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BOR - Papers in Press, published online ahead of print September 28, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043752
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BIOLOGY OF REPRODUCTION 74, 185–194 (2006)
DOI: 10.1095/biolreprod.105.043752
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Distinct Expression Patterns of Different Subunit Isoforms of the V-ATPase in the Rat Epididymis1

C. Pietrement 3, G-H. Sun-Wada 5, N. Da Silva 3, M. McKee 3, V. Marshansky 34 , D. Brown 34 , M. Futai 6, and S. Breton 2 34 

Program in Membrane Biology,3 Massachusetts General Hospital, Boston, Massachusetts 02114 Department of Medicine,4 Harvard Medical School, Boston, Massachusetts 02215 Department of Biochemistry,5 Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyoto 610-0332, Japan Futai Special Laboratory,6 Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency, Shinagawa-ku, Tokyo 141-0021, Japan

ABSTRACT

In the epididymis and vas deferens, the vacuolar H+ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.

epididymis, vas deferens

INTRODUCTION

The vacuolar-H+ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton pumping across membranes. It is ubiquitously expressed in eukaryotic cells, where it participates in the acidification of highly differentiated organelles, including the Golgi apparatus, lysosomes, endosomes, and secretory vesicles [1–4]. In addition, the V-ATPase is also found at high density in the plasma membrane of specialized epithelial cells that are involved in active proton transport and pH regulation of extracellular compartments, such as narrow and clear cells in the epididymis and vas deferens [5–8], renal intercalated cells [9–11], osteoclasts [12], or some cell types in the inner ear, including interdental cells, which resemble renal intercalated cells [13, 14]. In the epididymis and proximal vas deferens, plasma membrane V-ATPase in narrow and clear cells [6, 8, 15] is involved in the establishment of an acidic luminal pH that contributes to maintaining spermatozoa in a quiescent state during their maturation and storage in these organs [16, 17]. In the kidney, expression of the V-ATPase in the plasma membrane of collecting duct intercalated cells is critical for the regulation of systemic acid/base balance [1, 4, 18]. In osteoclasts, the V-ATPase plays a vital role in bone resorption [12, 19], and in the inner ear it is involved in maintaining the high K+ level of the endolymph that is essential for hearing [13, 14, 20]. Thus, in each individual cell type, the V-ATPase functions in a variety of distinct cellular processes.

The V-ATPase is a complex enzyme that is composed of many subunits and is divided into two distinct sectors [1–4, 21, 22]. The V0 sector is responsible for proton translocation and is composed of the transmembrane subunits previously designated by the lowercase letters a, c, c', c'', and d. The V1 sector forms a large cytosolic domain composed of eight subunits, designated by the uppercase letters A–H in a stoichiometry of A3B3CDEFG2H and is anchored to the plasma membrane via its interaction with the V0 domain [23]. In view of the complexity of this enzyme, a new nomenclature for each of its subunits has been recently presented and approved by both the Human and the Mouse Gene Nomenclature Committees [22]. This revised nomenclature is shown in Table 1. For simplicity, the abbreviated, single-letter subunit identification will be used throughout the text. The precise function of many of the V-ATPase subunits remains unknown, although subunit deletion studies, mainly in yeast, have described a variety of effects on assembly and function of the holoenzyme [24–26]. In addition, some of these subunits have more than one isoform encoded by different genes and with different tissue and subcellular expression patterns [4, 27–31]. Only one form has been identified for the A subunit of the human V-ATPase [31]. This ubiquitous subunit has a nucleotide-binding site and is thought to be the catalytic subunit of the V-ATPase. Subunit B is present in two isoforms: the B1 isoform, originally called the kidney isoform of the 56-kDa subunit, and the B2 isoform, originally called the brain isoform of the 56-kDa subunit [32, 33]. Subunits C, G, and E are part of the peripheral stalk that connects the V1 and V0 domains of the V-ATPase [34]. Two human isoforms are known for the C subunit. C1 is ubiquitously expressed, whereas C2 has so far been detected only in kidney and lung [27, 30]. Three G subunit isoforms have been described: G1, G2, and G3. G1 is ubiquitously expressed, G2 is brain specific, and G3 has been localized in the kidney [27, 30, 35, 36]. Two E subunit isoforms were identified: the sperm-specific E1 isoform and the ubiquitously expressed E2 isoform (originally called the 31-kDa subunit, or the E subunit) [37, 38]. Subunit a is the largest subunit of the V-ATPase and is part of the V0 domain of the pump. Four a subunit isoforms have been identified in the human V-ATPase: a1, a2, a3, and a4 [4]. Subunit a1 is expressed ubiquitously [39, 40]; subunit a2 was detected in the kidney, lung, and spleen [41]; and subunit a3 was localized in osteoclasts [42, 43], where it is essential for bone resorption [44]. Subunit a4 was detected in the kidney, inner ear, and the murine epididymis [45, 46]. Two d subunit isoforms have been identified for the human V-ATPase: d1 is ubiquitous, whereas d2 is present in kidney, lung, and osteoclast [27].


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  		TABLE 1. Revised nomeclature of Human V-ATPase subunit isoforms (based on Smith et al. [22])

Recent studies suggested that the diverse physiological functions of the V-ATPase in different membranes are established through the utilization of specific subunit isoforms [30, 36, 37, 43]. Selective interactions between these different isoforms were proposed to govern the subcellular V-ATPase targeting and to determine whether the pump will be located either in intracellullar structures or in the plasma membrane.

Our laboratory has shown expression of subunits B1, B2, and E2 in narrow and clear cells of the rat and human epididymis and vas deferens [6, 8, 15, 47, 48]. More recently, localization of subunit a4 was reported in the murine epididymis [28]. Staining of narrow and clear cells in the mouse epididymis was also obtained using an antibody raised against the 67-kDa subunit (subunit A) of the V-ATPase expressed in the fungus Neurospora crassa [49]. However, very little is known about the localization of other subunits of the V-ATPase in the epididymis and vas deferens, and the present study is aimed at characterizing the cellular and subcellular localization of subunits A, C1, C2, G1, G3, a1, a2, a4, d1, and d2 in epithelial cells of these organs.

MATERIALS AND METHODS

Antibodies

Affinity-purified rabbit polyclonal antibodies against the V-ATPase C1, C2, G1, G3, a1, a2, a4, d1, and d2 subunit isoforms were used. These antibodies have been characterized previously [29]. An affinity-purified polyclonal antibody raised in chicken against the V-ATPase E2 subunit was also used to identify narrow and clear cells [47, 50, 51]. A novel affinity-purified rabbit polyclonal antibody against the last 10 amino acids (CMQNAFRSLE) of the C-terminal tail of the V-ATPase A subunit was also used and was characterized in this study. A monoclonal mouse anti-TGN38 antibody was purchased from BD Transduction Laboratories to identify the trans-Golgi network. The following affinity-purified secondary antibodies were used, as appropriate: 1) a goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) (Kirkegaard and Perry Laboratories, Gaithersburg, MD), 2) a donkey anti-chicken IgY conjugated to indocarbocyanine (Cy3) (Jackson ImmunoResearch Laboratories, West Grove, PA), 3) and a donkey anti-mouse IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories).

Immunofluorescence: Conventional and Confocal Microscopy

Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were acquired, retained, and used in compliance with the National Research Council's recommendations. Sexually mature male rats were anesthetized with Nembutal (0.5 ml i.p.; Abbott Laboratories, North Chicago, IL) and perfused via the left ventricle with PBS (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) followed by fixative containing 4% paraformaldehyde, 10 mM sodium periodate, 75 mM lysine, and 5% sucrose in 0.1 M sodium phosphate buffer (PLP), as described previously [6, 7, 52]. Epididymis and vas deferens were cryoprotected in 30% sucrose/PBS, mounted for cryosectioning in Tissue-Tek OCT compound 4583 (Sakura Finetek USA, Inc., Torrance, CA), and quick-frozen. Sections were cut at 5 µm using a Reichert-Jung 2800 Frigocut cryostat (Leica Microsystems, Inc., Bannockburn, IL) and picked up onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). For indirect immunofluorescence microscopy, sections were hydrated 15 min in PBS and treated for 4 min with 1% SDS—an antigen retrieval procedure that we have previously described [53]. Sections were washed in PBS (3x for 5 min) and then blocked in PBS containing 1% BSA for 15 min. Primary antibodies were applied in a moist chamber for 90 min at room temperature or overnight at 4°C. Sections were washed in high-salt PBS (2.7% NaCl) twice for 5 min and once in normal PBS. Secondary antibody was then applied for 1 h at room temperature followed by washes as described above. Slides were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA). Some sections were double-stained by subsequent incubation with another primary antibody raised in a different species, followed by an appropriate secondary antibody.

Digital images were acquired using a Nikon Eclipse 800 epifluorescence microscope (Nikon Instruments, Inc., Melville, NY) using a Hamamatsu Orca 100 CCD camera (Hamamatsu, Bridgewater, NJ), analyzed using IPLab scientific image processing software (Scanalytics, Inc., Fairfax, VA), and imported into Adobe Photoshop image editing software (Adobe Systems Inc., San Jose, CA). Some images were taken using a Radiance 2000 confocal microscopy system (Bio-Rad Laboratories, Hercules, CA) using LaserSharp 2000 version 4.1 software and were imported into Adobe Photoshop software as TIFF files.

Protein Extraction and Western Blotting

Epididymis was harvested from anesthetized rats. Tissue was cut into small pieces and rinsed several times in PBS/protease inhibitors to remove most of the sperm. Tissue was homogenized in 10 ml/g of buffer containing 250 mM sucrose, 18 mM Tris, 1 mM EDTA, and complete protease inhibitor (Roche Applied Science, Indianapolis, IN), adjusted to pH 7.4 with HEPES, using a PRO 200 homogenizer followed by 20 strokes in a glass potter fitted with a Teflon pestle (Thomas Scientific, Swedesboro, NJ). The protein concentration was determined with the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) using albumin as a standard.

Protein extracts were diluted in Laemmli sample buffer, boiled for 1 min, and loaded onto Tris-glycine polyacrylamide 4%–20% gradient gels (PAGEr Duramide Precast Gels, 4%–20% Tris-glycine Gels; Cambrex, Rockland, ME). After SDS-PAGE separation, proteins were transferred onto an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories). Membranes were blocked in Tris-buffered saline (TBS) containing 5% nonfat dry milk and then incubated overnight at 4°C with the primary antibodies at a concentration of 0.5 µg/ml in TBS containing 2.5% milk. After three washes in TBS with 0.1% Tween 20 and 15-min block in TBS with milk, membranes were incubated with a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After five further washes, antibody binding was detected with the Western Lightning Chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA) and Kodak X-Omat blue XB-1 films.

Immunogold Staining for Electron Microscopy

Rats were perfused as described above with a solution containing 4% PLP and 0.01% glutaraldehyde in PBS. Small pieces of tissue were cryoprotected in 2.3 M sucrose in PBS. Ultrathin cryosections were cut on a Leica EM FCS at –80°C and collected onto formvar-coated gilded 200-mesh grids. Sections were blocked for 10 min on drops of 5% (v/v) normal goat serum plus 1% (w/v) bovine serum albumin in PBS. The anti-subunit A antibody was applied at a final concentration of 1:100 in DAKO diluent (DAKO Corp., Carpinteria, CA) for 1 h at room temperature. Following several washes in PBS, the antibody was labeled with goat anti-rabbit IgG conjugated to 15-nm gold particles (Ted Pella, Inc, Redding, CA) at a final concentration of 1:20 in DAKO diluent for 1 h at room temperature. After several rinses in distilled water, the sections were stained on ice-cold drops of uranyl acetate/tylose solution for 10 min and allowed to air-dry. The sections were examined in a Philips CM 10 transmission electron microscope at 80 kV.

RESULTS

Specificity of the V-ATPase Antibodies

Each of the V-ATPase subunit antibodies used in this study, except the novel anti-subunit A antibody, has been characterized previously [29, 47, 50, 51]. The specificity of these antibodies in epididymis samples was further confirmed by Western blotting. As shown in Figure 1, all antibodies gave one single band at the appropriate molecular weight, showing their purity. A more complete analysis was performed for the novel anti-A antibody by repeating the Western blot after preincubation of the antibody with the immunizing peptide. This antibody revealed a single band at the expected molecular weight of 70 kDa on Western blots from epididymis homogenates (Fig. 1 lane A), and no signal was detected after preincubation of the antibody with the A subunit peptide.


Figure 1
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  		FIG. 1. Detection of V-ATPase subunits by Western blot in total homogenate from rat epididymis. All antibodies used in the present study showed one single band at their appropriate molecular weight (A, 70 kDa; C1 and C2, 42 kDa; G1 and G3, 13 kDa; a1, a2, a4, 116 kDa; d1 and d2, 38 kDa). The signal was completely abolished after preincubation of the anti-subunit A antibody with the immunizing peptide, showing specificity of this novel antibody

Localization of theA Subunit of the V-ATPase

Immunofluorescence using our affinity-purified anti-subunit A antibody revealed a strong apical staining in the apical pole of narrow cells of the initial segments (Fig. 2A). Double labeling for the A (Fig. 2B) and E2 (Fig. 2C) subunits revealed their colocalization in narrow cells (yellow staining in Fig. 2D). A strong apical staining was observed for subunit A in clear cells of all regions of the epididymis, including the cauda (Fig. 3A), as well as the vas deferens (data not shown). Similarly to narrow cells, clear cells were identified by their coexpression of the E2 subunit (Fig. 3B). A weak cytosolic staining was also detected in these cells, as we have previously reported for other subunits of the V1 domain of the pump, including subunit E2 [6, 47, 48]. Figure 3C shows a complete colocalization of subunits A and E2 in subapical vesicles and apical microvilli (yellow staining). Immunogold electron microscopy confirmed the localization of subunit A in apical microvilli in addition to its subapical localization (Fig. 3D). Interestingly, this subunit was also detected in intracellular structures of principal cells located in the proximal regions of the epididymis (Fig. 4A). Double labeling for TGN38, a protein located in the trans-Golgi network, revealed that subunit A is present in structures that are closely associated with, but are at least partially distinct from, the TGN38-stained trans-Golgi (Fig. 4, B and C).


Figure 2
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  		FIG. 2. Localization of the V-ATPase A subunit in narrow cells. A 5-µm section of rat initial segments was stained with antibodies against the A and E2 subunits of the V-ATPase. Subunit A (A; green) is located in the apical pole of a subpopulation of epithelial cells. Double labeling for the A (B; green) and E2 (C; red) subunits showed that these subunits colocalize in the apical pole of narrow cells (C; yellow). Bar = 15 µm (A) and 10 µm (B–D)


Figure 3
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  		FIG. 3. Localization of the V-ATPase A subunit in clear cells. A 5-µm rat cauda epididymidis section was stained with antibodies against the A and E2 subunits of the V-ATPase. Subunit A (A; green) is located in the apical pole of clear cells identified by their E2 expression (B; red). The merged image of C shows colocalization of A and E2 in apical microvilli and subapical endosomes (C and inset; yellow staining). A faint staining was also detected in the cytosol of clear cells. No staining was detected in principal cells in this distal portion of the epididymis. The arrows indicate the border between apical microvilli and subapical vesicles. D) Immunogold electron microscope localization of subunit A in a clear cell from the cauda epididymidis. Subunit A is located in apical microvilli as well as in the subapical pole of the cell. Bar = 50 µm (A–C), 5 µm (insets in A–C), and 500 nm (D)


Figure 4
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  		FIG. 4. Localization of the A subunit in principal cells in the proximal epididymis. A 5-µm section of rat caput epididymidis stained for subunit A (A; green) and TGN-38 (B; red). Subunit A is located in intracellular structures in principal cells. Double labeling showed that these structures are closely associated but do not completely overlap with the trans-Golgi network, labeled with TGN-38 (C). Inset shows that intracellular organelles positive for subunit A (green) run in parallel with TGN-38-positive structures (red). Bar = 5 µm

Localization of theC Subunit of the V-ATPase: IsoformsC1 andC2

Both C1 (Fig. 5, A and A') and C2 (Fig. 5, D and D') were detected in clear cells of the epididymis. Epididymal narrow cells and clear cells from the vas deferens were also stained (data not shown). Strong apical and weaker cytosolic immunofluorescence staining was seen for these two V1 domain isoforms. However, the cytosolic staining for C1 seemed stronger compared to that of C2. Double labeling for the E2 subunit (Fig. 5, B, B', E, and E') revealed colocalization of both C isoforms with E2 in subapical vesicles (Fig. 5, C, C', F, and F'; yellow staining). In contrast, C1 and C2 were barely detected in apical microvilli (Fig. 5, C, C', F, and F'; red staining). No significant staining was detected in principal cells.


Figure 5
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  		FIG. 5. Localization of the V-ATPase C1 and C2 subunit isoforms. Rat cauda epididymidis was stained with antibodies against subunit isoforms C1 (A and A'; green) and C2 (D and D'; green) and were double-stained for E2 (B, B', E, and E'; red). C and C' and F and F' show merged images for C1 and E2 and for C2 and E2, respectively. Both C1 and C2 are located in clear cells, where they colocalize with E2 in subapical vesicles (yellow staining in C and F). No significant C1 and C2 staining in E2-labeled apical microvilli was detected (red staining in C' and F'). Weaker intracellular staining was also detected in the cytoplasm of clear cells, with a stronger staining for C1 compared to C2. No staining was detected in principal cells. Bar = 15 µm (A–F) and 5 µm (A'–F')

Localization of theG Subunit of the V-ATPase:G1 andG3 Isoforms

Similarly to the A and C subunits, strong apical and weaker cytosolic staining for both G1 and G3 were detected in narrow cells (data not shown), and clear cells of the epididymis (Fig. 6, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 6, C and D) showed colocalization of G1 and G3 with E2 in clear cells (Fig. 6, E and F). No significant staining was seen in principal cells. Interestingly, epididymal sperm showed immunoreactivity for the G3 isoform (Fig. 6B) but not for the G1 isoform (Fig. 6A). The localization of the brain-specific G2 isoform was not examined.


Figure 6
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  		FIG. 6. Localization of the G1 and G3 isoforms of the V-ATPase. Rat cauda epididymidis was stained with antibodies against subunit isoforms G1 (A; green) and G3 (B; green) and were double-stained for E2 (C and D; red). Both G1 and G3 are located in the apical pole of clear cells. The merged images shown in E and F show colocalization of G1 (E; yellow-orange staining) and G3 (F; yellow-orange staining) with E2 in subapical vesicles, with no apparent colocalization in E2-labeled microvilli (red staining in E and F). No staining is seen in principal cells. Epididymal sperm are stained for G3 but not for G1. Bar = 15 µm

Localization of thea Subunit of the V-ATPase:a1,a2, anda4 Isoforms

Isoforms a1 and a4 were detected in the apical pole of narrow cells (data not shown) and in clear cells of the epididymis (Fig. 7, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 7C) revealed that a1 colocalized with E2 in subapical vesicles but was absent from the microvilli (arrows in Fig. 7, A, C, and E). Subunit a4 showed a complete colocalization with E2 in subapical vesicles and microvilli of clear cells (Fig. 7, B, D, and F). Expression of the osteoclast-specific a3 isoform was not examined.


Figure 7
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  		FIG. 7. Immunolocalization of the V-ATPase a1 and a4 subunit isoforms. Rat cauda epididymidis was stained with antibodies against subunits a1 (A; green), a4 (B; green), and E2 (C and D; red). a1 and a4 are located in the apical pole of clear cells but show different patterns of staining. a1 colocalizes with E2 in subapical vesicles (E; yellow) but is absent from E2-labeled microvilli (E; red; arrows). A bright staining was observed for a4, which colocalizes with E2 in both subapical vesicles (F; yellow) and E2-labeled microvilli (F; yellow-orange staining; arrows). Bar = 15 µm

The distribution of the a2 isoform was quite distinct from that of the a1 and a4 isoforms. Whereas no significant staining was detected in the apical pole of narrow and clear cells (data not shown), subunit a2 was detected in intracellular structures of both clear and principal cells, with a weaker staining detected in clear cells. The staining was much stronger in the proximal regions of the epididymis, including caput, corpus, and proximal cauda (Fig. 8A), and was not detectable in the distal cauda (data not shown). Similarly to the A subunit, double labeling for TGN38 revealed that a2 is present in structures that are closely (but not exactly) associated with the TGN38-stained trans-Golgi network (Fig. 8, B–D).


Figure 8
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  		FIG. 8. Intracellular localization of the V-ATPase a2 subunit isoform in the proximal cauda epididymidis. A) Conventional immunofluorescence localization of a2 (A; green) in intracellular structures of principal cells. Nuclei are stained with DAPI (blue). B, C, and D) Confocal microscopy showing a proximal cauda epididymidis section stained for a2 (B; green) and TGN38 (C; red). a2-labeled intracellular structures are closely associated but are not identical to the TGN38-labeled trans-Golgi network (D; merged panel; red and green staining). Bar = 5 µm

Localization of thed Subunit of the V-ATPase:d1 andd2 Isoforms

The d1 isoform was detected in the apical pole of narrow cells (data not shown) and of clear cells in the epididymis (arrows in Fig. 9A) and vas deferens (data not shown), where it colocalizes with the E2 subunit (arrows in Fig. 9, C and E). The staining for d1 in clear cells was weaker compared to other subunits and did not allow for the exact localization of this subunit in apical microvilli and/or subapical endosomes. Immunogold electron microscopy will be required to determine with precision the subcellular localization of this subunit in clear cells. Interestingly, a strong apical d1 staining was also observed in the apical membrane of principal cells (arrowheads in Fig. 9A). This is the first description of one of the subunits of the V-ATPase being highly expressed in the plasma membrane of principal cells. In contrast, the d2 isoform was exclusively found in narrow (not shown) and clear cells (arrows in Fig. 9B), identified by their E2 expression (arrows in Fig. 9, D and F). Whereas d2 was located in subapical endosomes, it was absent from E2-labeled microvilli (middle arrow in Fig. 9F; red staining).


Figure 9
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  		FIG. 9. Localization of the V-ATPase d1 and d2 subunit isoforms. Rat cauda epididymidis was stained for d1 (A; green) and d2 (B; green). Double labeling for E2 was performed (C and D; red) to identify clear cells. In clear cells (arrows), a strong apical staining for d2 (B) and a weaker apical staining for d1 (A) were detected. The merged picture shown in E indicates colocalization of d1 with E2 in the subapical pole of clear cells (yellow-orange staining). F) Colocalization of subunit d2 with E2 in subapical vesicles (yellow staining) but no colocalization in E2-labeled microvilli (red staining in cell located in the middle of the panel). In addition, a strong d1 staining is seen in the apical membrane of principal cells (A and E; green; arrowheads), whereas principal cells are negative for d2 (B and F). Bar = 20 µm

DISCUSSION

Immunofluorescence was used to examine the expression and the cellular and subcellular localization of five subunits of the V-ATPase and some of their respective isoforms in rat epididymis and vas deferens: A, C1, C2, G1, G3, a1, a2, a4, d1, and d2. All five subunits were detected in epithelial cells of these organs but showed specific patterns of expression (Fig. 10).


Figure 10
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  		FIG. 10. Diagram illustrating the subunit composition of the V-ATPase and their expression in the epididymis. Subunits that form the membrane-bound V0 domain are labeled in lowercase letters, and subunits of the cytosolic V1 domain are labeled in uppercase letters. Based on Kawasaki-Nishi et al. [23]

Narrow and Clear Cell Localization

The V1 sector subunits A, C1, C2, G1, and G3 were all expressed in narrow and clear cells where they were located in their apical pole. These results are in agreement with the previously reported expression of other V1 subunits, B1, B2, and E2, in these cells [5, 6, 54] and further confirm the high expression of the V-ATPase in acidifying clear cells. Our laboratory has shown that these cells exhibit a significant bafilomycin-dependent proton secretion and are responsible for the majority of proton secretion in the rat vas deferens [5, 6]. Net proton secretion in these cells is regulated via recycling of V-ATPase-containing vesicles between apical microvilli and intracellular vesicles [54–56]. The precise colocalization of subunit A with subunit E2 in apical microvilli and subapical vesicles is consistent with the assembly of these unique subunit isoforms to form the V-ATPase complex [4]. This result indicates that clear cells use the A subunit for net luminal proton secretion in the epididymis. Both the ubiquitous isoform C1 and the more restricted C2 isoform were expressed in clear cells. Two C2 isoforms, C2a and C2b, resulting from alternative mRNA splicing, have been identified [29]. C2a was detected in the lung and C2b in the kidney. In this study, an antibody that recognizes both C2a and C2b was used [29]. We postulate that the C2 isoform that was recognized in epididymal clear cells is C2b because of the similarity between clear cells and kidney intercalated cells [57] and because of the common embryological origin of the kidney and epididymis. Similarly, the ubiquitous G1 subunit isoform as well as the more restricted G3 isoform were detected in the apical pole of narrow and clear cells. G3 mRNA has been detected in the kidney [27, 30], and its expression in epididymal clear cells could also be attributed to the fact that kidney and epididymis share the same embryological origin. The presence of G2 in the epididymis was not investigated here because this subunit has been previously shown to be specific for the brain and absent from the kidney [36]. In addition to their localization in the apical plasma membrane and subapical vesicles, a faint cytosolic staining was also observed for A, C1, C2, G1, and G3, similarly to the previously reported localization of other subunits of the V1 domain, including subunit E2, in the soluble fraction of clear cells [6, 47, 48].

Many subunits of the V0 sector, including a1, a4, d1, and d2, were also expressed in narrow and clear cells. a1 and a4 were located in their apical pole but showed distinct subcellular distribution. a4 showed the brightest staining and the closest colocalization with subunit E2 in subapical vesicles and apical microvilli. Expression of subunit a4 has also recently been described in the mouse epididymis [28]. Thus a4 appears to be the predominant isoform in narrow and clear cells. The ubiquitously expressed a1 isoform was present in subapical vesicles but was absent from apical microvilli. Both d1 and d2 were found in the apical pole of narrow and clear cells, where they might participate in endosomal acidification and/or proton secretion. Because of its brighter apical staining in clear cells and its high expression in kidney intercalated cells [27], d2 appears to be the predominant isoform in proton-secreting narrow and clear cells.

Principal Cell Localization

Interestingly, subunit A was detected in principal cells, where it was localized in intracellular structures closely associated with but not identical to the TGN38-labeled trans-Golgi network (TGN). Immunogold electron microscopy will be required to identify precisely these intracellular structures. Clear cells also showed a similar staining pattern for subunit A, but the staining was much weaker compared to principal cells. In addition, isoform a2 was detected in similar intracellular structures in principal cells with a weaker staining detected in clear cells. Staining of serial sections with an anti ß-COP antibody showed no apparent colocalization of a2 with ß-COP in the Golgi apparatus (data not shown). Localization of a2 protein in both clear and principal cells correlates with the previously described ubiquitous distribution of a2 mRNA [28, 42, 58, 59]. These results indicate that subunits A and a2 may be involved in the acidification of intracellular organelles in both clear and principal cells. These results are in agreement with the notion that TGN-derived vesicles are acidic and that the V-ATPase plays an active role in this process [2, 4, 60–62]. Vesicle acidification induces the recruitment of coat proteins and small GTPases of the ADP-ribosylation factor (ARF) family [63–65], a process that is important for the appropriate targeting and recycling of various membrane proteins, including GLUT4 and AQP2 [4].

It was particularly surprising to find subunit d1 in the apical membrane of principal cells. So far, no other V-ATPase subunits have been described in the apical membrane of principal cells, consistent with the absence of bafilomycin-sensitive proton secretion that has been previously reported in these cells [6]. In yeast, subunit d remains tightly associated with the V0 domain following dissociation of the V1 domain [23]. Because this subunit has no predicted transmembrane domain, its presence in the apical membrane of principal cells, where no other subunits of the V0 domain have been described, is puzzling. Further studies will be required to determine whether subunit d1 may associate with other integral membrane proteins and may participate in a function other than the proton transport activity of the V-ATPase. A similar function has been previously suggested for the 56-kDa subunit (B1), which was detected in nonacidifying endosomes isolated from kidney collecting duct principal cells [66]. Alternatively, some unidentified V0 domain subunit isoforms may be expressed in principal cells and may serve to anchor d1 in their apical membrane.

Potential Role of Different Subunit Isoforms of the V-ATPase in Epididymal Epithelial Cells

The present study shows that different isoforms of the same subunit of both the V0 and the V1 domains colocalize in the apical pole of narrow and clear cells (C1 and C2, G1 and G2, a1 and a4, d1 and d2). In yeast and osteoclasts, targeting of the V-ATPase is controlled by the assembly of different a subunit isoforms into the holoenzyme [25, 43]. The three a isoforms detected in clear cells may, therefore, be involved in differential targeting of the V-ATPase to specific membrane domains: a2 in TGN-associated structures, a1 and a4 in endosomes, and a4 in apical microvilli. Previous studies have shown that subunits d, C, and G participate in the stability of the V-ATPase holoenzyme [4, 23, 67], and these subunits may also play a role in stabilizing the V-ATPase on specific membrane domains in narrow and clear cells. Alternatively, various subunits of the V-ATPase, including subunits a, d, A, and C, have been shown to control the activity of the V-ATPase by modulating the coupling of proton transport to ATP hydrolysis [4, 23–26, 68]. The presence of various isoforms of subunits a, d, and C in clear cells may, therefore, reflect different levels of V-ATPase activity in the membrane domains and intracellular compartments in which they are expressed.

We have previously shown in rat and mouse epididymis that clear cells express both the B1 and the B2 isoform [48]. While B1 completely colocalized with E2 in apical microvilli and subapical vesicles, B2 was located only in E2-positive vesicles and was absent from microvilli. Coimmunoprecipitation assays from kidney extracts showed that C1, G1, and E2 interact preferentially with B2 and that C2b, G3, and E2 interact with B1 [29]. Out of the V0 domain subunits, d1 interacts with both B1 and B2, whereas d2 associates with B1 exclusively. Subunits a1, a2, and a4 all bind to B2, but only a4 can associate with B1. The localization of B2, therefore, correlates with the present localization of isoforms a1, d1, C1, and G1 in subapical vesicles and their absence in apical microvilli. However, the absence of a2 from subapical vesicles, where B2 is located, was not expected. Abundant a1 and a4 was detected in these vesicles and might compete away interaction between B2 and a2 in this compartment.

Some isoforms could serve as a possible alternative mechanism for the active role played by the predominant one in the V-ATPase holoenzyme. Mutations of some subunits of the V-ATPase, including B1 and a4, are linked to renal distal tubular acidosis and deafness [13, 20]. Our finding that a4 and B1 are highly expressed in narrow and clear cells of the epididymis raised the possibility that fertility might be altered in patients harboring these mutations. Long-term clinical follow-up of these young patients will be required to address this question. However, mice lacking the B1 subunit [69, 70] are not infertile (Karen E. Finberg, personal communication and our observations), indicating that their epididymal luminal acidification, which is necessary for sperm maturation and storage, is not critically impaired. These studies suggest that another B isoform, possibly B2, can compensate for the lack of B1 to maintain active proton secretion. Similarly, a1 might represent a possible backup or alternative mechanism for the active role played by the a4 isoform in luminal proton secretion in clear cells.

In summary, the expression of various isoforms of the same V-ATPase subunit in narrow and clear cells strongly indicates that these cells are fully equipped for active proton secretion and that they possess potentially important backup mechanisms that may compensate for the lack of one or more V-ATPase subunit isoforms. This underlies the important role that the V-ATPase plays in acidifying the lumen of the epididymis, a process that is important for sperm maturation and storage. The presence of various isoforms of one given subunit in narrow and clear cells may also be important for the regulation of V-ATPase activity in the different subcellular compartments in which it is expressed (e.g., endosomes vs. plasma membrane). In addition, the localization of subunits a2 and A in intracellular organelles of both clear and principal cells indicates their potential role in regulating intracellular traffic in these cells. Finally, expression of the d1 isoform in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits was an unexpected finding that may indicate other, as yet unidentified role(s) for this isoform.

FOOTNOTES

1 Supported by National Institutes of Health grants HD40793 (to S.B.), DK38452 (to D.B. and S.B.), and DK42956 (to D.B.); grants from the Committee of American Memorial Hospital of Reims, France; the Conseil Régional de Champagne-Ardenne, France; and the Ministère des Affaires Etrangères (Concours Lavoisier), France (to C.P.); and grants-in-aid from the Ministry of Education, Science and Culture of Japan (to M.F. and G-H.S-W.). The work performed in the Microscopy Core Facility of the Massachusetts General Hospital Program in Membrane Biology was supported by Center for the Study of Inflammatory Bowel Disease grant DK43351 and Boston Area Diabetes and Endocrinology Research Center award DK57521. Back

2 Correspondence: Sylvie Breton, Massachusetts General Hospital, Simches Research Center, Program in Membrane Biology, 185 Cambridge St., CPZN 8202, Boston, MA 02114. Fax: 617 643 3182; sbreton{at}partners.org Back

Received: 13 May 2005.

First decision: 31 May 2005.

Accepted: 23 September 2005.

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T. G. Paunescu, L. M. Russo, N. Da Silva, J. Kovacikova, N. Mohebbi, A. N. Van Hoek, M. McKee, C. A. Wagner, S. Breton, and D. Brown
Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice
Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1915 - F1926.
[Abstract] [Full Text] [PDF]