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J. Biol. Chem., Vol. 277, Issue 20, 18098-18105, May 17, 2002

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A Proton Pump ATPase with Testis-specific E1-Subunit Isoform Required for Acrosome Acidification^*

Ge-Hong Sun-Wada, Yoko Imai-Senga, Akitsugu Yamamoto^§, Yoshiko Murata, Tomoyuki Hirata, Yoh Wada, and Masamitsu Futai^¶

From the  Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corp., Osaka 567-0047, Japan and ^§ Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan

Received for publication, December 5, 2001, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The vacuolar-type H⁺-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K_m^ATP than yeast V-ATPase. Consistent with the temperature-sensitive growth of vma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vacuolar H⁺-ATPase (V-ATPase),¹ an ATP-dependent proton pump, is one of the ubiquitous eukaryotic enzymes. It is present in endomembrane organelles such as vacuoles, lysosomes, endosomes, Golgi apparatus, chromaffin granules, and coated vesicles (1-3) and is also found in the plasma membranes of specialized cells including osteoclasts and renal epithelial cells (4, 5). V-ATPase is required for diverse cellular processes including receptor-mediated endocytosis, renal acidification, bone resorption, neurotransmitter accumulation, and activation of acid hydrolases (2).

V-ATPase (V₁V₀) has a similar structure and mechanism to F-type ATPase (ATP synthase, F₁F₀), and their ATP-dependent conformational changes are transmitted between the peripheral complex (V₁ or F₁) and the proton pore (V₀ or F₀) through a number of subunits forming a stalk (1-3). Deletion of mammalian V_o subunit c, encoded by a single gene (6, 7), has been shown to cause an embryonic lethal phenotype (8, 9). One of the most important questions is how a ubiquitous V-ATPase can function in a wide variety of physiological processes.

The diverse functions of V-ATPase may be established by utilizing a specific subunit isoform(s), its basic functional structure being maintained. Multiple subunit isoforms have been found for the largest subunit, a, of the V₀ sector in nematode (10), chicken (11), mouse (12-15), and man (16). a4 is specifically expressed in renal intercalated cells (14, 17), and its mutations cause renal acidosis (17), whereas a defect of a3, a component of the osteoclast plasma membrane enzyme (12), results in osteopetrosis (18). The only reported isoforms of the mammalian V₁ sector is the B subunit. B1 is specifically expressed in kidney and cochlea (19), and B2 is expressed ubiquitously.

In this study, we have isolated a mouse gene, Atp6e1, coding for a novel isoform (named E1) of the V-ATPase E subunit. E1 exhibits high similarity to ubiquitously expressed E2 (Atp6e2) and is expressed exclusively in the germ cells of testis. We found that a V-ATPase with E1 and a2 isoforms is located specifically in the acrosomal membrane of sperm. This unique V-ATPase is required for an acidic intra-acrosomal pH of 5.3 + 0.1 (20), which is necessary for processing protease zymogen essential for fertilization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of E1 and E2 cDNAs-- poly (A)⁺ RNAs from mouse testis were prepared using TRIZOL^® (Invitrogen) and Oligotex-dT30 (Takara) by standard methods. The cDNA encoding for E1 was amplified by reverse transcription-PCR (High Fidelity RNA PCR kit, Takara) with primers 5'-cctgcctctgcccccgtttctgagagcagccatg-3' (GH93, forward) and 5'-gaggaagatgacaccccacccccaccccag-3' (GH94, reverse), designed from available mouse expressed sequence tags (EST). The resulting PCR product was cloned into pBluescript, and then the sequences of two independent clones were verified by ABI Prism® BigDye^TM Terminator cycle sequencing (Applied Biosystems). The full-length cDNA was isolated by means of rapid amplification of cDNA ends. The cDNA encoding the E2 isoform was obtained from mouse EST clone accession number BF385623 and then sequenced.

Northern Blot Analysis, Southern Analysis, and in Situ Hybridization-- Aliquots of poly(A)⁺ RNA (2 µg) from testis, ovary, or uterus were subjected to gel electrophoresis and then transferred to a nylon membrane (Hybond N; Amersham Biosciences) (21). The mouse multiple tissue blots were purchased from CLONTECH. The probes corresponding to the +491 to +957-bp region of E1 and the +716 to +1148-bp region of E2 were used for hybridization. Southern analysis was performed as previously described (14), with probes corresponding to the +491 to +813-bp region of E1 and the +716 to +1148 bp region of E2 cDNAs.

Digoxigenin (DIG)-11-UTP-labeled single-stranded RNAs were prepared with the DIG RNA-labeling mixture and the corresponding T3 or T7 RNA polymerase (Roche Molecular Biochemicals). The 466- and 432-bp fragments from the 3'-untranslated regions of E1 and E2, respectively, were cloned into pBluescript (Stratagene) and used for preparing probes. In situ hybridization was performed on 8-µm cryo-sections from the testes of ICR male mice (8~10 weeks old) (22). Alkaline phosphatase was detected after incubation for 2-24 h at 37 °C, which was terminated with Tris-EDTA. The slides were rinsed in water, counterstained with methyl green and then mounted with Crystal Mount (Biomeda).

Complementation of Yeast vma4 with E1 or E2 cDNA and Preparation of Heterologous Vacuolar Vesicles-- The entire open reading frames of E1 and E2 were amplified by PCR with HiFi^TM polymerase (Roche Molecular Biochemicals) and cloned into the pKT10 or pKT10-N-myc vector (23). The resulting plasmids, pKT-E1 (or pKT-N-myc-E1) and pKT-E2 (or pKT-N-myc-E2), were transformed into RH403 (MAT, vma4::TRP1, leu2-1, ura3-52, trp1-63, his3-200, lys2-801 ade2-101) generated by inserting the HincII-SmaI fragment of the TRP1 into the EcoT14 I site of the VMA4. The transformants were streaked onto yeast extract/peptone/dextrose plates (pH 5.0 or 7.5) (24). Strain RH403 harboring pKT10-E1 or pKT10-E2 was grown in SCD medium (0.67% yeast nitrogen base, 0.2% casamino acid, and 2% glucose) buffered to pH 5.0 with 50 mM succinate, 50 mM K₂HPO₄ (25). 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 (26) and then 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). They were precipitated, resuspended in buffer C, and then stored at 80 °C.

Measurement of ATPase Activity-- ATPase activity was measured using a coupled spectrophotometric assay as described previously (27) with several modification. To determine the K_m^ATP and V_max for V-ATPase complexes containing the yeast Vma4p or mouse E1 and E2, ATPase activity was measured over a range of ATP concentrations from 0.05-1.5 mM. Vacuolar membrane vesicles isolated from the vma4 strain expressing Vma4p, E1, or E2 (10 µg 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% dimethyl sulfoxide or 1 µM concanamycin A at 25 °C for 10 min. The assay was started by addition of ATP, and the absorbance at 340 nm was measured continuously using a Hitachi UV-visible recording spectrophotometer (UV-2500 PC). V-ATPase was defined as the fraction of the ATPase activity inhibited by 1 µM concanamycin A. ATP-dependent proton transport activity was measured by quenching of acridine orange fluorescence using a Hitachi F-3000 fluorescence spectrophotometer (28).

Antibodies against V-ATPase Subunits-- Isoform-specific rabbit antibodies against synthetic peptides (E1, QYMRLCQKHLEVQVDQTEHLPS, positions 154-175; E2, MYKIATKKDVDVQIDQEAYLPEE, positions 154-176) were generated and then purified with peptide affinity columns (Sulfolink kit, Pierce). The anti-a subunit isoform antibodies used were described previously (12). The anti-A antiserum was purchased from WAKO. The anti-c antiserum was kindly provided by Dr. S. Ohkuma (29).

Preparation of Membrane Fractions and Western Blotting-- Tissues were dissected out from two 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 Molecular Biochemicals), and then homogenized with a Wheaton homogenizer. The lysates, obtained on centrifugation at 1,000 × g for 5 min, were centrifuged at 8,000 × g for 10 min. The supernatants were centrifuged at 100,000 × g for 60 min, and the pellets suspended in the above buffer were used as membrane fractions. Yeast cells (YPH499) transformed with either pKT-N-myc-E1 or pKT-N-myc-E2 were lysed as previously described (30), the total lysates being used as positive controls.

Solubilization and Immunoprecipitation of Testis V-ATPase-- The immunoprecipitation was performed as previously described (14). Briefly, the membrane fraction obtained from testes was incubated at 4 °C for 2 h in solubilization buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2% n-octyl--glucopyranoside and 200 mM NaCl) and then centrifuged at 100,000 × g for 30 min. The supernatant (solubilized V-ATPase) was incubated with 1 µg of anti-a2 or preimmune IgG at 4 °C overnight and then with protein A-Sepharose beads for 2 h at room temperature. After washing the beads six times with the above buffer, the immunoprecipitates were boiled in the sample buffer (31) and subjected to Western blotting.

Immunohistochemistry, Fluorescence Microscopy, and LysoTracker Staining-- Adult male ICR mice were used as the source for the isolation of testes, spermatogenic cells, and sperm (32, 33). Immunohistochemistry of testis cryo-sections (6 µm) was performed as previously described (14). Sperms or spermatogenic cells were attached to a poly-L-lysine-coated slide glass in 1 ml of Krebs-Ringer bicarbonate buffer (32) and then fixed for 1 h in 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.2 (PBS). The cells were rinsed three times in PBS and then permeabilized for 30 min at room temperature in 0.2% Triton X-100 in PBS. After blocking for 1 h with 10% goat serum and 0.2% gelatin in PBS, the cells were incubated with primary antibodies at 4 °C overnight. After three rinses with PBS containing 0.02% Tween 20 (PBST), the samples were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 40-60 min at room temperature. The samples were washed three times with PBST and then stained with 10 ng/ml 4,6-diamidino-2-phenylindole (Molecular Probes) for 1 min. The slides were washed and mounted in a drop of Vectashield mounting medium (Vector Labs) and then examined under a Zeiss Axioplan2 epifluorescence microscope.

For double immunostaining, the fixed sperm were first labeled with anti-a2 antibodies as described above. After three rinses with PBST, the samples were incubated with Cy3-conjugated secondary antibodies (Jackson Immuno Research) for 40-60 min at room temperature, extensively washed with PBST, and blocked again with 10% goat serum and 0.2% gelatin in PBS for 1 h. The samples were then incubated for 2 h with FITC-labeled anti-E1 antibodies (FluoReporter® FITC protein labeling kit, Molecular Probes). The double-labeled sperm were then washed, stained with 4,6-diamidino-2-phenylindole, and mounted for microscopy as described above.

For LysoTracker staining, sperm were isolated as above and then incubated with 75 nM LysoTracker Red DND-99 (Molecular Probes) in buffer for 30 min at 33 °C under 5% CO₂. After washing three times with Krebs-Ringer bicarbonate buffer, the samples were observed by fluorescence microscopy.

Electron Microscopy-- The pre-embedded silver enhancement immunogold method was used as described previously (14). The seminiferous tubules were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Cryo-sections (6 µm) were reacted with primary antibodies overnight followed by incubation with colloid gold (1.4-nm diameter)-conjugated secondary antibodies. The gold labeling was intensified using a silver enhancement kit (Nano Probes).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Isolation of a Novel Isoform, E1, of V-ATPase E Subunit-- Similarity searches with the yeast Vma4p (E subunit) amino acid sequence revealed the presence of related mouse EST clones in GenBank^TM data bases and the RIKEN full-length cDNA project data base (34). In particular, the mouse EST clones fall into two categories that are homologous to Vma4p, indicating the presence of two genes. Because one class of the EST was detected solely in testis, we have isolated the full-length cDNA from the adult mouse testis and named the encoded protein the E1 isoform. E2 cDNA was obtained by sequencing EST clone accession number BF385623. We designated the genes coding for E1 and E2 as Atp6e1 and Atp6e2, respectively. According to the FANTOM data base of RIKEN, we found that the Atp6e1 cDNA has been mapped to chromosome 17 (~47.15 centimorgans). This region is part of a segment syntenic with the location of human Atp6e1 on 2p16-p21.²

The E1 and E2 isoforms comprise 226 amino acid residues, exhibit more than 70% identity to each other, and show ~33% identity to Vma4p (Fig. 1a). Conserved residue Asp-139 corresponds to Asp-145 in VMA4, in which mutations affect the stability of Vma4p (35) (Fig. 1a). Southern analysis confirmed that mouse E1 and E2 are encoded by two distinct genes (Fig. 1b).

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Alignment of the amino acid sequences of subunit E isoforms and Southern analysis of Atp6e1 and Atp6e2 genes. a, the amino acid sequences of the mouse E1 and E2 isoforms and yeast Vma4p are aligned. The numbers represent the positions of amino acid residues. Identical residues are indicated by shadowing, and the regions used for antibody preparation are underlined. Asp-145 in Vma4p is indicated (asterisk). b, genomic Southern blot analysis of the Atp6e1 and Atp6e2 genes. Mouse genomic DNA (10 µg) was digested with various endonucleases. After electrophoresis and blotting, the filter was hybridized with radioactive probe of E1 or E2. kb, kilobases.

Mouse E1 and E2 Are Able to Complement the Yeast vma4 Mutant-- The open reading frame of E1 or E2 was placed under the control of the yeast TDH3 constitutive promoter, and the resulting plasmids were introduced into cells lacking endogenous Vma4p. Expression of mouse E1 or E2 complemented the vma4 mutation (Fig. 2a). This result indicates that mouse E1 and E2 are functional V-ATPase subunits. Interestingly, we found that yeast cells expressing E1 showed a temperature-sensitive growth phenotype, whereas E2 functioned at a high temperature similar to authentic yeast VMA4 (Fig. 2b).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Complementation of the yeast vma4 mutation by mouse E1 and E2. a, a yeast mutant (vma4) harboring the vector, E1 or E2 expression plasmid, or the VMA4 gene on a single copy plasmid (VMA4) was streaked onto a yeast extract/peptone/dextrose plate buffered to pH 5.0 or 7.5 and then incubated for 4 days at 30 °C before photographing. b, the same amount of vma4 cells (5 × 104) harboring pKT10-E1 or pKT10-E2 was removed from the culture and serially diluted 5-fold 7 times (right to left for each dilution), and then 4 µl of the diluted mixture was spotted on a yeast extract/peptone/dextrose plate, pH 7.5, and incubated at 30 °C or 37 °C for 3 days. c, electrochemical H+ gradient (pH) formation in yeast vacuolar vesicles was assayed with fluorescence dye acridine orange (28). Vesicles (10 µg of protein) were incubated in 1 ml of 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, and 1 µM acridine orange with 0.1% dimethyl sulfoxide or 1 µM concanamycin A at 25 or 37 °C for 20 min and assayed for pH formation at the corresponding temperature. ATP (0.5 mM sodium-salt, open arrowheads) and 5 mM NH4Cl (closed arrowheads) were added at the times indicated.

To compare the kinetic properties of V-ATPase complexes containing Vma4p, E1, or E2, vacuolar membranes were isolated from vma4 cells expressing the E1, E2, or Vma4p. The K_m^ATP was ~310 µM for the complex containing Vma4p and ~150 µM for the complex containing E1 or E2 (Table I). No significant kinetic difference between the E1- and E2-containing enzymes was observed. Because the yeast cells expressing E1 or E2 showed different temperature sensitivity (Fig. 2b), we examined the ATPase activities of the complex containing E1 or E2 at 25 °C and 37 °C. The ATPase activities of both complexes increased approximately 2-fold when the assay temperature was shifted from 25 to 37 °C (Table II), indicating that their catalytic activities are not temperature-sensitive.

We then measured the ATP-dependent proton transport activities using quenching assay of a fluorescence dye acridine orange. The ATP-dependent proton transport activity at 25 °C in vacuolar membranes isolated from E1- or E2-expressing cells was almost the same as that observed for Vma4p (Fig. 2c). The ratio of proton transport to ATP hydrolysis for either E1- or E2-containing complex was 65% that of Vma4p-containing enzyme at (Table I). However, we found that the E1-containing complex was completely inactive in proton transport after incubation at 37 °C for 20 min, whereas the E2- or Vma4p-containing complex still retained significant proton transport activity (Fig. 2c). These results suggest that the V-ATPase-containing E1 isoform exhibited temperature-sensitive uncoupling of proton transport and ATP hydrolysis.

Expression of E1 mRNA Is Developmentally Regulated during Spermatogenesis-- As shown in Fig. 3, the 1.4-kilobase E2 transcript was detected from all tissues examined, whereas on the contrary, the E1 transcript (1.2 kilobases) was only expressed in testis. E2 was expressed from early embryonic stages, whereas E1 expression was only detectable in adult testis (Fig. 3a). In contrast to the expression in testis, E1 was not expressed in female reproductive organs (Fig. 3b). Because the testis undergoes postnatal development during which progressively more mature germ cells appear, we examined the E1 expression during development. E1 mRNA was not observed in the testis up to 2 weeks after birth but became detectable at 3 weeks, gradually increased, and reached a plateau level at 8 weeks (Fig. 3c).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of E1 and E2 transcripts in mouse tissues. Mouse multiple tissue and embryo stage Northern blots (CLONTECH) (2 µg of poly (A)+ RNA/lane) were hybridized with the 32P-labeled E1 or E2 probes (a). Two µg of poly(A)+ RNA/lane from ovary, uterus, and testis (b) or testes of mice of different ages (weeks (w); c) was subjected to electrophoresis, blotted onto a membrane, and then hybridized with a radioactive probe. The sizes of the E1 (1.2 kilobases (kb)) and E2 transcripts (1.4 kilobases) correspond to the sizes of the isolated cDNAs (arrows).

We then investigated whether or not E1 expression is specific to restricted germ cells and regulated during spermatogenesis. Frozen sections of 8-10-week-old mouse testes were hybridized with E1 antisense probes (Fig. 4). E1 mRNA was expressed in the middle zone between the center and the basal lamina of seminiferous tubules (Fig. 4, c and e), where late spermatocytes and early round spermatids are abundant. E2 mRNA was detected at a high level in spermatogonia, the mitotic dividing cells, and a low level in spermatocytes (Fig. 4f). E1 mRNA was not detectable in Sertoli cells, spermatogonia, or spermatozoa. These results clearly indicate that the expression of E1 and E2 is regulated differently in testis.

View larger version (163K): [in this window] [in a new window]   Fig. 4.   In situ localization of E1 and E2 in testis sections. Adult testis sections (8 µm) stained with toluidine blue are shown as controls (a and b). RNA in situ hybridization in sections with E1- and E2-specific antisense riboprobes is shown in c, e, and f, respectively. Sections were also hybridized with the corresponding E1- (d) and E2-specific sense riboprobes (data not shown). The sections were counterstained with methyl green. ST, seminiferous tubule; sg, spermatogonium; sc, spermatocyte; st, spermatid; sp, spermatozoon; AS, antisense; S, sense. Bar, 25 µm.

E1 Is Specifically Expressed in the Acrosomal Membrane-- The expression pattern of E1 suggests its involvement in germ cell-specific function(s). We then examined the localization of the isoforms using antibodies raised against specific peptides of E1 and E2 (see Fig. 1 for sequences). Immunoblotting with the purified antibody against E1 revealed a 33,000-M_r protein specifically in testis, whereas the E2 antibody recognized a band (32,000 M_r) for all tissues examined (Fig. 5). The molecular weights of both bands correspond to those predicted from the respective cDNAs.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Western blotting analysis of E1 and E2 expression in mouse tissues. Membrane fractions from murine brain, kidney, spleen, liver, and testis together with ones from yeast expressing E1-myc and E2-myc fusion proteins were subjected to gel electrophoresis in the presence of sodium dodecyl sulfate. Immunoblotting using the purified polyclonal rabbit IgG against each isoform is shown. The arrows indicate the positions of E1 and E2 estimated from their cDNA sequences.

For testicular spermatogenic cells, three typical staining patterns were observed for E1 (Fig. 6, a-c). The E1 isoform was located specifically in the developing acrosomes of elongated spermatids (Fig. 6a, arrows) and proacrosomal granules of round spermatids (Fig. 6b, arrows). Weak staining of E1 was observed in ~ 20% of the seminiferous tubules (Fig. 6c). This difference is possibly due to the different maturation stages of the seminiferous tubules. The E2 isoform was located in the perinuclear region of all cells except spermatozoa (Fig. 6d).

View larger version (174K): [in this window] [in a new window]   Fig. 6.   Localization of E and a isoforms in testis seminiferous tubules. Six-µm cryo-sections were immunostained with antibodies against E1 (a-c), E2 (d), a1 (e), a2 (f), and a3 (g). Three typical staining patterns were observed for E1 (a-c). The E1 isoform localized in the acrosome regions of spermatocytes, spermatids, and sperm is indicated (arrows). The acrosomal localization of a2 and the Sertoli cell-specific localization (arrows) of a3 are also shown (f and g, respectively). Bar, 25 µm.

We observed the localization of three a subunit isoforms (a1, a2, and a3) that are expressed in testis, as we have shown previously (12). Immunostaining with the specific antibodies revealed that these isoforms exhibited different distributions in seminiferous tubules. The a1 isoform was expressed in almost all cells except the spermatozoa (Fig. 6e). Interestingly, the isoform localized in the acrosomes of elongated spermatids was a2 (Fig. 6f, arrows), and a3 was expressed specifically in Sertoli cells (Fig. 6g, arrows), which produce the nutrients required for the completion of spermatogenesis (36).

In epididymal sperm, the E1 isoform was located specifically in the acrosomes, which can be visualized by LysoTracker staining because of their luminal acidic pH (Fig. 7). LysoTracker staining was completely abolished when sperm were treated with 10 nM bafilomycin A₁, a specific inhibitor of V-ATPase (Fig. 7c), indicating that acrosome acidification is carried out solely by V-ATPase. The V-ATPase A subunit and the a2 isoform were observed in acrosome (Fig. 7, e and f). Other a subunit isoforms (a1 and a3) were not detectable in mature acrosomes (Fig. 6, e and g).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Localization of E1 isoform in sperm. A phase-contrast image (a) is shown together with a living epididymal sperm stained with LysoTracker Red (b). A sperm was stained with LysoTracker after incubation for 30 min in Krebs-Ringer bicarbonate buffer containing 10 nM bafilomycin A1 (c). For immunochemical identification of the E1, A, and a2 subunits, sperm were fixed and labeled with anti-E1 (d), anti-A (e), and anti-a2 (f) antibodies. Merged images with DNA visualized using the fluorescent dye 4,6-diamidino-2-phenylindole are also shown (g-i). The slight differences observed in the acrosome staining were due to the orientation of individual sperm attached to the slide glasses. The double immunolabeling is also shown with FITC-conjugated anti-E1 (j), anti-a2 (k), merged image of j and k with 4,6-diamidino-2-phenylindole staining (l), and the corresponding phase-contrast image (m). Electron microscopic localization of E1 isoform in acrosome membranes of testis sperm (n) is shown by electron dense silver-enhanced immunogold particles (see arrows, for examples). N, nucleus; IM, inner membrane; OM, outer membrane of acrosome. Bar, 500 nm.

The localization of the E1 in acrosomal membrane was confirmed by immunoelectron microscopy. The testes were fixed and processed for immunogold labeling with the anti-E1 antibodies. The gold particles (arrows) were observed on the outer and inner acrosomal membranes (Fig. 7n).

The co-localization of E1 and a2 in acrosomal membrane was further examined by double-staining experiments using anti-a2 and FITC-labeled anti-E1 antibodies. The staining signals for E1 and a2 in the acrosome were completely merged (Fig. 7, j-m). The result indicated that the V-ATPase with isoforms E1 and a2 is required for acrosome acidification.

In addition, immunoprecipitation experiment was carried out to confirm the presence of both E1 and a2 isoforms in the same V-ATPase complex. The octylglucoside-solubilized V-ATPase fraction from testis membranes was incubated with anti-a2 IgG, and the immunoprecipitate was subjected to polyacrylamide gel electrophoresis. The precipitate contained the E1 isoform and subunit c of the V₀ sectors, indicating that both E1 and a2 are present in the same enzyme (Fig. 8).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Co-immunoprecipitation of E1 and a2 from solubilized testis membrane. The octylglucoside-solubilized membrane fraction was incubated with anti-a2 (-a2) or control IgG (IgG) in the buffer used for the solubilization of V-ATPase (IP). The immunoprecipitates were subjected to 12 and 15% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose sheets, and incubated with anti-E1 and anti-c antibodies, respectively. The positions of the E1 and c subunits are indicated by arrows. As a control, the brain membrane fraction was applied to the electrophoresis (membrane).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified a novel isoforms (E1and E2) of the mouse V-ATPase E subunit, and the corresponding genes, Atp6e1 and Atp6e2, respectively. In contrast to the ubiquitously expressed E2, E1 was expressed specifically in testis and localized in proacrosomal granules in spermatocytes and acrosomes in developing spermatids and mature sperm. We also found that the A subunit of V₁ and a2 of V₀ were localized in the same membranes (Figs. 6 and 7), indicating that a unique V-ATPase is present in acrosomes. The E1 as well as the E2 isoform functionally complemented the yeast counterpart vma4 null mutation, indicating that mouse isoforms can assemble with other V-ATPase subunits to form a functional enzyme complex.

The acrosome is known to be an acidic secretory vesicle containing hydrolytic enzymes that are involved in the passage of the sperm across the zona pellucida. Thus, the assembly of a proton pump is an essential step for the biogenesis of this unique organelle. The presence of V-ATPase in acrosomes was predicted in a recent review (1) and suggested by a preliminary experiment (37). The present results clearly indicate that a V-ATPase with specific isoforms E1 and a2 is solely responsible for acrosome acidification. Our results show that E1 is induced concomitantly with the onset of acrosome biogenesis, whereas the ubiquitous E2 isoform is present in spermatocytes but completely absent in late spermatids and mature sperm.

Interestingly, the a subunit that is localized on the acrosomal membrane is a2, an isoform localized specifically to Golgi apparatus, not other a isoforms.³ This is consistent with the previous morphological observation that the acrosome is formed through the fusion of vesicles derived from the Golgi apparatus (38, 39). Thus, the assembly of V-ATPase with E1 and a2 isoforms is acrosome-specific and essential for its physiological function.

Previous studies indicated that the E subunit is essential for the assembly and activity of the yeast V-ATPase (40). Tomashek et al. (41) demonstrated that the stability of the yeast E subunit protein depended on the presence of the G subunit (VMA10) and proposed that the two V₁ subunits may interact as a part of a "stalk" in the V-ATPase. It was reported recently that the E subunit interacts with aldolase and may be involved directly in the coupling of V₁V₀ assembly and glucose metabolism (42). Therefore, E isoforms may have different roles in V-ATPase activity and assembly. The yeast vma4 cells expressing the E1 isoform were temperature-sensitive, whereas those with the E2 could grow at 37 °C. Unexpectedly, the yeast V-ATPase containing the E1 isoform exhibited a temperature-sensitive defect in coupling between proton transport and ATP hydrolysis. The temperature sensitivity may be consistent with the lower optimal temperature (~33 °C) for the culture of spermatid cells (32, 33). The present results also indicate that E subunit is essential for energy coupling, and V-ATPase became temperature-sensitive because the yeast Vma4p was replaced by mouse E1. This notion is also supported by the structure model of V-ATPase which located E subunit at the second stalk connecting catalytic V₁ and proton pathway V₀ sectors (43).

The presence of E isoforms (named E1 and E2) was observed recently in plants including pea, mung bean, and Arabidopsis thaliana (44). The two isoforms of had different K_m and V_max values. The plant E1 was hardly detectable in leaves and cotyledons, whereas E2 was expressed ubiquitously. Their expression is different, but neither of them shows highly restricted expression similar to that of mouse E1.

We have shown that a unique V-ATPase with E1 and a2 isoforms is involved in the acidification of the acrosomal lumen. Our results also indicate that apparently ubiquitous enzymes become acrosome-specific upon assembly with subunit isoforms E1 and a2. a3 and a4 may play similar roles in osteoclasts and renal intercalated cells (12, 14, 15). Studies on subunit isoforms are important for understanding the physiological functions of V-ATPase in various acidic compartments.

	ACKNOWLEDGEMENTS

We thank Ryogo Hirata for yeast strain RH403. We are grateful to A. Fukuyama for the expert technical assistance in the histochemistry. We also thank S. Shimamura and M. Nakashima for preparation of the manuscript.

	FOOTNOTES

^* This research was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and the Hayashi and Naito Foundations.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB074757 (Atp6e1) and AB074758 (Atp6e2).

^¶ To whom correspondence should be addressed. Tel.: 81-6-6879-8480; Fax: 81-6-6875-5724; E-mail: m-futai@sanken.osaka-u.ac.jp.

Published, JBC Papers in Press, February 28, 2002, DOI 10.1074/jbc.M111567200

² Imai-Senga, Y., and Sun-Wada, G. H. (2002) Gene (Amst.), in press.

³ T. Toyomura and M. Futai, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar H⁺-ATPase; EST, expressed sequence tag; bp, base pair; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PBST, PBS containing 0.02% Tween 20; FITC, fluorescein isothiocyanate; E1, testis-specific V-ATPase subunit E isoform; E2, ubiquitous V-ATPase subunit E isoform.

	REFERENCES

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