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J. Biol. Chem., Vol. 278, Issue 45, 44843-44851, November 7, 2003
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From the
Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047 and theDepartment of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
Received for publication, July 5, 2003 , and in revised form, August 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and 
intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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V-ATPase has a membrane peripheral V1 sector for ATP hydrolysis and an integral VO sector for proton translocation and exhibits similarity to F-ATPase (ATP synthase) in both structure and catalytic mechanism. The ATP-dependent conformational changes are transmitted between the peripheral complex (V1 or F1) and the proton pore (VO or FO) through a number of subunits forming a stalk (1, 3, 5). We have demonstrated that the catalytic mechanism involving subunit rotation is conserved in V- and F-ATPases (12-16). Deletion of mammalian VO subunit c, which is encoded by a single gene (17, 18), has been shown to cause an embryonic lethal phenotype (19), indicating that the enzyme is essential in early development. The subunits in the stalk region are required for activity and assembly in yeast (20) and mammals (11, 21).
Recent studies suggested that the diverse physiological functions of V-ATPase are established through the utilization of specific subunit isoforms(s) (21-25), the basic functional enzyme structure being maintained. Multiple isoforms have been found for the largest subunit, a, of the VO sector in nematode (26), chicken (27), mouse (6, 28-30), and humans (31). The mammalian a4 is specifically expressed in renal intercalated cells (29, 32). Consistently, a4 mutations cause renal acidosis (32), whereas a defect of a3, a component of the osteoclast plasma membrane V-ATPase (6, 25), results in osteopetrosis (33). The a1, a2, and a3 subunit isoforms exhibit different subcellular localizations (25). V-ATPases with kidney-(d2, G3, and C2) (22, 34), testis-(E1) (11, 21), and brain-(G2) (24) specific V1 subunit isoforms are involved in renal acidification, fertilization, and neurotransmitter accumulation, respectively.
We have reported that the mouse V-ATPase C subunit has two isoforms, C1 being expressed ubiquitously, and C2 specifically in kidney and lung (22). In this study, we found that C2 shows further diversity (C2-a and C2-b) due to alternative mRNA splicing. They exhibited different expression in kidney and lung. C2-a was specifically expressed in type II alveolar epithelial cells, and localized to the lamellar bodies specialized for the storage and secretion of surfactant phospholipids (35), whereas C2-b was found predominantly in the plasma membrane of renal intercalated cells. Immunoprecipitation revealed that the kidney-specific isoforms, including B1, G3, d2, a4, and C2-b, were present in the same complex, whereas the ubiquitously expressed B2, C1, G1, and other a isoforms were found in different complex. These results indicated a selective assembly of V-ATPase subunit isoforms in vivo. In addition, a chimeric yeast V-ATPase with the mouse C2-a or C2-b isoform was functional in yeast vacuoles but showed a lower Km(ATP) value and lower proton transport activity than that with C1 or yeast C subunit (Vma5p).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Northern Blot Analysis and in Situ Hybridization—Mouse multiple-tissue blots were purchased from Clontech. The PCR products amplified with primers GH76/GH88 and the kidney cDNA as the template, or with primers GH175/GH176 and lung cDNA as the template were used as probes.
(Digoxigenin)-11-UTP-labeled single-stranded RNAs were prepared with a digoxigenin RNA labeling mixture and the corresponding T3 or T7 RNA polymerase (Roche Applied Science). The 712- and 1192-bp fragments of the C2-a isoform were subcloned into pBluescript (Stratagene) and used to prepare probes. In situ hybridization was performed on 8-µm cryosections from the lungs of ICR male mice (8-10 weeks old) (11). Alkaline phosphatase was detected after incubation for 2-24 h at 37 °C. The slides were rinsed in water, counter-stained with methyl green, and then mounted with Crystal Mount (Biomeda).
Complementation of Yeast 
vma5 with C1, C2-a, or C2-b cDNA, and Preparation of Vacuolar Vesicles with Chimeric V-ATPase—The entire open reading frames of C1, C2-a, and C2-b were subcloned into pKT10 (36). The resulting plasmids, pKT-C1, pKT-C2-a, and pKT-C2-b, were transformed into SF838-1D vma5 (MAT, leu2-3,112, ade6, his4-519, ura3-52, pep4-3, gal2, vma5::LEU2) (gift from Dr. T. Stevens).
Spheroplasts were generated and further incubated in 2% glucose, 1% yeast extract, 2% polypeptone, and 0.8 M sorbitol for 15 min at 30 °C. Vacuoles were prepared with discontinuous Ficoll gradients (37) and converted into vesicles with 10 volumes of buffer C (25 mM MES-Tris, pH 6.9, containing 25 mM KCl, 10% glycerol, and 5 mM MgCl2). The vesicles were precipitated, suspended in buffer C, and stored at -80 °C.
Measurement of ATPase Activity—ATPase activity was measured using a coupled spectrophotomeric assay, as described previously with several modifications (38). To determine Km(ATP) and Vmax for V-ATPases containing yeast Vma5p, mouse C1, C2-a, or C2-b, ATPase activity was measured over a range of ATP concentrations, from 0.05 to 1.5 mM. Vacuolar membrane vesicles isolated from the vma5 strain expressing Vma5p, C1, C2-a, or C2-b (10 µg of protein) were incubated in ATPase assay buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, 1 mM MgCl2, 4 mM MgSO4, 1.5 mM phosphoenolpyruvate, 0.35 mM NADH, 20 units/ml pyruvate kinase, and 10 units/ml lactate dehydrogenase) with 0.1% Me2SO or 1 µM concanamycin A at 25 °C for 10 min. The assay was started by the addition of ATP, and the absorbance at 340 nm was followed continuously with a Hitachi UV-visible recording spectrophotometer (UV-2500 PC). V-ATPase was defined as the fraction of the ATP hydrolyzing activity inhibited by 1 µM concanamycin A. ATP-dependent proton transport activity was measured by AMCA fluorescence quenching (39).
Preparation of Antibodies—Isoform-specific rabbit antibodies against synthetic peptides (Table I) were generated and purified with affinity columns conjugated with the corresponding recombinant proteins. The antibodies against subunit a and c were described previously (6). The anti-A antiserum was purchased from WAKO. The monoclonal antibody against ABCA3 (P-180) was purchased from Covance. Because the molecular weights of several isoforms are close to that of heavy chain or the whole complex of immunoglobulin, we used biotinylated antibodies in immunoblot analysis to avoid the interference from the antibody bands. The biotinylation was carried out using a commercially available kit (Pierce).
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Preparation of Membrane Fractions—Tissues were dissected out from ICR male mice (10 weeks old) at 4 °C, suspended in 5 mM MOPS buffer (pH 7.0) containing 0.25 M sucrose, 0.1 mM MgSO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture (Roche Applied Science) and then homogenized with a Wheaton homogenizer. The lysate, obtained after centrifugation of the homogenate at 1,000 x g for 5 min, was centrifuged at 8,000 x g for 10 min. The supernatant was centrifuged at 100,000 x g for 60 min, and the pellet suspended in the above buffer was used as the membrane fraction.
Kidney membrane fraction used in immunoprecipitation was prepared as follows. Kidneys were obtained from 20 mice (ICR, 8 weeks old), and suspended in 10 mM HEPES-KOH (pH 7.4) containing 0.25 M sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture (Roche Applied Science). The suspension was homogenized in a Wheaton homogenizer. The supernatant fraction, obtained by centrifugation at 500 x g for 5 min, was further centrifuged at 10,000 x g for 15 min. The supernatant was centrifuged at 100,000 x g for 60 min. The precipitate was suspended in PBS containing 1 mM EDTA and 10% glycerol and stored at -80 °C until use. A total lysate of yeast cells (YPH499) transformed with either pKT-C1 or pKT-C2-a was prepared as previously described (40) and used as a positive control in Western blot analysis.
Immunoprecipitation and Western Blot Analysis—Kidney membranes (2 mg of protein/ml) were incubated at 4 °C for 2 h in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2% n-octyl--glucopyranoside, and 200 mM NaCl (solubilization buffer). Supernatant fraction, obtained after centrifugation at 100,000 x g for 30 min, contained solubilized V-ATPase. The solubilized fraction (100 µg of protein) was incubated for 1 h with purified antibodies (1 µg) against B1 or B2 isoforms or preimmune IgG in solubilization buffer. The immunocomplex was incubated with protein A-Sepharose beads at 4 °C for 2 h with constant rotation. Beads were pelleted by centrifugation, washed six times with the solubilization buffer, resuspended in the sample buffer (2% SDS, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris-HCl (pH 6.8)), and boiled for 2 min. Immunoprecipitates and membrane fractions were subjected to PAGE and blotting onto polyvinylidene difluoride membranes. They were incubated with biotinylated primary antibodies against subunit isoforms and followed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin (DAKO) for detection.
Immunohistochemistry and Immunofluorescence Microscopy—ICR mice (8 weeks old) were anesthetized, perfused with PBS to remove the blood, and then fixed with 4% paraformaldehyde. The lungs and kidneys were dissected out, fixed in paraformaldehyde overnight, and embedded in paraffin. Serial sections were stained immunochemically and counterstained with hematoxylin.
Electron Microscopy—The pre-embedded silver enhancement immunogold method was used (41). A lung was fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Cryo-sections (6 µm) were incubated with primary antibodies overnight, followed by incubation with the secondary antibodies conjugated with colloidal gold (1.4-nm diameter). The gold labeling was intensified using a silver enhancement kit (Nano Probes).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Tissue Variations in Expression of Alternatively Spliced Forms of C2—The tissue distributions of alternatively spliced forms of the C2 messenger RNA were examined by RT-PCR with primers (GH175 and GH176) corresponding to sequences within the insertion region. The C2-b transcript was found mainly in kidney, whereas C2-a was found predominantly in lung (Fig. 1B). This was confirmed by Northern analysis. A C2-a-specific probe (probe 1, Fig. 1C) recognized a single band for lung (Fig. 1D, left panel), whereas the probe corresponding to the entire open reading frame of C2-a (probe 2, Fig. 1C) detected signals for both kidney and lung (Fig. 1D, right panel).
Antibodies for C1 and C2 were generated against isoform-specific synthetic peptides Glu12 to Lys28 and Asp12 to Ser29 (Table I), respectively, and affinity-purified with columns conjugated with the corresponding recombinant proteins. The epitope regions are highly conserved in various mammals (Fig. 2A). Immunoblotting with the purified antibody against C2 revealed 38,000 and 40,000 molecular weight proteins in kidney and lung, respectively (Fig. 2B, lower panel), the molecular weights of both bands corresponding to those predicted from the corresponding cDNAs. We did not detect a protein band for lung with a molecular weight corresponding to C2-b. These results indicated that the C2-a isoform is the major form expressed in lung and that C2-b is kidney-specific. On the other hand, the C1 antibody recognized a 38,000 molecular weight band for all tissues examined (Fig. 2B, upper panel), confirming the previous Northern analysis results (22).
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Localization of C2-a in Lung—In situ hybridization with the C2-a antisense probe clearly demonstrated the localization of C2-a mRNA in mouse alveolar corner cells usually localized at the branching region of an alveolar wall (Fig. 3A). Type I alveolar cells with the typical flattened shape were not stained. The sense probe did not reveal any significant signals (Fig. 3B).
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Immunochemical staining with the C2-specific antibody revealed that the C2-a isoform is localized specifically to type II alveolar epithelial cells having large and rounded nuclei with a prominent nucleolus and vacuolated cytoplasm (Fig. 4A). Type II cells secrete surfactants to reduce the surface tension within the alveoli to prevent alveolar collapse during expiration (35), and their cytoplasm is filled with vesicles containing phospholipids in the form of lamellar bodies. Staining with antibodies against C2 and ABCA3 transporter, a 180-kDa lamellar body membrane protein, clearly showed that C2-a was expressed specifically in type II cells (Fig. 4A, arrows). The A subunit of V-ATPase was also detected at a high level in type II alveolar cells. In contrast, the C1 isoform was expressed at a similar level in airway epithelial cells, alveolar macrophages, pulmonary vessels, airway smooth muscle cells, and connective tissue cells (Fig. 4A, C1). We also examined the intracellular localization of C2-a by confocal microscopy and found that the staining signals of C2-a were superimposed on those of ABCA3 (Fig. 4B).
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Localization of C2-a in type II cells was also confirmed by immunoelectron microscopy. The gold particles for anti-C2 antibodies labeled the membranes of lamellar bodies, whereas the C1 antibodies mainly stained the membranes of other organelles (Fig. 5, arrows). These results indicated that a V-ATPase with the C2-a isoform is localized in the membranes of lamellar bodies, suggesting a unique V-ATPase is involved in the acidification of lamellar bodies in type II alveolar cells.
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Localization of C2-b and Other Kidney-specific Subunit Isoforms—Immunohistochemical analysis was carried out to identify the renal cells expressing the C2-b isoform. No significant signal was observed for glomeruli or proximal and distal convoluted tubules (Fig. 6A). The C2-b isoform was strongly expressed in the cortical and medulla collecting ducts (Fig. 6, A and B). In the cortical collecting ducts, and intercalated cells are responsible for proton and bicarbonate secretion, respectively (7, 42). The and cells have a V-ATPase localized on their apical and basolateral plasma membranes, respectively (7). Peanut lectin agglutinin is associated with the apical membranes of cells but not with those of cells (43). Double immunostaining indicated that all intercalated cells expressing the C2-b isoform on the basolateral surface were stained with the peanut lectin at the apical region, indicating that cells have the basolateral C2-b isoform (Fig. 6C). The cells have 
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exchanger (anion exchanger 1, AE1) on their basolateral membranes (44-46). All intercalated cells having the apical C2-b isoform were basolateral AE1-positive, indicating that cells have apical C2-b (Fig. 6D). These results indicate that a V-ATPase with the C2-b isoform is localized specifically on the apical and basolateral surfaces of and intercalated cells, respectively. The localization of C2-b was similar to those of other kidney-specific B1 and a4 isoforms of V-ATPase (29), suggesting that these isoforms are involved in the same functions. In addition, immunohistochemical staining with isoform-specific antibodies (Table I) revealed that the other kidney-specific subunit isoforms (d1 and G3) exhibited exactly the same localization in intercalated cells as B1, a4, and C2-b (Fig. 6E).
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Selective Interactions of Subunit Isoforms in Mouse Kidney— The intercalated cell-specific localization of kidney-specific isoforms prompted us to examine whether they were assembled selectively to form a unique V-ATPase. We tested each antibody to immunoprecipitate the enzyme complex from solubilized kidney membrane fraction and found that both anti-B1 and anti-B2 antibodies could efficiently precipitate the entire family V-ATPase complexes. We have used the biotinylated antibodies followed by horseradish peroxidase (HRP)-conjugated streptavidin to detect the corresponding isoform in immunoprecipitated complex. No proteins in kidney lysate reacted with HRP-streptavidin (data not shown), indicating that no endogenous kidney proteins could be recognized by HRP-streptavidin. The specificities of all the antibodies (Table I) were confirmed and used below (data not shown).
As described above, the V1 sector contained three kidney-specific isoforms, B1, C2-b, and G3. Immunoprecipitation with anti-B1 or anti-B2 antibody revealed that C2-b and G3 were co-precipitated with the kidney isoform B1 (Fig. 7, A and B), whereas the ubiquitous counterparts C1 and G1 were mostly detected in the precipitates of the ubiquitous B2 (Fig. 7, C and D). These results indicated that kidney-specific subunits B1, C2-b, and G3 were present in the same V1 sector of the V-ATPase complex. The G1 isoform was also co-precipitated with the B1, although the amount was significantly less than that with B2 (Fig. 7D). It should be mentioned that the G2 isoform is not expressed in kidney (24). The E subunit contained two isoforms, and the E2 is the only isoform expressed in kidney. Immunoprecipitation revealed that the E2 is co-precipitated with both B subunit isoforms (Fig. 7E).
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The ubiquitously expressed d1 isoform was co-immunoprecipitated with both B1 and B2 (Fig. 8A), whereas, the kidney-specific d2 was associated mainly with the B1 containing complex (Fig. 8B). Meanwhile, the ubiquitously expressed a1, a2, and a3 were co-immunoprecipitated predominantly with B2, but not with B1 (Fig. 8, C-E). The kidney-specific a4 was co-precipitated with both the B1 and B2 (Fig. 5F), consistent with its localization at intercalated cells and brush border (29, 30). The subunit c proteolipid, which is encoded by a single gene in mouse (18, 19), was co-immunoprecipitated with either B1 or B2 (Fig. 8G). These results indicated that the kidney-specific isoforms of VO sector subunits, together with those of V1 sector, were present in the same V-ATPase.
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Kinetic Analyses of V-ATPase Containing C Subunit Isoforms—C1 and C2-a could functionally replace the yeast counterpart, Vma5p (22). When expressed under the control of the yeast TDH3 constitutive promoter, C2-b was also able to complement a deletion mutation (vam5) similar to C1 and C2-a, indicating that the chimeric yeast V-ATPases with C1, C2-a, and C2-b can be compared functionally. Vacuolar membranes were isolated from vma5 cells expressing each mouse isoform or Vma5p. Western blot analysis revealed that the amounts of the A subunit in the cells expressing the chimeric V-ATPases with C1, C2-a, and C2-b were about 50%, 35%, and 30% of that observed in vacuolar membranes with Vma5p (Table II, relative expression), indicating that the expression level of the chimeric V-ATPase was lower than that of the yeast enzyme. Corresponding to the expression levels, the specific activities of the V-ATPases with C1, C2-a, and C2-b were also lower than that for Vma5p (Table II). The Km(ATP) values of the V-ATPases with C1 and Vma5p were similar, whereas those with C2-a and C2-b were significantly lower.
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We found that the ATP-dependent proton transport activity in vacuolar membranes expressing the V-ATPases with C1, C2-a, and C2-b were lower than that observed for the enzyme with yeast Vma5p. The apparent proton transport activity of the V-ATPase with C2-b was about 10% of that with Vma5p. Furthermore, the efficiency of energy coupling for C2-b was significantly low, when compared with the chimeric V-ATPases with C1 and C2-a (Table II). These results suggest that the C subunit is related to the energy coupling of V-ATPase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Subunit C is believed to be a peripheral stalk component that may not be essential for enzyme activity in a reconstituted system (47) but is important for assembly of the V1 complex (20). When VMA5 (C subunit gene) was deleted from the yeast genome, the vma5 mutant was unable to grow at neutral pH, however, both a fully assembled VO complex and a core V1 sub-complex were formed (48). These results indicated that the C subunit is required, at least, for assembly of the V1 and VO sectors.
We found that a deletion mutation of vma5 could be complemented by expression of Vma5p or mouse C subunit isoforms under the control of the TDH3 promoter, thus, the role of the C subunit could be analyzed using chimeric yeast V-ATPase with mouse isoforms. The V-ATPase with C2-a or C2-b exhibited a lower Km value or coupling efficiency between ATPase hydrolysis and proton transport, suggesting that the C isoforms may be involved in regulation of the V-ATPase activity required in different tissues.
C2-a is the only lung-specific V-ATPase subunit isoform so far identified. Histological staining with specific antibodies revealed that the V-ATPase with C2-a is localized to the lamellar body membranes in type II alveolar epithelial cells. Lamellar bodies have been shown to have V-ATPase to maintain an acidic internal pH of 
5.5 (49-51). The luminal acidic pH may be required for packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein-dependent lipid aggregation (50). Our results indicated that the V-ATPase in lamellar bodies contains a unique C2-a isoform with a 46-amino acid insertion. This insertion was not found in the C1 isoform or in the C subunits of other organisms, including yeast (20), plant (52), and nematode (53). It is of interest to examine the role of this insertion required for regulation of lamellar body acidification.
In contrast to C2-a, C2-b was expressed in kidney and localized to the plasma membranes of and intercalated cells in renal collecting tubules, similar to other kidney-specific subunit isoforms, including d1, G3, a4, and B1 (7, 29, 30). Immunoprecipitation revealed that these kidney-specific isoforms are selectively assembled into the same complex to form a unique proton pump on plasma membrane of renal intercalated cells. The ubiquitous isoforms, on the other hand, were associated with V-ATPases that are required for acidification of intracellular organelles and endocytic vesicles in proximal tubules. Meanwhile, there were minor but interesting combinations observed. The kidney-specific a4 was also localized to the endosomal membranes in proximal tubule and formed enzyme complex with ubiquitous subunit isoforms. On the other hand, the ubiquitous G1 or d1 was also observed on the plasma membranes of intercalated cells, although at low frequency when compared with the kidney-specific counterpart.
It has been shown that the plasma membrane V-ATPases have several features distinguishable from those of intracellular organelles: (a) they reside at about 1000 times higher densities than those in intracellular membranes (54); (b) their amplified expression occurs in a cell-specific manner (55, 56); and (c) they have a polarized plasma membrane distribution that allows vectorial proton secretion (for a review, see Ref. 57). The kidney produces cytosolic regulatory proteins capable of interacting directly with the V-ATPase to modify its activity (58, 59). These proteins may regulate the plasma membrane V-ATPase of intercalated cells through interaction with its kidney-specific isoforms. The kidney-specific isoforms, including C2-b, may also have a targeting signal different from the ubiquitous forms, leading to their expression in the plasma membranes. Future studies on the tissue-specific isoforms will probably provide evidence as to how the specialized V-ATPases are assembled and targeted to different destinations.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 81-6-6879-8480; Fax: 81-6-6875-5724; E-mail: m-futai{at}sanken.osaka-u.ac.jp.
1 The abbreviations used are: V-ATPase, vacuolar H+-ATPase; EST, expressed sequence tag; RT-PCR, reverse-transcribed PCR; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; AMCA, 9-amino-6-chloro-2-methoxyacridine.
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ACKNOWLEDGMENTS |
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vma5 and vma6 strains. We are also grateful to A. Fukuyama for the expert technical assistance in the histochemistry. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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