound quantitatively to most of the vacant sites left by the eluted HKalpha (data not shown).


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Fig. 4.   SDS separates alpha - and beta -subunits and exposes 2/2E6 epitope. H-K-ATPase was solubilized in 1% Triton X-100 and immobilized on a series of wheat germ agglutinin (WGA) columns. Individual columns were exposed to graded amounts of SDS, and respective eluants were probed by Western blotting for alpha -subunit (effluent HKalpha ). MAb 2/2E6 was subsequently passed through columns, and, after thorough washing, bound 2/2E6 was released by 2% SDS and probed with horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (2/2E6 bound). Locations of molecular mass standards are indicated on right. Densitometric quantitation of blots (bottom) reveals that release of effluent HKalpha by SDS was roughly parallel to exposure of 2/2E6 epitope on WGA-bound beta -subunit of H-K-ATPase (HKbeta ). Values are expressed relative to %bound at 1% SDS.

We also used urea and the cationic detergent DTAB as alternative agents to dissociate the alpha - and beta -subunits. Similar to the results obtained with SDS, there was good correlation between the amount of HKalpha liberated and the amount of the MAb 2/2E6 bound to HKbeta as the concentration of these agents was increased (data not shown).

The 2/2E6 epitope is identified as S226LHY229. The heptapeptide phage display library was used as described in METHODS AND MATERIALS to screen the peptide sequence corresponding to the epitope for MAb 2/2E6 binding. Seven individual clones of the phage were selected for amplification and DNA sequencing. The corresponding peptide sequences are shown in Table 1. Inspection of these sequences indicates that the common sequence for the epitope is SXHY. The most likely site is SLHY, corresponding to amino acids 226-229 of the rabbit HKbeta sequence.

                              
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Table 1.   Amino acid sequences that bind to MAb 2/2E6 revealed by the phage peptide display system

To confirm that SLHY is the epitope, we used a synthetic peptide corresponding to amino acids 224-239 to characterize the interaction. Culture medium containing 2/2E6 was mixed with increasing amounts of the peptide and then used for Western blots of the HKbeta . As shown in Fig. 5, this synthetic peptide competitively inhibited reaction of 2/2E6 with HKbeta . Moreover, the synthetic peptide binds to 2/2E6 coupled to protein G-agarose, but not to 2G11 coupled to protein G-agarose (data not shown). Thus the epitope recognized by 2/2E6 is located at the quadrapeptide S226LHY229.


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Fig. 5.   The 16-mer peptide corresponding to amino acids 224-239 of rabbit gastric beta -subunit competes with MAb 2/2E6 in binding to HKbeta . Culture medium containing 2/2E6 was mixed with 0-50 µg of peptide, and mixtures were used for Western blots of H-K-ATPase-rich microsomes. Broad band between 60 and 80 kDa is strongly visible in control with no peptide present, whereas signal was correspondingly decreased as peptide concentration was increased.

MAb 2G11 interacts with the NH2-terminal eight amino acids of the beta -subunit. Earlier work showed that MAb 2G11 binds to the cytoplasmic domain of HKbeta (7). Through the use of site-specific proteases, we now provide a more definitive binding site. Figure 6, A and B, shows that, after digestion of microsomal vesicles with Arg-specific clostripain and subsequent deglycosylation with PNGase F, distinctly different peptides are recognized by our two antibody probes: a peptide of ~30 kDa is recognized by MAb 2/2E6 and a peptide of ~2 kDa is recognized by MAb 2G11. The NH2-terminal cytoplasmic domain of the rabbit HKbeta has potential Arg cleavage sites at Arg-13 and Arg-18, which would be predicted to yield NH2-terminal peptides of ~2.2 and 1.5 kDa, respectively, as indicated by the following sequence: MAALQEK7K8SCSQR13MEEFR18HYCWN PDT. . . . .


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Fig. 6.   Epitope for MAb 2G11 is within 8 most NH2-terminal amino acids of HKbeta . H-K-ATPase-rich microsomal vesicles (100 µl containing 0.2 mg of protein) were digested with 10 µg of clostripain (Arg-C) or lysyl endoproteinase C (Lys-C) for 16 h at 37°C. Digestions were terminated by boiling in 0.5% SDS for 5 min. Samples were diluted 10- to 20-fold, 5 U of peptide N-glycosidase F (PNGase F) were added to deglycosylate, and samples were incubated at room temperature overnight. Samples were separated on 10% or 12% tricine gels and blotted to nitrocellulose. A: digestion by Arg-specific Arg-C produced a large beta -subunit fragment of ~30 kDa recognized by MAb 2/2E6 and a small fragment of ~2 kDa recognized by MAb 2G11. Lys-C digestion also produced a similar ~30-kDa fragment recognized by MAb 2/2E6, but it completely eliminated any peptide recognition by MAb 2G11. B: 2nd experiment of Arg-C digestion, but because of incomplete deglycosylation by PNGase F, some faint staining with 2/2E6 (but not with 2G11) can be seen at 60-80 kDa for fully glycosylated beta -subunit.

The MAb 2G11-positive band would be consistent with either arginyl peptide, given the limited resolution of the gel. The 30-kDa MAb 2/2E6-positive band from clostripain digestion would be consistent with the remaining portion representing the transmembrane and extracellular domains of the 32-kDa deglycosylated core HKbeta . When the microsomal vesicles were digested with Lys C, which is specific for Lys residues, the NH2-terminal peptide was not recognized (Fig. 6A), suggesting the possibility that cleavage at Lys-7 or Lys-8 eliminated the site of 2G11 interaction, although the positive response with MAb 2/2E6 reveals that the bulk of the beta -subunit is still present. Taken together, these data demonstrate that the epitope for interaction of MAb 2G11 is proximal to the eight NH2-terminal amino acids in HKbeta .

Immunostaining of HKbeta expressed in LLC-PK1 cells. Gottardi and Caplan (18) stably transfected a kidney cell line, LLC-PK1 cells, with a gene for HKbeta and subsequently showed that HKbeta is synthesized in the endoplasmic reticulum (ER) and sorted to the apical plasma membrane without an accompanying alpha -subunit. When we employed this same stably transfected LLC-PK1 cell line, we observed a strong reaction of MAb 2/2E6 with the apical plasma membrane, even when the antibody was added to live cells (Fig. 7A). That is, neither fixation nor permeabilization was required for surface staining of the 2/2E6 epitope, although these treatments, without denaturation, were effective in exposing additional beta -subunit sites within an intracellular membrane compartment(s) (Fig. 7B). On the other hand, the MAb 2G11 epitope is exposed after only permeabilization of transfected cells (Fig. 7, C and D). Thus, in the LLC-PK1 expression system, reaction of HKbeta with 2/2E6 did not require denaturation, and since HKalpha was not being coexpressed, the results are consistent with the interpretation that the epitopic site for MAb 2/2E6 is in a region of alpha -beta association.


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Fig. 7.   HKbeta on cell surface of LLC-PK1 cells is immunostained by MAb 2/2E6 without fixation or permeabilization. Cultures of LLC-PK1 cells, stably transfected with HKbeta , were probed with MAb 2/2E6 (A and B) or MAb 2G11 (C and D). In A and C, live cells were probed, washed, and labeled with secondary antibody. MAb 2/2E6 (A) clearly has access to beta -subunit on plasma membrane; MAb 2G11 (C) does not. In B and D, cells were fixed with Formalin and permeabilized with Triton X-100 before they were probed. Labeling can be seen for both antibodies, on surface and within cells. Scale bar, 10 µm for all low-power panels and 5 µm for high-power insets.

Immunostaining of HKbeta in transfected MDCK cells. A second cultured epithelial cell system in which to study the expression and trafficking of HKbeta was generated by expressing HKbeta in MDCK cells (MDCK-HKbeta cells). MDCK-HKbeta cells were fixed in formaldehyde and stained with MAb 2/2E6 without (Fig. 8A) or with (Fig. 8B) postfixation treatment with methanol. Surprisingly, unlike the case with transfected LLC-PK1 cells (Fig. 7), but similar to that seen for isolated rabbit gastric glands (Fig. 1), methanol or high concentrations of urea are required after fixation to effect staining of HKbeta in MDCK-HKbeta cells with MAb 2/2E6. Also, in contrast to data generated from the LLC-PK1 model in this study and from Gottardi and Caplan (18), attempts to surface immunolabel HKbeta by prebinding MAb 2/2E6 were unsuccessful (data not shown), as were attempts to surface immunoprecipitate metabolically radiolabeled HKbeta (data not shown). These results imply that, in transfected MDCK cells, access of MAb 2/2E6 to its extracellular epitope on HKbeta is restricted unless HKbeta is further denatured. These results parallel those obtained with native H-K-ATPase in glands or isolated gastric microsomes. In the case of oxyntic cell membranes, access may be limited by association of HKbeta with HKalpha ; however, because HKbeta alone was transfected into MDCK-HKbeta cells, another mechanism by which the MAb 2/2E6-binding epitope is masked must exist in MDCK-HKbeta cells.


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Fig. 8.   Immunostaining of HKbeta -transfected Madin-Darby canine kidney (MDCK) cells requires denaturation to expose epitope for MAb 2/2E6. Cultures of transfected MDCK cells were fixed in formaldehyde and permeabilized with 1% Triton X-100. After fixation and permeabilization, cells were directly incubated with MAb 2/2E6 and then with FITC-labeled secondary antibody (A) or briefly exposed to methanol before immunostaining with 2/2E6 (B). C: cells directly probed with MAb 2G11 after fixation and permeabilization. D: cells briefly exposed to methanol before they were probed with MAb 2G11. Scale bar, 10 µm.

HKbeta is expressed at the cell surface of MDCK-HKbeta cells. Analysis of the distribution of anti-HKbeta immunofluorescence in MDCK-HKbeta cells suggested that HKbeta may be present at the cell surface. To assay for HKbeta at the cell surface of transfected MDCK cells, the apical and basolateral plasma membranes of MDCK-HKbeta cells were separately biotinylated. Cell lysis was performed under denaturing conditions, and lysates were incubated with streptavidin-agarose to precipitate biotinylated proteins. Western blots of biotinylated proteins were probed with MAb 2/2E6. As seen in Fig. 9A for two different clones of MDCK-HKbeta cells, HKbeta appears to be expressed at the cell membrane, and cell surface expression of HKbeta is apparently predominantly apical. In addition, in SDS gels, biotinylated HKbeta migrates as a broad band of apparent molecular mass of 60-80 kDa, indicating that its oligosaccharide chains have been modified from the high-mannose core oligosaccharides (18, 23, 30). Alternatively, cell surface HKbeta was biotinylated, and cell lysis was performed under denaturing conditions. HKbeta was immunoprecipitated with anti-HKbeta cytoplasmic domain MAb 2G11, and the immunoprecipitate was probed on Western blots with streptavidin-HRP. By this approach, cell surface HKbeta was also observed to be predominantly apical (data not shown).


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Fig. 9.   Biotinylation of HKbeta at cell surface of transfected MDCK cells. A: apical (a) or basolateral (b) membranes of 2 clones of transfected MDCK cells were biotinylated. Cell lysis was performed under denaturing conditions. Biotinylated HKbeta was precipitated with streptavidin-agarose and detected on Western blots with MAb 2/2E6 and enhanced chemiluminescence (ECL). B: association of HKbeta with an endogenous 94-kDa protein at cell surface. After cell surface biotinylation, cell lysates were prepared under nondenaturing conditions. HKbeta was immunoprecipitated with MAb 2G11. Biotinylated HKbeta and any noncovalently associated, biotinylated proteins were detected on Western blots with streptavidin-HRP and ECL. Positions of prestained molecular mass markers are as follows: ovalbumin (55 kDa), BSA (86 kDa), and beta -galactosidase (120 kDa).

The percentage of HKbeta at the apical surface at steady state was estimated from densitometric analysis of Western blots. At steady state, 0.6 ± 0.24% (mean ± SE, n = 4) of HKbeta is expressed at the apical surface. These results, together with the cellular distribution of HKbeta determined by immunofluorescent labeling, suggest that most of HKbeta may be sequestered in the secretory pathway or in an intracellular membrane compartment.

HKbeta is associated with an endogenous 94-kDa MDCK cell protein at the cell surface of MDCK-HKbeta cells. To determine whether HKbeta was expressed alone at the cell surface in MDCK-HKbeta cells, as was observed in other transfected cells (12, 14, 18, 23), the cell surface was biotinylated and cell lysis was effected under nondenaturing conditions. HKbeta was subsequently immunoprecipitated with the anticytoplasmic domain MAb 2G11, and the immunoprecipitates were analyzed on Western blots by probing with streptavidin-HRP. As shown in Fig. 9B, a biotinylated endogenous 94-kDa MDCK cell protein coimmunoprecipitated with HKbeta , suggesting that at least a fraction of HKbeta at the cell surface is associated with another protein. This 94-kDa protein did not appear in immunoprecipitates from cells lysed under denaturing conditions (Fig. 9A), suggesting that the 94-kDa protein and HKbeta are noncovalently associated.

Noncovalent association of HKbeta with the 94-kDa protein occurs early in the biosynthetic pathway. Pulse labeling of transfected MDCK-HKbeta cells with radiolabeled Cys and Met for 15 min and subsequent immunoprecipitation of pulse-labeled HKbeta under nondenaturing conditions with MAb 2G11 resulted in the coimmunoprecipitation of two radiolabeled proteins: a 94-kDa protein and a protein migrating at ~50-55 kDa (Fig. 10A). The protein migrating at ~50-55 kDa is presumably a core-glycosylated, high-mannose ("immature") form of HKbeta . This core-glycosylated form of HKbeta has been previously identified in other heterologous expression systems (19, 23, 25, 26, 30), and the oligosaccharide chains are sensitive to hydrolysis by Endo H (Fig. 10B). Therefore, the association between these two proteins apparently occurs early in the secretory pathway.


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Fig. 10.   Immunoprecipitation of metabolically radiolabeled HKbeta . A: coimmunoprecipitation of a 94-kDa protein with pulse-labeled HKbeta . Transfected MDCK-HKbeta cells were metabolically radiolabeled with [35S]Cys and [35S]Met for 15 min and lysed under nondenaturing conditions. Lysates were subsequently immunoprecipitated with MAb 2G11. Samples were run on SDS-PAGE, and radiolabeled proteins were visualized by fluorography. B: metabolically pulse-labeled HKbeta immunoprecipitated by MAb 2G11 is core glycosylated. An immunoprecipitate of HKbeta was obtained as described for A and incubated without (lane 1) or with (lane 2) endoglycosidase H (Endo H). Oligosaccharides on pulse-labeled HKbeta immunoprecipitated by MAb 2G11 are sensitive to hydrolysis by Endo H. After prolonged digestion with Endo H, although 94-kDa protein is still slightly visible, a noticeable loss of 94-kDa protein often occurred. C: association between HKbeta and 94-kDa protein is noncovalent. Lanes 1 and 2, cells were lysed under denaturing conditions (SDS) and immunoprecipitated with MAb 2/2E6 (lane 1) or 2G11 (lane 2). From cells lysed under denaturing conditions, either MAb immunoprecipitates only HKbeta . Lanes 3 and 4, sequential immunoprecipitation (seq) of pulse-labeled HKbeta with MAbs 2/2E6 and 2G11. Cells were pulse labeled and lysed under nondenaturing conditions. Lysates were immunoprecipitated first with MAb 2/2E6 (lane 3) and then with MAb 2G11 (lane 4). Under nondenaturing conditions, MAb 2/2E6 immunoprecipitates only HKbeta , whereas MAb 2G11 immunoprecipitates a complex of HKbeta and a 94-kDa protein. Migration of molecular mass standards is indicated.

The association between core-glycosylated HKbeta and the 94-kDa protein is noncovalent, since immunoprecipitation of HKbeta after lysis under denaturing conditions results in the isolation of only pulse-labeled HKbeta (Fig. 10C, SDS, lanes 1 and 2). In addition, when metabolically radiolabeled HKbeta in cell lysates produced under nondenaturing conditions was sequentially immunoprecipitated first with MAb 2/2E6 and then with MAb 2G11, only uncomplexed HKbeta was immunoprecipitated by MAb 2/2E6, whereas the 94-kDa protein was observed only in the subsequent MAb 2G11 immunoprecipitates (Fig. 10C, seq, lanes 3 and 4).

In pulse-chase experiments, various amounts of three major radiolabeled proteins from the pulse-labeling period and subsequent chase times were immunoprecipitated using MAb 2G11 under nondenaturing conditions (Fig. 11). The apparent molecular masses of the three proteins are 50-55, 60-80, and 94 kDa. The two proteins with the lower apparent masses correspond to the high-mannose form (50-55 kDa) and the mature, complex carbohydrate form (60-80 kDa) of HKbeta , respectively. These forms are similar to those reported for HKbeta expressed in other heterologous expression systems (19, 23, 25, 26, 30) and suggest that the modification of oligosaccharides on HKbeta to the 60- to 80-kDa form is consistent with its transport through the late secretory pathway. The relative mobility of mature 60- to 80-kDa HKbeta in SDS gels of immunoprecipitates from these pulse-chase experiments is very similar to that of HKbeta detected at the cell surface by biotinylation (Fig. 9). These data suggest that at steady state the oligosaccharides on HKbeta expressed at the cell surface are predominantly the mature form. Thus, in this system, the acquisition of complex carbohydrates and cell surface expression of newly synthesized HKbeta appear to be correlated.


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Fig. 11.   Pulse-chase analysis of metabolically radiolabeled HKbeta in transfected MDCK-HKbeta cells. MDCK-HKbeta cells were pulse labeled and chased for 0-23 h. Cells were lysed under nondenaturing conditions, and HKbeta was immunoprecipitated with MAb 2G11. Immunoprecipitates were separated on SDS-PAGE and visualized by fluorography. Positions of molecular mass markers are indicated.

Pulse-chase experiments revealed that the association of HKbeta with the 94-kDa protein is not only rapid but also long lived. The HKbeta -94-kDa complex can be coimmunoprecipitated after 23 h of chase (Fig. 11). After 23 h, essentially only the 94-kDa band is visible; the high-mannose and mature forms of HKbeta are no longer visible. The cohort of unassembled, high-mannose HKbeta may have been degraded, whereas the mature form of HKbeta may not be visible because of a combination of its apparently extensive glycosylation (with its consequent migration as a relatively diffuse band on SDS gels) and its loss of radioactive signal because of its finite half-life.

The 94-kDa protein associated with HKbeta is endogenous NaKalpha . The identity of the 94-kDa protein associated with HKbeta at the cell surface was revealed when the coimmunoprecipitating proteins from MDCK-HKbeta cells were probed with anti-NaKalpha antibodies on Western blots. NaKalpha clearly coimmunoprecipitates with HKbeta under nondenaturing conditions (Fig. 12A). The coimmunoprecipitation does not occur from cell lysates harvested under denaturing conditions, confirming that the association of HKbeta with NaKalpha is noncovalent. Moreover, when the surface-biotinylated complex of HKbeta and 94-kDa protein is probed on Western blots, the surface-biotinylated 94-kDa protein clearly reacts with the anti-NaKalpha antibodies (Fig. 12B). Thus, in MDCK-HKbeta cells, HKbeta can apparently assemble with endogenous NaKalpha , and a fraction of this complex can be targeted to the apical plasma membrane.


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Fig. 12.   Endogenous alpha -subunit of Na-K-ATPase (NaKalpha ) coimmunoprecipitates with HKbeta under nondenaturing conditions from MDCK-HKbeta cells (A and B) and transfected LLC-PK1 cells (C and D). A: cells were lysed under nondenaturing (lane 1) or denaturing conditions (lane 2), and HKbeta was immunoprecipitated with MAb 2G11. Immunoprecipitates were run with separation by SDS-PAGE, transferred to nitrocellulose, and blotted with an MAb against NaKalpha . B: 94-kDa surface-biotinylated protein associated with HKbeta reacts with polyclonal anti-NaKalpha antibodies. After cell surface biotinylation, cell lysates were prepared under nondenaturing conditions. HKbeta was immunoprecipitated with MAb 2G11. Biotinylated HKbeta and any noncovalently associated, biotinylated proteins were detected on Western blots with streptavidin-HRP (lane 1) or polyclonal anti-NaKalpha antibodies (lane 2) and ECL. Sample in lane 1 is same sample in Fig. 10B and is included for comparison with sample in lane 2, which was produced in same experiment and run on same gel and Western blot. C: coimmunoprecipitation of HKbeta and a 94-kDa protein from metabolically radiolabeled, transfected LLC-PK1 cells. Cells were metabolically radiolabeled, and HKbeta was immunoprecipitated by MAb 2G11. D: NaKalpha is detected in Western blots of immunoprecipitates of HKbeta . Transfected MDCK (lane 1), untransfected MDCK (lane 2), or transfected LLC-PK1 (lane 3) cells were lysed under nondenaturing conditions. Lysate was immunoprecipitated with MAb 2G11 and probed on Western blots with a monoclonal anti-NaKalpha antibody. Positions of prestained molecular mass markers are shown.

These results suggest that, in these MDCK-HKbeta cells, exit of HKbeta from the biosynthetic pathway and its delivery to the cell surface may depend on its association with endogenous NaKalpha . These results also provide a mechanism for the masking of the MAb 2/2E6-binding epitope on HKbeta transfected into MDCK cells: at steady state, most of the HKbeta may be assembled with endogenous NaKalpha , particularly HKbeta expressed at the cell surface.

HKbeta assembles with endogenous NaKalpha in transfected LLC-PK1 cells. With the observation that HKbeta assembles with endogenous NaKalpha in MDCK-HKbeta cells, we sought to reevaluate the assembly competence of HKbeta expressed in LLC-PK1 cells. From LLC-PK1 cells, metabolically radiolabeled HKbeta could be coimmunoprecipitated with a 94-kDa protein with MAb 2G11 (Fig. 12C). In addition, immunoprecipitation of HKbeta with MAb 2G11 from cells lysed under nondenaturing conditions clearly results in the coimmunoprecipitation of endogenous NaKalpha , as shown in the Western blot in Fig. 12D. These results suggest that HKbeta may be generally competent to assemble with NaKalpha in renal epithelial cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two anti-HKbeta MAbs have been further characterized. The epitope to MAb 2/2E6 appears to include the amino acids S226LHY229 in the extracellular domain of HKbeta . In addition to evidence presented here, we have observed that MAb 2/2E6 reacts with all known HKbeta containing the SLHY motif, whereas there is no recognition of those beta -subunits, dog (SLRY) or chicken (NLHY), where substitutions have occurred (8). The SLHY epitope appears to become masked when assembled with HKalpha or NaKalpha . The masking of this epitope in assembled, but not unassembled, H-K-ATPase suggests that SLHY may be part of a site for alpha -beta interaction. Given that this site appears to be masked in heterodimers of HKalpha -HKbeta as well as NaKalpha -HKbeta hybrid heterodimers, this epitope may be a region that is involved in alpha -beta association in all heterodimeric P-type ATPases. Alternatively, it is possible that the loss of recognition by MAb 2/2E6 may result from the "burying" of its epitope as a consequence of the intrinsic folding of the extracellular domain of HKbeta , rather than assembly with an alpha -subunit. We do not favor this interpretation, since HKbeta , presumably alone at the cell surface of LLC-PK1 cells, can bind MAb 2/2E6 without requiring denaturation or reduction of disulfide bonds. In these cells, HKbeta should have satisfied all the quality control mechanisms, including proper folding, as a prerequisite to its exit from the ER for its appearance at the cell surface in the apparent absence of an alpha -subunit.

Additional evidence suggests that MAb 2/2E6 binds to an epitope that is formed at an interface between alpha - and beta -subunits. Two amino acids in the 2/2E6-binding epitope, L227 and Y229, are identical in all three isoforms of the beta -subunit of the Na-K-ATPase (beta 1, beta 2, and beta 3) as well as HKbeta , and the adjacent region Y229Y(or F)P231YYG is conserved throughout all known beta -subunits (8, 31, 39). The Y229Y(or F)P231YYG sequence is absolutely conserved in all beta -subunits of heterodimeric P-type ATPases characterized to date and overlaps with the SLHY motif in HKbeta . The importance of this region in alpha -beta assembly/association is further underscored by the work of Geering et al. (17) showing that the ability of the beta -subunit to assemble with the alpha -subunit is virtually abolished by mutating the proline residue of YY(or F)PYYG. Thus these two overlapping motifs may be one part of HKbeta that is responsible for the association of HKbeta with NaKalpha .

The second anti-HKbeta MAb, MAb 2G11, was previously shown to bind to the cytoplasmic domain of HKbeta (7). In this study we further defined the epitope to reside near the eight most NH2-terminal amino acids. This region must be at or near an important site for functional activity, and possibly a cytoplasmic K+ binding site, inasmuch as association with MAb 2G11 inhibits H-K-ATPase activity and alters the KA for activation of p-nitrophenyl phosphatase activity by K+ (7). It is also noteworthy that all mammalian species where HKbeta is recognized by MAb 2G11 (mouse, rat, human, rabbit, and dog) have the same eight NH2-terminal amino acids, whereas those beta -subunits with different NH2-terminal sequences, including chicken HKbeta and all isoforms of the beta -subunit of the Na-K-ATPase, are not recognized by MAb 2G11 (8).

We also showed here the utility of MAb 2G11 for immunoprecipitating assembled as well as unassembled HKbeta . We used MAb 2G11 in conjunction with MAb 2/2E6 to evaluate the assembly of HKbeta in transfected MDCK-HKbeta and LLC-PK1 cells. The simplest interpretation of our results is that there are apparently two cellular pools of HKbeta . One pool is unassembled HKbeta . In LLC-PK1 cells, unassembled HKbeta appears to transit to the plasma membrane; in MDCK-HKbeta cells, unassembled HKbeta largely appears to remain in the ER. The other pool of HKbeta assembles with endogenous NaKalpha in MDCK and LLC-PK1 cells. Although the assembly of NaKalpha -HKbeta has been convincingly demonstrated in expression systems such as Xenopus oocytes (23, 25) and yeast (14, 15, 43), our results represent the first demonstration of assembly of HKbeta with NaKalpha in epithelial cells, a significant result for several reasons. First, published studies on the trafficking of transfected HKbeta in LLC-PK1 and MDCK cells used the assembly-sensitive MAb 2/2E6 (18). Moreover, several studies have shown that HKbeta appears to transit to the plasma membrane in the absence of an alpha -subunit in other cell types (12, 18, 19, 38). All these studies used MAb 2/2E6; thus analysis of these trafficking data would be restricted to that of unassembled HKbeta . For MDCK-HKbeta cells, we showed that a significant fraction of HKbeta appears to be assembled with NaKalpha at steady state; thus a significant population of HKbeta was likely overlooked with respect to the analysis of trafficking of HKbeta in these earlier studies. Because HKbeta also assembles with NaKalpha in LLC-PK1 cells, the conclusions regarding HKbeta trafficking in transfected epithelial cell lines may suffer from the same limitations (18, 38). In both previously published studies, it was stated that HKbeta assembly with NaKalpha was not detected. These differences in results regarding HKbeta assembly are not likely the result of clonal variations, since the LLC-PK1 cell line transfected with HKbeta is common to both studies. The differences, however, are likely due to the different MAbs used by each investigator. In the light of the present data, it will be important to reevaluate the trafficking of HKbeta with respect to assembled and unassembled HKbeta in these epithelial cell lines. In addition, our preliminary unpublished data suggest that the trafficking of HKbeta in MDCK-HKbeta cells is more complex than formerly believed and may be regulated not only by assembly of HKbeta , but also by time of culture or after induction of polarity (unpublished observations). Similarly, the targeting of endogenous Na-K-ATPase in MDCK cells as a function of the establishment of polarity is well documented (13, 32).

Second, these two transfected epithelial cell lines appear to display different phenotypes relative to the relationship between assembly of HKbeta and its appearance at the cell surface. In LLC-PK1 cells, assembly of HKbeta with an alpha -subunit does not appear to be a prerequisite for its exit from the ER and appearance at the cell surface. The mechanism by which HKbeta appears to be able to reach the plasma membrane alone remains to be determined; alternatively, it remains to be determined whether HKbeta in LLC-PK1 cells assembles with another protein that is not detectable by the assays used in this study and whether the 2/2E6 epitope remains exposed in such a protein complex. Assembly with such a surrogate may then facilitate exit of this complex from the ER for delivery to the plasma membrane. On the other hand, MDCK-HKbeta cells characterized here appear to function in a manner that is more consistent with the present views of quality control mechanisms for membrane protein assembly in the ER. That is, HKbeta does not appear to exit the ER without assembly with an alpha -subunit. In this case, it is the alpha -subunit of the endogenous Na-K-ATPase. Conversely, numerous studies have shown that the exit of nascent alpha -subunits from the ER is dependent on the presence of a beta -subunit (for review see Refs. 16 and 33).

Third, isoforms of the H-K-ATPase and Na-K-ATPase are clearly coexpressed in cells other than the gastric oxyntic cell, such as colonic (4, 11, 39) and renal medullary cells (27). The data presented here suggest that the potential for the assembly of hybrid heterodimers in epithelial cells is significant. Thus, in some instances, cells must possess regulatory mechanisms to prevent hybrid heterodimer assembly, as in the gastric oxyntic cell. One possible mechanism is to regulate subunit expression, hence assembly, at the level of mRNA translation of ATPase subunits, as shown recently in MDCK cells (20). Alternatively, there may be other intrinsic posttranslational mechanisms in place to prevent such hybrid assembly, inasmuch as subunits of the H-K-ATPase and Na-K-ATPase appear to have a high preference for their own respective alpha -subunits, as demonstrated in an Sf9 insect cell expression system (26). On the other hand, hybrid heterodimer assembly may need to be facilitated. For example, recent evidence suggests that colonic HKalpha assembles with the beta 1-subunit of the Na-K-ATPase in kidney and distal colon (9). Another study has localized an Na-K-ATPase alpha 1-beta 2 heterodimer at the apical membrane of cells in developing kidney tubules (3). The regulation of assembly of P-type ATPase in cells expressing more than one heterodimeric P-type ATPase may ultimately involve a hierarchy of mechanisms, as has been demonstrated for the intracellular trafficking of Na-K-ATPase in epithelial cells (32).

One limitation of this study is that we are unable to assess functional competence of NaKalpha -HKbeta heterodimers. Although functional assembly of NaKalpha -HKbeta heterodimers has been demonstrated in heterologous expression systems such as Xenopus (23, 25) and yeast (14, 15), the two transfected epithelial cell lines in this study appear to express HKbeta at only immunologic, rather than functional, levels. The development of an epithelial cell culture system to test concomitantly the assembly, trafficking, and function of hybrid and/or chimeric P-type ATPases would be extremely valuable. Another feature of the LLC-PK1 and MDCK-HKbeta cells to consider is that the assembly (or lack thereof) of HKbeta and its trafficking in heterologous systems may be a consequence of the relative overexpression of HKbeta as a result of the removal of the expression of HKbeta from appropriate cellular controls due to transfection. Nevertheless, the two epithelial cell lines expressing HKbeta and the reagents characterized further here may play an important role in the characterization of the regulation of P-type ATPase assembly in epithelial cells.


    ACKNOWLEDGEMENTS

The authors acknowledge the expert technical assistance of Young Y. Jeng.


    FOOTNOTES

*  C. T. Okamoto and D. C. Chow contributed equally to this work.

This work was supported in part by a grant-in-aid from the former Greater Los Angeles Affiliate of the American Heart Association (C. T. Okamoto) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51588 (C. T. Okamoto), DK-38972 (J. G. Forte), and DK-10141 (J. G. Forte).

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 and other correspondence: C. T. Okamoto, Dept. of Pharmaceutical Sciences, 1985 Zonal Ave., University of Southern California, Los Angeles, CA 90089-9121 (E-mail: cokamoto{at}hsc.usc.edu).

Received 8 June 1999; accepted in final form 2 November 1999.


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DISCUSSION
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