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J. Biol. Chem., Vol. 277, Issue 23, 20671-20677, June 7, 2002

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Mutational Study on the Roles of Disulfide Bonds in the -Subunit of Gastric H⁺,K⁺-ATPase^*

Tohru Kimura, Yoshiaki Tabuchi^§, Noriaki Takeguchi, and Shinji Asano^§¶

From the  Faculty of Pharmaceutical Sciences and ^§ Molecular Genetics Research Center of Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

Received for publication, January 17, 2002, and in revised form, March 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The gastric proton pump, H⁺,K⁺-ATPase, consists of the catalytic -subunit and the non-catalytic -subunit. Correct assembly between the - and -subunits is essential for the functional expression of H⁺,K⁺-ATPase. The -subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the -subunit with serine individually and in several combinations. The mutant -subunits were co-expressed with the -subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the / assembly, stability, and cell surface delivery of the - and -subunits and H⁺,K⁺-ATPase activity was studied. Mutant -subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H⁺,K⁺-ATPase activity. All the mutant -subunits with replacement(s) of the extracellular cysteines did not assemble with the -subunit, resulting in loss of H⁺,K⁺-ATPase activity. These mutants did not permit delivery of the -subunit to the cell surface. Therefore, each of these disulfide bonds of the -subunit is essential for assembly with the -subunit and expression of H⁺,K⁺-ATPase activity as well as for cell surface delivery of the -subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The gastric proton pump, H⁺,K⁺-ATPase, consists of two kinds of subunits. One is the catalytic -subunit, which has 10 transmembrane domains and contains sites for ATP binding (1, 2) and its acylphosphorylation (3), binding sites of proton pump inhibitors (4-8), and sites responsible for ion recognition (6, 9-13). The other is the non-catalytic -subunit, which is heavily glycosylated. The -subunit is also essential for the functional expression of H⁺,K⁺-ATPase (9, 14, 15) and involved in stabilization as well as targeting the -subunit to the plasma membrane (16). In fact, the -subunit alone can leave the endoplasmic reticulum (ER)¹ and travel to the cell surface when expressed in mammalian cell lines, whereas the -subunit cannot leave the ER without the -subunit (17, 18).

The -subunit is a type II transmembrane protein and has a small amino-terminal cytoplasmic domain, a single transmembrane domain, and a large ectodomain (80% of the whole molecule) containing its carboxyl terminus (19). This structure is conserved between gastric H⁺,K⁺-ATPase and Na⁺,K⁺-ATPases. Gastric H⁺,K⁺-ATPase -subunit contains nine cysteine residues that are conserved among different animal species; two are located in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain (19-23). Among them, the six cysteine residues in the ectodomain form three disulfide bonds that are well conserved between H⁺,K⁺- and Na⁺,K⁺-ATPase -subunits. These disulfide bonds are important for protein folding and for the maintenance of ATPase function, because the ATPase activities of both enzymes were abolished by reduction with dithiothreitol or 2-mercaptoethanol (24-26). Na⁺,K⁺-ATPase activity was also abolished or severely suffered when any one of the three disulfide bonds of the -subunit was disrupted by replacing the extracellular cysteine residue(s) by serine, as assessed by expressing the mutant Na⁺,K⁺-ATPases in Xenopus oocytes (27, 28). However, there have been no reports regarding the roles of each disulfide bond in the functional expression and cell surface targeting of Na⁺,K⁺-ATPase in mammalian cells. There also have been no reports demonstrating the precise role of each disulfide bond of the gastric H⁺,K⁺-ATPase -subunit.

In this study, we replaced each of the nine cysteine residues of gastric H⁺,K⁺-ATPase -subunit by serine, transiently co-expressed the mutant -subunits together with the wild-type -subunit in HEK-293 cells (human embryonic kidney cell line), and studied the roles of each of the cysteine residues in the expression of the - and -subunits and in H⁺,K⁺-ATPase activity. We also abolished one, two, or all of the disulfide bonds by progressively introducing mutations in the extracellular cysteine residues, constructed stable cell lines co-expressing these mutant -subunits together with the -subunit, and examined the role of the disulfide bonds in the expression, stability, and cell surface delivery of the - and -subunits, / assembly, and H⁺,K⁺-ATPase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- HEK-293 cells were a kind gift from Prof. Jonathan Lytton (University of Calgary, Calgary, Canada). pcDNA3 (G418) and pcDNA3.1/ZEO(+) vectors were obtained from Invitrogen. Pfu DNA polymerase was from Stratagene (La Jolla, CA). 2-Methyl-8-(phenyl-methoxy)imidazo[1,2-a] pyridine-3-acetonitrile (SCH 28080) was a kind gift from Dr. Peter Chiu (Schering-Plough Co., Kenilworth, NJ). Restriction enzymes and other DNA- and RNA-modifying enzymes were from Toyobo (Osaka, Japan) and New England Biolabs (Beverly, MA). Anti-gastric H⁺,K⁺-ATPase -subunit monoclonal antibody 1H9 and anti--subunit monoclonal antibody 2B6 were obtained from Molecular Biological Laboratories (Nagoya, Japan). All other reagents were of molecular biology grade or the highest grade of purity available.

cDNAs of - and -Subunits of H⁺,K⁺-ATPase-- H⁺,K⁺-ATPase - and -subunit cDNAs were prepared from rabbit gastric mucosae and cloned as described elsewhere (9). The - and -subunit cDNAs were digested with EcoRI and XhoI. The obtained fragments were each ligated into pcDNA3 or pcDNA3.1/ZEO(+) vector treated with EcoRI and XhoI.

Site-directed Mutagenesis-- Introduction of site-directed mutations was carried out by sequential PCR steps as described elsewhere (6), in which appropriately mutated -subunit cDNAs (segments between nucleotide 1 (EcoRI site) and 302 (AflII site) or between nucleotide 302 (AflII site) and 1036 (Eco47III site)) were prepared. For PCR amplification of the segment between the EcoRI and AflII sites, the 5'-flanking sense and 3'-flanking antisense primers were 5'-GCAATTAACCCTCACTAAAGG-3' (T3 primer) and 5'-CGTGAACTTGCTGGAGAACTT-3' (nucleotides 499-518). For PCR amplification of the segment between the AflII and Eco47III sites, the 5'-flanking sense and 3'-flanking antisense primers were 5'-GCTGAAGTCGCCAGGCGTAAC-3' (nucleotides 281 to 301) and 5'-CCACGGGAAGCAGCGGACGC-3' (nucleotides 1049-1068). Sense and antisense synthetic oligonucleotides, each 21 bases long containing one mutated base near the center, were designed. The cDNA of H⁺,K⁺-ATPase -subunit in pBluescript SK() was used as a PCR template. PCR was routinely carried out in the presence of 200 µM each dNTP, 500 nM primers, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 20 mM Tris-HCl, pH 8.8, 0.1% Triton X-100, 100 µg/ml bovine serum albumin, and 2.5 units of Pfu DNA polymerase for 30 cycles. DNA sequencing was done by the dideoxy chain termination method using Autoread and Autocycle DNA sequencing kits and an ALFexpress DNA sequencer (Amersham Biosciences). After sequencing, the fragment amplified in the final PCR was digested with EcoRI plus AflII or AflII plus Eco47III and ligated back into the relevant position of the wild-type H⁺,K⁺-ATPase -subunit construct.

Cell Culture and Transient Expression-- Cell culture of the HEK-293 cell line was carried out as described previously (9). For transient expression, - and -subunit cDNA transfection was performed by the calcium phosphate method with 10 µg of cesium chloride-purified DNA/10-cm dish. Cells were harvested 2 days after the DNA transfection.

Establishment of Stable Cell Lines-- HEK-293 cells were first transfected with the wild-type or mutant H⁺,K⁺-ATPase -subunit cDNA cloned in pcDNA3 (G418). Cells were selected with a selection medium containing 1 mg/ml Geneticin for 24 h after transfection and split 1:4 to 1:10. The cells were cultured for 1-2 weeks in the selection medium. Colonies resistant to Geneticin were isolated and screened for protein expression by immunofluorescence. Cells were re-cloned by limited dilution followed by screening for protein expression by Western blot and immunofluorescence. Established stable cell lines expressing the -subunit were maintained in culture medium containing 0.5 mg/ml Geneticin. Cell lines expressing the -subunit were then subjected to the second transfection with the H⁺,K⁺-ATPase -subunit cDNA cloned in pcDNA3.1/Zeo(+).

Cells were selected in a selection medium containing 0.5 mg/ml Geneticin and 0.2 mg/ml Zeocin and cloned twice by limited dilution. Established stable cell lines expressing both the - and -subunits were maintained in a culture medium containing 0.5 mg/ml Geneticin plus 0.2 mg/ml Zeocin.

Preparation of Membrane Fractions, SDS-Polyacrylamide Gel Electrophoresis, and Western Blot-- Membrane fractions of HEK cells were prepared as described previously (9). SDS-polyacrylamide gel electrophoresis was carried out as described elsewhere (29). Membrane preparations (30 µg of protein) were incubated in a sample buffer containing 2% SDS, 2% -mercaptoethanol, 10% glycerol, and 10 mM Tris-HCl, pH 6.8, at room temperature for 10 min and applied to the SDS-polyacrylamide gel. Western blot was carried out as described previously (9).

Quantification of Expressed H⁺,K⁺-ATPase in the Membrane-- The content of expressed H⁺,K⁺-ATPase - and -subunits was quantified by comparing with the subunits in a pig gastric vesicle preparation (30). The content of -subunit was quantified after treatment with N-glycosidase F (17). The membrane fractions of HEK cells were run on the same SDS-polyacrylamide gel as a series of diluted gastric vesicle preparations and blotted. The blots were scanned using an optical scanning image system. The content of H⁺,K⁺-ATPase in the membrane fractions was estimated from the standard curve of the gastric vesicle preparation.

Antibody-- Anti-gastric H⁺,K⁺-ATPase -subunit antibody Ab1024 was previously raised against the carboxyl-terminal peptide (residues 1024-1034) of the H⁺,K⁺-ATPase -subunit (PGSWWDQELYY) (31). Anti--subunit monoclonal antibody 2B6 and anti--subunit monoclonal antibody 1H9 were derived from the splenocytes of mice with autoimmune gastritis (23). The epitope of 2B6 was located on the carboxyl-terminal portion of the -subunit (32).

Pulse-Chase Labeling and Immunoprecipitation-- Stable cell lines were cultured on collagen-coated 6-well plates. Cells were washed and incubated at 37 °C for 30 min in methionine-free medium. Cells were labeled for 60 min with [³⁵S]Met, Cys-labeling mixture (EXPRESS) (PerkinElmer Life Sciences) and chased in a complete Dulbecco's modified Eagle's medium for indicated periods. Cells were washed with washing buffer containing 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, and incubated in 500 µl of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4 °C for 30 min. After centrifugation at 16,000 × g for 20 min, the supernatant was incubated with an anti--subunit antibody, 1H9, or an anti--subunit, 2B6, at a 1:100 dilution and 10 µl of ImmunoPure immobilized protein A (Pierce) at 4 °C for 12 h. After centrifugation, the pellet was washed 4 times with the lysis buffer followed by 2 washes in 0.1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4. The pellet was solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis and incubated at room temperature for 10 min. The proteins separated on SDS-polyacrylamide gel were visualized by digital autoradiography of dried gels using Bio Imaging Analyzer BAS 2000 (Fuji Photo Film, Tokyo).

Immunoprecipitation-- Membrane fractions of HEK cells stably expressing the complex were incubated in 1 ml of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4 °C for 30 min as described previously (32). The solubilized fraction was incubated with anti -subunit antibody Ab1024 and ImmunoPure immobilized protein A at 4 °C for 12 h. After centrifugation, the pellet was washed 4 times with the lysis buffer followed by 2 washes in 0.1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4. The pellet was solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis and incubated at room temperature for 10 min. The -subunit in the blot was detected by anti--subunit antibody 2B6 in combination with a peroxidase-conjugated anti-mouse antibody, which was preabsorbed with rabbit serum.

Immunohistochemistry-- Stable cell lines were fixed for 10 min in cold methanol (20 °C) and washed three times with PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS(+)). Cells were permeabilized in a permeabilization buffer containing 0.3% Triton X-100 and 0.1% bovine serum albumin in PBS(+) for 15 min at room temperature. Nonspecific antibody binding was blocked by preincubation of cells in a goat serum dilution buffer solution (16% goat serum, 0.3% Triton X-100, 0.9% NaCl, and 20 mM NaP_i, pH 7.4) for 30 min. All antibody incubations were carried out using the goat serum dilution buffer solution. Cells were incubated for 1 h at room temperature with anti-H⁺,K⁺-ATPase  (Ab1024) or anti-H⁺,K⁺-ATPase  (2B6) antibody followed by three washes with the permeabilization buffer. Fluorescein isothiocyanate-conjugated anti-rabbit IgG and rhodamine-conjugated anti-mouse IgG secondary antibodies were used for 1 h at room temperature at a 1:100 dilution. After washing with PBS(+) three times, immunofluorescence images were visualized using a Zeiss LSM 510 laser-scanning confocal microscope. When indicated, cells were fixed with 3.5% formaldehyde at room temperature for 30 min instead of methanol, and the antibody incubation was carried out without permeabilization.

Assay of H⁺,K⁺-ATPase Activity-- H⁺,K⁺-ATPase activity was measured from the decrease in the amount of NADH coupled with regeneration of ATP from ADP (coupled-enzyme assay) in 1.2 ml of a reaction mixture containing 50 µg of membrane protein, 3 mM MgCl₂, 800 µM ATP, 160 µM NADH, 0.8 mM phosphoenolpyruvate, 3 units/ml pyruvate kinase, 2.75 units/ml lactate dehydrogenase, 5 mM NaN₃, 1 mM ouabain, 15 mM KCl, and 40 mM Tris-HCl, pH 7.4. The decrease in the amount of NADH was measured at 37 °C from absorbance at 340 nm in a Beckman spectrophotometer as described elsewhere (33). H⁺,K⁺-ATPase activity, defined as the SCH 28080-sensitive K⁺-ATPase, was calculated as the difference between the K⁺-ATPase activities in the presence and absence of 50 µM SCH 28080. Protein was measured using the BCA protein assay kit from Pierce with bovine serum albumin as a standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Introduction of Mutations in the Cysteine Residues of H⁺,K⁺-ATPase -Subunit-- Rabbit gastric H⁺,K⁺-ATPase -subunit contains nine cysteine residues, Cys¹⁰ and Cys²¹ in the cytoplasmic domain, Cys⁵⁸ in the transmembrane domain, and Cys¹³¹, Cys¹⁵², Cys¹⁶², Cys¹⁷⁸, Cys²⁰¹, and Cys²⁶³ in the ectodomain. The six cysteine residues located in the ectodomain form three disulfide bonds between Cys¹³¹ and Cys¹⁵² (loop 1), Cys¹⁶² and Cys¹⁷⁸ (loop 2), and Cys²⁰¹ and Cys²⁶³ (loop 3) (Fig. 1). In Table I, we summarized a series of mutant -subunits prepared in the present study. First, we prepared nine single mutants by replacing each of these cysteine residues with serine: C10S, C21S, C58S, C131S, C152S, C162S, C178S, C201S, and C263S. Next, three double mutants were prepared in which each pair of the extracellular cysteines forming disulfide bonds were replaced by serines: C131S/C152S (termed L-1), C162S/C178S (termed L-2), and C201S/C263S (termed L-3). In the following step, one quadruple mutant was prepared; C131S/C152S/C162S/C178S (termed L-1,2). Finally, a mutant -subunit in which all of the extracellular cysteine residues were replaced by serines was prepared: C131S/C152S/C162S/C178S/C201S/C263S (termed L-1,2,3). The resulting -subunit was expected to have no disulfide bond. Each of these mutant -subunits or the wild-type -subunit was transiently or stably co-expressed with the wild-type H⁺,K⁺-ATPase -subunit in HEK-293 cells.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic illustration of the H+,K+-ATPase -subunit. Rabbit gastric H+,K+-ATPase -subunit contains one transmembrane segment. The amino terminus is located in the cytoplasm, and the carboxyl terminus is located in the ectodomain. The -subunit contains nine cysteine residues, Cys10 and Cys21 in the cytoplasmic domain, Cys58 in the transmembrane domain, and Cys131, Cys152, Cys162, Cys178, Cys201, and Cys263 in the ectodomain. These six extracellular cysteine residues form three disulfide bonds. The -subunit is modified with seven N-linked carbohydrate chains at Asn99, Asn103, Asn130, Asn146, Asn161, Asn193, and Asn222.

View this table: [in this window] [in a new window]   Table I -Subunit mutants used in this work

Expression of the -Subunits in the Membrane Fractions-- In the Western blots, the expression level of the -subunit in the membrane fraction was higher when it was transiently co-expressed with the wild-type -subunit rather than expressed alone (Fig. 2A, lanes 1 and 5). This was due to the stabilization of the -subunit in the membrane (9, 32). -Subunit mutants C10S, C21S, and C58S also significantly increased the expression of the -subunit compared with that in the absence of the -subunit (Fig. 2A, lanes 2-4). However, -subunit mutants L-1, L-2, L-3, L-1,2, and L-1,2,3 did not increase the expression level of the -subunit (Fig. 2B, lanes 2-6). Mutants C131S, C152S, C162S, C178S, C201S, and C263S also did not increase the expression of the -subunit (data not shown). These results indicate that each free cysteine residue located in the cytoplasmic and transmembrane segments is not involved in the stabilization of the -subunit, whereas each disulfide bond is important for the stabilization of the -subunit. Precise study on the role of each disulfide bond of the -subunit in the stabilization of the -subunit was performed by pulse-chase-labeling experiments of stable cell lines, which is presented later in the present paper.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Western blots with an anti-gastric H+,K+-ATPase -subunit antibody (Ab1024) of membrane fractions prepared from HEK cells transiently co-expressing the wild-type H+,K+-ATPase -subunit plus wild-type or mutant -subunit. A, HEK-293 cells were co-transfected with the -subunit cDNA plus wild-type -subunit cDNA (lane 1) or mutant -subunit C10S (lane 2), C21S (lane 3), or C58S cDNA (lane 4) or they were transfected with the -subunit cDNA alone (lane 5). B, HEK-293 cells were co-transfected with the -subunit cDNA plus wild-type H+,K+-ATPase -subunit cDNA (lane 1) or mutant -subunit L-1 (lane 2), L-2 (lane 3), L-3 (lane 4), L-1,2 (lane 5), or L-1,2,3 cDNA (lane 6), or they were transfected with the -subunit cDNA alone (lane 7). These cell membrane fractions (30 µg) were separated on an SDS-polyacrylamide gel and subjected to Western blotting with antibody Ab1024. Bands representing the H+,K+-ATPase -subunit are shown by the bold arrows on the right side.

H⁺,K⁺-ATPase Activity of the Mutants-- H⁺,K⁺-ATPase activity found in the membrane fractions of cells transiently co-expressing the -subunit plus wild-type or mutant -subunit was measured (Fig. 3). When the cells were co-transfected with the -subunit plus -subunit mutant C10S, C21S, or C58S cDNAs, H⁺,K⁺-ATPase activity was retained. In Western blot analysis, the -subunit content in 30 µg of membrane fractions from cells expressing the /C10S, /C21S, and /C58S mutants and the wild-type / was equivalent to the -subunit content present in 0.55, 0.44, 0.48, and 0.57 µg of a gastric vesicle preparation, respectively. Because the H⁺,K⁺-ATPase -subunit comprises ~60% of the protein content of the gastric vesicle preparation, we estimate that 1 mg of each membrane fraction contains 10.9, 8.7, 9.5, and 11.5 µg of the -subunit, respectively. Therefore, the specific activities of these mutants were calculated to be 95, 119, and 113 µmol/mg of -subunit/h, respectively, and are almost comparable with that of the wild type (115 µmol/mg of -subunit/h). However, H⁺,K⁺-ATPase activity was not observed in the cells expressing the -subunit plus -subunit mutant L-1, L-2, L-3, L-1,2 or L-1,2,3 (Fig. 3). Similarly, H⁺,K⁺-ATPase activity was not observed when the cells were co-transfected with the -subunit plus each single mutant cDNA, C131S, C152S, C162S, C178S, C201S, or C263S (data not shown). Therefore, each free cysteine residue located in the cytoplasmic and transmembrane domains is not directly involved in the function of H⁺,K⁺-ATPase, whereas each disulfide bond is important for the maintenance of the H⁺,K⁺-ATPase activity. This behavior of each mutant in the expression of H⁺,K⁺-ATPase activity is comparable with that in the stabilization of the -subunit; mutant -subunit that is able to stabilize the -subunit retained the H⁺,K⁺-ATPase activity.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   H+,K+-ATPase activity of membrane fractions from cells transiently co-expressing the H+,K+-ATPase -subunit plus wild-type or mutant -subunit or the -subunit alone. H+,K+-ATPase activity was calculated as the difference between the K+-ATPase activities in the presence and absence of 50 µM SCH 28080. The values are the mean ± S.E. for three transfections.

Intracellular Localization of - and -Subunits in HEK Cells-- To study precisely the roles of the disulfide bonds on the intracellular localization and stability of the - and -subunits and the / assembly, we used stable cell lines expressing the -subunit with the mutant -subunits. Here, we studied intracellular localization of the - and -subunits using the immunofluorescence technique and a confocal laser-scanning microscope. The -subunit cannot attain a cell surface localization without the -subunit (data not shown). Fig. 4 shows that in the cells co-expressing the wild-type - and -subunits, both subunits attained a cell surface distribution in addition to exhibiting intracellular expression, indicating that the -subunit permitted the -subunit to reach the cell surface. In fact, the wild-type -subunit was observed at the cell surface in the immunofluorescence studies without permeabilization treatment. However, there was apparently no cell surface expression of both the - and -subunits in the cells co-expressing the -subunit together with the L-2, L-3, L-1,2, or L-1,2,3 mutant. The expression was restricted to perinuclear regions. These mutant -subunits were not observed at the cell surface in the absence of permeabilization treatment. On the other hand, low levels of the L-1 mutant were observed at the cell surface under non-permeabilized conditions. These results indicate that both of the two carboxyl-terminal disulfide bonds of the -subunit (loop 2 and 3) are essential for cell surface delivery of the - and -subunits.

View larger version (90K): [in this window] [in a new window]   Fig. 4.   Immunolocalization of the H+,K+-ATPase - and -subunits expressed in stable cell lines. HEK-293 cells were stably transfected with the cDNAs encoding the H+,K+-ATPase - and wild-type or mutant -subunit (L-1, L-2, L-3, L-1,2, or L-1,2,3). Immunofluorescence analysis was performed using a polyclonal rabbit antibody directed against the -subunit (Ab1024) and a mouse monoclonal antibody directed against the -subunit (2B6) under permeabilized conditions. The - (A) and -subunits (B) were detected with secondary antibodies conjugated to rhodamine and fluorescein isothiocyanate, respectively. The merge patterns are also presented (C). Immunofluorescence analysis was also performed for the -subunits under non-permeabilized conditions (D).

Expression of the - and -Subunits in the Membrane Fractions of Stable Cell Lines-- The expression levels of the - and -subunits in the membrane fractions were studied by Western blots with anti- and -antibodies, respectively (Fig. 5). The expression level of the -subunit in the membrane fraction was 5-9 times higher in the cells co-expressing the wild-type - and -subunits compared with those co-expressing the - and mutant -subunits, L-1, L-2, L-3, L-1,2, or L-1,2,3 (Fig. 5A). These results are in good agreement with the similar experiments in the transient expression as shown in Fig. 2B.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   Western blots with an anti- or -subunit antibody of membrane fractions from stable cell lines co-expressing the wild-type -subunit plus wild-type or mutant -subunit. The cell membrane fractions (30 µg) were separated on an SDS-polyacrylamide gel and subjected to Western blotting with anti--subunit antibody Ab1024 (A) or anti--subunit antibody 2B6 (B). m and c represent the -subunit with complex-type (mature) carbohydrate chains and that with high mannose-type (core) carbohydrate chains, respectively.

In the cells co-expressing the wild-type - and -subunits, two -subunit bands reacting with antibody 2B6 were observed, one with a molecular mass of 60-70 kDa (_m) representing the -subunit modified with complex-type carbohydrate chains and the other with a molecular mass of 48 kDa (_c) representing the -subunit modified with high mannose-type carbohydrate chains as shown in the previous study (32) (Fig. 5B, lane 1). The _m constituted a large fraction of the -subunit present in the stable cell line expressing the wild-type - and -subunits. This pattern was different from that found in the /-expressing samples in the transient expression, in which the band representing the _c was more dense than that representing the _m (32). These results indicate that a major portion of the -subunit in the /-expressing stable cell line leaves the ER to attain the trans-Golgi as shown in Fig. 4.

In the cells co-expressing the -subunit plus -subunit mutant L-1, L-2, L-3, L-1,2, or L-1,2,3, the expression level of these mutant -subunits was 5-9 times lower compared with that of the wild type (Fig. 5B), although such a clear difference in the expression level was not observed in the immunohistochemical results in Fig. 4. In the L-2, L-3, L-1,2, and L-1,2,3 mutants, only one band representing the _c was observed. In the L-1 mutant, a small amount of the _m was observed in addition to the _c (Fig. 5B, lane 2). These results indicate that -subunit mutants L-2, L-3, L-1,2, and L-1,2,3 were retained in the ER and did not reach the trans-Golgi, as shown in Fig. 4. In the case of the L-1 mutant, some fraction of the -subunit appears to reach the trans-Golgi or the cell surface.

Assembly between the -Subunits and Mutant -Subunits-- To study the / assembly, membrane fractions of the stable cell lines were immunoprecipitated with the anti--subunit antibody followed by Western blotting with anti--subunit antibody 2B6 (Fig. 6). In membrane fractions prepared from cells expressing the wild-type - and -subunits (lanes 1 and 2), the anti--antibody co-precipitated the -subunit with a molecular mass of 70 kDa (_m), indicating that the - and -subunits were assembled in the membrane. The co-precipitating -subunit was clearly observed in experiments employing 100 and 500 µg of membrane fractions prepared from the cells expressing the wild-type - and -subunits. However, mutants L-1, L-2, L-3, L-1,2, and L-1,2,3 were not co-precipitated with the -subunit at all from 1 mg of the membrane fractions. Therefore, the lack of co-precipitation in these mutant enzymes is not solely due to the lower expression levels. These results indicate that each disulfide bond of the -subunit is important for the / assembly.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Western blots with an anti--subunit antibody of anti--subunit immunoprecipitates from membrane fractions of stable cell lines. The membrane fractions (100 and 500 µg for lanes 1 and 2, respectively, and 1 mg for lanes 3-8) of stable cell lines expressing the wild-type -subunit plus wild-type (lanes 1 and 2), mutant L-1 (lane 3), L-2 (lane 4), L-3 (lane 5), L-1,2 (lane 6), or L-1,2,3 -subunit (lane 7), or mock-transfected HEK-293 cells (lane 8) were incubated in 1 ml of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4 °C for 30 min. The solubilized membrane fractions were incubated with anti--subunit antibody Ab1024 and protein A-coated beads. The precipitated preparations were separated on an SDS-polyacrylamide gel and subjected to Western blotting with anti--subunit antibody 2B6. m and c represent the -subunit with complex-type (mature) carbohydrate chains and that with high mannose-type (core) carbohydrate chains, respectively.

Stability of the Mutant -Subunits-- To compare the stability of the wild-type and mutant -subunits (L-1, L-2, L-3, L-1,2, and L-1,2,3), we performed pulse-chase labeling of the stable cell lines expressing the wild-type or mutant -subunits together with the -subunit followed by immunoprecipitation with anti--subunit antibody 2B6 (Fig. 7A). After a 60-min pulse period, the wild-type and mutant -subunits were observed as a band(s) representing the -subunit modified with the high mannose-type carbohydrate chains (_c) (chase time 0 in Fig. 7A). It should be noted that the bands of the mutant -subunits appeared smeary and dense compared with that of the wild type. The number and/or pattern of carbohydrate chains attached to the mutant -subunits may be different from that of the wild type. At chase times of 1-6 h, the wild-type -subunit was observed as a smeary band with a higher molecular mass, which represented the -subunit modified with the complex-type carbohydrate chains (_m) followed by gradual degradation within 12 h chase. On the other hand, the molecular mass of the mutant -subunits did not change in the chase time, indicating that they were not modified with complex-type carbohydrate chains. These results are in good agreement with the findings that these mutant -subunits (except for mutant L-1) were not targeted to the cell surface as shown in Fig. 4. They were gradually degraded within 3-6 h. Therefore, the mutant -subunits, especially the L-1,2,3 mutant, are less stable compared with the wild-type -subunit, indicating that the disulfide bond(s) in the -subunit is involved in the stabilization of the -subunit.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Pulse-chase labeling of stable cell lines co-expressing the wild-type -subunit plus wild-type, mutants L-1, L-2, L-3, L-1,2, or L-1,2,3 -subunit followed by immunoprecipitation with an anti--subunit (A) or an anti--subunit antibody (B). Stable cell lines were labeled for 60 min with [35S]Met, Cys-labeling mixture (EXPRESS) (PerkinElmer Life Sciences) in methionine-free, cysteine-free Dulbecco's modified Eagle's medium followed by the chase with the complete medium for indicated periods. Cells were washed with washing buffer containing 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, and incubated in 500 µl of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4 °C for 30 min. After centrifugation at 16,000 × g for 20 min, the supernatant was incubated with anti--subunit antibody 2B6 (A) or anti--subunit antibody 1H9 (B), and ImmunoPure immobilized protein A (Pierce) at 4 °C for 12 h. After centrifugation, the pellet was washed 4 times with the lysis buffer followed by 2 washes in 0.1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4. The pellet was solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis, separated on a gel, and visualized by digital autoradiography.

Stability of the -Subunits Co-expressed with the Mutant -Subunits-- To compare the stability of the -subunit co-expressed with the wild-type and mutant -subunits, we also performed pulse-chase labeling of the stable cell lines expressing the wild-type or mutant -subunits together with the -subunit followed by the immunoprecipitation with anti--subunit antibody 1H9. Fig. 7B shows that the -subunit co-expressed with the wild-type -subunit was stable in 3-6 h followed by gradual degradation, whereas the -subunit co-expressed with the mutant -subunits was much less stable. When expressed with mutant -subunit protein, degradation of the -subunit was apparent within 1 h and was almost complete within 3 h. Therefore, the disulfide bonds, which are important for the / assembly, are also involved in the stabilization of co-expressed -subunit.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the process of protein maturation, correct folding is very important for the acquisition of stability and physiological function. Misfolded proteins are retained in the ER by the quality control system, transferred to the cytoplasm through the translocon, and degraded by proteasomes located in the cytoplasm (34, 35).

Formation of intra- or intermolecular disulfide bonds is one of the major posttranslational modification processes for protein folding. Some specific disulfide bonds play a key role(s) for protein folding and stabilization, and others play a key role for the acquisition of catalytic activity. Influenza virus hemagglutinin, with a single transmembrane segment, contains six disulfide bonds in its ectodomain. They are important for efficient folding, stabilization, and cell surface expression (36). A secretory protein, bovine pancreatic trypsin inhibitor, contains three disulfide bonds, which are important for efficient stabilization and secretion (37). Escherichia coli alkaline phosphatase loses its intracellular stability and/or catalytic activity when its specific disulfide bonds are cleaved (38).

Gastric H⁺,K⁺-ATPase contains 30 cysteine residues in the -subunit and 9 cysteine residues in the -subunit (20, 39). There are no disulfide bonds in the -subunit (24). Several luminal cysteine residues of the -subunit (Cys⁸¹⁵, Cys⁸²⁴, Cys⁸⁹⁴, and Cys³²³) are the binding sites of proton pump inhibitors (5, 7). On the other hand, the -subunit contains six conserved cysteine residues in the ectodomain, which form three disulfide bonds (24). These disulfide bonds are overall thought to be essential for protein folding and for maintenance of the ATPase function, because the ATPase activity was abolished by reduction with dithiothreitol or 2-mercaptoethanol at high concentrations or at high temperatures (24).

To further study the roles of each cysteine residue and disulfide bond of the H⁺,K⁺-ATPase -subunit in the assembly between the - and -subunits in the stability and cell surface delivery of the - and -subunits and in H⁺,K⁺-ATPase activity, we replaced individual, several, and all of the extracellular cysteine residues with serines and transiently and stably expressed them with the -subunit in HEK-293 cells.

When each cysteine residue on the cytoplasmic and transmembrane segments was replaced by a serine residue, mutant -subunits were assembled with the -subunit, and the resulting / complexes retained H⁺,K⁺-ATPase activity. However, when any one of three disulfide bonds of the H⁺,K⁺-ATPase -subunit was disrupted, the mutant -subunit did not assemble with the -subunit, resulting in the loss of the H⁺,K⁺-ATPase activity, loss of stabilization of the -subunit, and loss of cell surface expression of the -subunit. The loss of the disulfide bond(s) of the -subunit likely changes the conformation of the -subunit, resulting in a decrease in the stability of the -subunit and a decrease or loss of its cell surface delivery. Therefore, each disulfide bond of the H⁺,K⁺-ATPase -subunit is important for / assembly and cell surface expression and stability of the - and -subunits as well as for H⁺,K⁺-ATPase activity. The functional importance of the disulfide bonds of the -subunit shown here differs from that of the carbohydrate chains on the -subunit. H⁺,K⁺-ATPase -subunit contains seven carbohydrate chains (40), each of which is not essential for / assembly, cell surface expression, and stability of the -subunit as well as H⁺,K⁺-ATPase activity (17). However, the effect of removing carbohydrate chains is cumulative; when all of the carbohydrate chains were removed from the -subunit, neither H⁺,K⁺-ATPase activity nor cell surface delivery was observed (17).

The present results found with the H⁺,K⁺-ATPase mutants are partly comparable with those found with the Na⁺,K⁺-ATPase mutants. In experiments using Na⁺,K⁺-ATPase -subunit mutants expressed in Xenopus oocytes, Noguchi et al. (27) report that abolition of any one of three disulfide bonds of the -subunit destroyed the Na⁺,K⁺-ATPase activity, whereas replacing the free cysteine in the transmembrane segment by serine retained the activity. Beggah et al. (28) also report that abolition of either of the two carboxyl-terminal disulfide bonds of the -subunit (loops 2 and 3) destroyed the Na⁺,K⁺-ATPase activity, whereas a small quantity of functional Na⁺,K⁺-pump was expressed at the cell surface of Xenopus oocytes when cRNAs for the wild-type -subunit and the loop 1 mutant -subunit were coinjected. In the present study for gastric H⁺,K⁺-ATPase, removal of any one of three disulfide bonds of the -subunit destroyed the H⁺,K⁺-ATPase activity.

The roles of the disulfide bonds of the -subunit in the / assembly are more clearly different between H⁺,K⁺-ATPase and Na⁺,K⁺-ATPase despite their structural similarity. In the previous study of the Na⁺,K⁺-ATPase expressed in Xenopus oocytes, the disruption of either of the two carboxyl-terminal disulfide bonds (loops 2 and 3) of the -subunit abolished the / assembly, whereas the / assembly was retained after cleavage of the most amino-terminal disulfide bond (loop 1) (27). On the other hand, our present results showed that the / assembly was lost after removal of any one of three disulfide bonds of the H⁺,K⁺-ATPase -subunit, indicating that each disulfide bond is important for the / assembly. However, it cannot be completely excluded that these differences are due to the difference in expression systems between Xenopus oocytes and mammalian stable cell lines.

In conclusion, all free cysteine residues of the -subunit in the intracellular and transmembrane segments are not necessary for the functional expression of H⁺,K⁺-ATPase. On the other hand, all three disulfide bonds in the ectodomain of the -subunit are necessary for the / assembly, H⁺,K⁺-ATPase activity, stability of the - and -subunits, and cell surface delivery of the -subunit. The two carboxyl-terminal disulfide bonds (loop 2 and 3) are necessary for cell surface delivery of the -subunit.

	ACKNOWLEDGEMENT

We are grateful to Prof. Michael Caplan for helpful discussion and careful revision of the manuscript.

	FOOTNOTES

^* This study was supported in part by grants-in-aid for Scientific Research (to S. A. and N. T.) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan and a fellowship from Fujisawa Research Foundation (to S. A.).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. Tel.: 81-76-434-7187; Fax: 81-76-434-5176; E-mail: shinji@ms.toyama-mpu.ac.jp.

Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M200523200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a] pyridine-3-acetonitrile; PBS, phosphate buffered saline; HEK, human embryonic kidney cells; Ab, antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES