Four Subunit a Isoforms of Caenorhabditis elegans Vacuolar H (cid:1) -ATPase CELL-SPECIFIC EXPRESSION DURING DEVELOPMENT*

We have identified four genes ( vha-5, vha-6, vha-7, and unc-32 ) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans , the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43–60% identity and 62–84% similarity with the bovine subunit a 1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activ-ity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717–6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to

across membranes coupled with ATP hydrolysis. Acidification caused by V-ATPases is responsible for intracellular processes such as activation of zymogen, release of ligands from receptors, degradation of macromolecules, accumulation of neurotransmitters in secretory vesicles, and sorting of nascent polypeptides (5,6). These enzymes are also found in the plasma membranes of osteoclasts (7), renal intercalated cells (8), and epithelial cells of seminal ducts (9).
V-ATPases have a peripheral sector (V1) for ATP hydrolysis and an integral sector (Vo) for proton translocation across membranes. Vo consists of at least five different subunits (a, d, c, cЈ, and cЉ). Subunits c, cЈ, and cЉ are often called proteolipids and function mainly to form a proton pathway (4). Subunit d is peripherally associated with other integral subunits (1). Subunit a is the largest protein (116 kDa) among the V-ATPase subunits and has been reported to have organelle-or cellspecific isoforms responsible for cellular processes (7, 10 -16). In yeast, two a isoforms (Vph1p and Stv1p) have been found in vacuolar and Golgi/endosomal membranes, respectively (10). In mammals, three a isoforms (a1, a2, and a3) exhibit cell-and tissue-specific expressions (7,14). In particular, the a3 isoform is highly expressed in osteoclasts and specifically localized in their plasma membranes for bone resorption (7,17). Furthermore, mutations of the a3 gene have been shown to cause severe osteopetrosis (17)(18)(19)(20)(21). However, functions of the a1 and a2 isoforms remain unclear.
We have focused on V-ATPase in Caenorhabditis elegans to elucidate the functional roles of acidic compartments in development and cellular processes. Analysis involving RNA interference indicated that V-ATPase is indispensable for early embryogenesis of the worm (22). In this study, we identified four genes (vha-5, vha-6, vha-7, and unc-32) for subunit a isoforms, the first example of four distinct subunit a isoforms in higher eukaryotes. These genes are expressed in a cell-specific manner during worm development. The unc-32 gene is essential for embryogenesis, and vha-5 and vha-6 are required for larval development, whereas vha-7 is dispensable. The results suggest that V-ATPases with these isoforms generate specific acidic compartments required for development.

Maintenance of Worm Strains and Preparation of Total Lysate and
Membranes-The strain used in this study, wild-type Bristol N2, was cultured and maintained as described previously (23). Transgenic worms were obtained by microinjection with the selectable marker gene, rol-6 (su1006) (24).
Mixed stage worms (5 g, wet weight) were suspended at 4°C in 25 ml of 10 mM HEPES-KOH (pH 7.4) containing 0.25 M sucrose, 10 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, and 1 mM dithiothreitol. The suspension was passed through a French Press (1200 kg/cm 2 ), and the total lysate was centrifuged at 10,000 ϫ g for 15 min. The supernatant was further centrifuged at 100,000 ϫ g for 30 min. The precipitate was suspended in 50 mM Tris-HCl (pH 7.5) and used for membrane fraction.

RESULTS
Identification of Four Isoforms of C. elegans V-ATPase Subunit a-We found that four putative genes (C26H9A.1, F35H10.4, VW02B12L.1, and ZK637.8) homologous to the bo-vine V-ATPase subunit a1 gene were present in the C. elegans genome (29). We determined the DNA sequences of the corresponding EST clones and found that they lacked the spliced leaders (SL1 or SL2) that are attached to the 5Ј end of almost all trans-spliced transcripts (30).
Using spliced leader and gene-specific primers, reverse transcription-PCR was carried out to obtain the 5Ј-terminal regions of the cDNA clones. All amplified products had the SL1 sequence exclusively, implying that the corresponding genes are localized upstream of polycistronic units because SL1 is not found in the downstream genes of the units (30,31).
RNA Interference of the vha-5, vha-6, vha-7, and unc-32 Genes-It is of interest to determine whether or not the four FIG. 1. Gene structures of the vha-5, vha-6, vha-7, and unc-32 genes. The vha-5, vha-6, vha-7, and unc-32 genes were mapped to chromosome IV, II, IV, and III, respectively. Closed and open boxes represent the coding and untranslated regions of these isoform genes, respectively. Arrows indicate the length and direction of the transcripts. The genomic fragment containing the indicated upstream region of each gene was fused with the GFP gene to examine expression (plasmid pHJ-V5P01, vha-5; plasmid pHJ-V6P03, vha-6; plasmid pHJ-V7P01, vha-7; and plasmid pHJ32P01, unc-32).
Other putative genes and the directions for their transcription are also indicated by shaded boxes and arrows, respectively. subunit a isoforms are expressed in the same cells and which isoforms are important for worm development. We addressed these questions by means of RNA interference, a powerful tool for silencing gene expression (33). We introduced dsRNAs of the isoform genes into adult worms to disrupt the expression of the corresponding genes in progenies. Introduction of unc-32 dsRNA arrested almost all progenies at an embryonic stage (ϳ100 cells) (Table II), indicating that the unc-32 gene is required for a specific process during embryogenesis.
The progenies of worms injected with vha-5 dsRNA were able to develop normally up to the larval 2 (L2) stage, but most of them died at this stage (Table II). In contrast, the progenies of worms injected with vha-6 dsRNA remained at the L1 stage for several days and finally died without further development. On the other hand, vha-7 dsRNA had no effect on the development, morphology, or behavior of the resulting progenies or the in-jected worms (Table II; data not shown). These findings indicate that V-ATPases with these subunit a isoforms may have critical functions at distinct developmental stages.
Cell-specific Expression of V-ATPase Subunit a Isoforms in C. elegans-We visualized the expression of these genes using GFP reporter genes. The genomic organization around these genes supports the argument that a promoter region for a polycistronic unit is present in front of the corresponding gene ( Fig. 1). Based on this observation, the upstream region and a part of the exons for each isoform were inserted in front of the GFP gene.
The vha-6::GFP transgenic worms showed signals exclusively in their intestinal cells between the L1 (Fig. 3, E and F) and adult (Fig. 3, G and H) stages. The vha-7::GFP construct was highly expressed in hypodermal cells (Fig. 3, I, arrowheads, and J) and the uterus (Fig. 3, K and L). In transgenic L1 larvae (Fig. 3, M and N), the unc-32::GFP signals were predominantly observed in their nerve ring and ventral nerve cord, consistent with the report that the unc-32 (e189) mutation causes uncoordinated movement (35). In the transgenic adult worms, the signal was detected in vulvae and spermathecaluterine valves (Fig. 3, O and P). Weak signals were also detected in the pharynx (Fig. 3, O and P). These results indicated that subunit a isoform genes are expressed specifically in distinct cells and that their expression patterns do not overlap.
Immunodetection of the vha-5 and vha-6 Gene Products-Reporter gene analysis revealed that the four isoforms were expressed in distinctly different cells. Thus, it became of interest to determine their intracellular localization during devel-opment. Of these genes, the vha-5 and vha-6 genes were essential for larval development, whereas the unc-32 (Table II), proteolipid, and C subunit genes (22) were required for embryogenesis. We focused on VHA-5 and VHA-6 because analysis of their locations may provide clues with regard to acidic compartments or cells required for larval development. Affinity-purified antibodies against VHA-5 clearly recognized a single band (106 kDa) for a lysate of a mixed-stage population (Fig. 4A, lane  1). Anti-VHA-6 antibodies bound specifically to a 100-kDa band exhibiting slight smearing (ϳ 106 kDa) (Fig. 4A, lane 2). These molecular masses were larger than the calculated molecular weights (99,311, VHA-5; 98,538, VHA-6), suggesting that VHA-5 and VHA-6 were posttranslationally modified. Because bovine subunit a is known to be an N-linked glycosylated protein (36), the two isoforms may also be N-linked glycosylated.
Immunoprecipitation of a Isoforms with Other V-ATPase Subunits-The membrane fraction of a mixed-stage population was incubated with octylglucoside, the solubilized fraction was treated with antibodies against each isoform, and 12% polyacrylamide gel electrophoresis was performed on the immunoprecipitate in the presence of sodium dodecyl sulfate. As shown by immunoblot (Fig. 4B), the immunoprecipitate obtained with antibodies against VHA-5, VHA-6, and UNC-32 contained subunit C of the membrane extrinsic V1 sector. These results, together with high similarities to the bovine a1 isoform, suggest that the VHA-5, VHA-6, and UNC-32 isoforms were as- sembled with other V-ATPase subunits. Thus, immunohistochemical studies using the antibodies may indicate the location of V-ATPases with individual isoforms.
Localization of VHA-5 in Embryos, Larvae, and Adults-VHA-5 was detectable in a dot-like compartment in every embryonic cell (Fig. 5, A, B, and E, arrowheads). The compartment was found exclusively around the nucleus and observed during embryogenesis (4-cell stage, Fig. 5A; 8-cell stage, Fig. 5, B and C; and ϳ100-cell stage, Fig. 5, E and F). A cup-shaped structure was often observed around nuclei instead of the dot-like compartment (Fig. 5D, arrows). On the other hand, VHA-5 expression changed dramatically at the larval stage (Fig. 5G) and became dominant in an H-shaped excretory cell (Fig. 5, H and  I), consistent with the results for transgenic worms carrying the vha-5::GFP fusion gene. VHA-5 expression was not observed in the pharynx, where the vha-5::GFP reporter was active, indicating that the expression may be due to an increased copy number of the reporter plasmid.
Distribution of VHA-6 in Embryos and the Intestine-VHA-6 was preferentially localized in dot-like structures in P2 cells of 4-cell stage embryos (Fig. 6, A, arrow, and B). Only single cells continued to express VHA-6 during further development (Fig.  6C, arrows). After the cleavage stage (ϳ 300 cells), when intestinal cells appear, VHA-6 was detected in two distinct cell types (Fig. 6D, arrows). These two cell types were aligned on both lateral sides at the comma stage (Fig. 6E, arrows). The expres-sion pattern of VHA-6 was similar to the cell-specific distribution of P granules, which are passed onto a germ line cell lineage from P2 cells (37).
After the comma stage in embryos, expression of VHA-6 changed. In 2-fold embryos, VHA-6 was limited to the intestine (Fig. 6F, arrowheads). Such intestinal expression was also observed in larvae (L1, Fig. 6G; L3, Fig. 6H) and adults (data not shown). The C. elegans intestine comprises a tube formed from 20 cells with microvilli on their apical surface (38). VHA-6 was clearly detectable on the apical surface of intestinal cells, forming two lines along the worm body (Fig. 6, G and I, arrowheads). Furthermore, VHA-6 was clearly found at the junction between the pharynx and the intestine (Fig. 6, H and I, arrows). These results indicate that VHA-6 on the apical surface of intestinal cells may function by taking up nutrients through microvilli. DISCUSSION We have identified four genes (vha-5, vha-6, vha-7, and unc-32) encoding subunit a isoforms of C. elegans V-ATPase. These gene products exhibit high identity and completely retain 11 amino acid residues essential for the activity, assembly, or intracellular sorting of yeast V-ATPase (32). We have found no homologues in the C. elegans genome except for these genes.
These results indicate that this is the first example of four distinct isoforms of V-ATPase subunit a being identified in higher eukaryotes. Pujol et al. (25) reported that the unc-32 gene gives rise to six different transcripts through alternative splicing. However, no alternatively spliced transcript for vha-5, vha-6, or vha-7 has been found in the EST data base. 2 Subunit a isoforms have been identified in human (11)(12)(13), mouse (7,14), cow (15), chicken (16), and yeast (10). We recently found a fourth isoform (a4) of mouse subunit a, similar to the case of C. elegans, 3 implying that at least four a isoforms are required in higher eukaryotic cells.
Transgenic analysis involving the GFP reporter gene showed that the four isoforms were expressed in a cell-specific manner. The vha-5, vha-6, vha-7, and unc-32 promoters were highly active in the H-shaped excretory cells, intestine, hypodermis, and nerve cells, respectively. It is noteworthy that the vha-7 and unc-32 genes were strongly expressed in the uteri of hermaphrodites. Thus, the two gene products may have maternal roles in embryonic development.
VHA-5 was expressed predominantly in H-shaped excretory cells in adult worms. The excretory cells are thought to function in osmoregulation and the excretion of toxic and metabolic waste (39 -42). We found that the cells preferentially express the genes for the 16-kDa proteolipid (vha-1, vha-2, and vha-3), the 23-kDa proteolipid (vha-4), and the C (vha-11) subunits of V-ATPase (22,26,34). These findings suggest that VHA-5 may be a critical isoform of V-ATPase with regard to its function in the H-shaped excretory cells. Furthermore, RNA interference analysis of the vha-5 gene showed that the progenies of worms injected with vha-5 dsRNA died specifically at the L2 stage. These results imply that further development of L2 larvae requires excretory functions driven by V-ATPase with the VHA-5 isoform.
V-ATPases localized on plasma membranes are known to generate an acidic environment outside the cells. They are found in osteoclasts (7), renal intercalated cells (8), epithelial cells of seminal ducts (9), and the bladder (43). Toyomura et al. (7) have clearly shown that the mouse subunit a3 isoform plays important roles in determining the V-ATPase localization in the plasma membrane. We have shown that VHA-6 is distrib-2 T. Oka and M. Futai, unpublished data. 3 T. Oka and M. Futai, manuscript in preparation.

FIG. 4. Association of subunit a isoform with V1 subunit.
A, immunoblot of VHA-5 and VHA-6. C. elegans total lysate (30 g) of a mixed-stage population was subjected to polyacrylamide-gel electrophoresis and blotted onto a nitrocellulose membrane. The membrane was incubated with affinity-purified antibodies against VHA-5 (lane 1) or VHA-6 (lane 2). Immunodetection was carried out using alkaline phosphatase-conjugated antibodies, 5-bromo-4-chloro-3-indolyl phosphate, and 4-nitro blue tetrazolium chloride. VHA-5 and VHA-6 were recognized as 106-kDa and 100 -106-kDa bands, respectively. B, association of VHA-5, VHA-6, and UNC-32 with the V1 subunit of V-ATPase. Membranes (500 g of protein) of a mixed-stage population were incubated at 4°C for 2 h in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2% octylglucoside, and 200 mM NaCl. The solubilized fraction (100 g of protein) was incubated for 1 h with antibodies against VHA-5, VHA-6, or UNC-32. The immunoprecipitate was subjected to 12% polyacrylamide gel electrophoresis and blotted onto a nitrocellulose paper. The position of C subunit is indicated by an arrowhead. The paper was incubated with antibodies against C subunit. The solubilized fraction precipitated with antibodies against C subunit (C subunit) and treated with preimmune IgG (IgG) are shown as controls. The other immunoprecipitation method was as described previously (4).
uted specifically on the apical surface of intestinal cells in larval and adult worms. The amino-terminal region (1-140 amino acid residues) of VHA-6 was sufficient for localization on the apical surface (data not shown). We speculate that VHA-6 is a key subunit for the sorting of V-ATPase to the plasma membrane to acidify the intestinal lumen.
RNA interference analysis showed that vha-6 dsRNA ar-rested larval development at the L1 stage. The L1 larvae did not die immediately but remained alive for up to a week. This phenotype is similar to that of L1 larvae starved after hatching. The L1 larvae of the vha-6 dsRNA-injected worms took up a fluorescence dye, DiO, into their intestinal lumen (data not shown), indicating that vha-6 dsRNA did not affect the food intake ability of the worms. Thus, it is reasonable to assume  B is a phase-contrast image of A: ABa, ABp, EMS, and P2 cells were identified by their morphology. D, after the cleavage stage (ϳ200 cells), VHA-6 was expressed in two kinds of cells (arrows). E, at the comma stage, the two types of cells highly expressing VHA-6 (arrows) were aligned on both lateral sides. F, in 2-fold embryos, VHA-6 was strongly observed on the luminal sides (arrowheads) of the intestine. G, in L1 larvae, VHA-6 was strongly expressed on the apical surface of intestinal cells (arrowhead). H and I, in addition to intestinal cells, VHA-6 was also detectable at the junction between the pharynx and the intestine (arrows) of adult worms. that defective proton transport into the intestinal lumen due to the lack of VHA-6 arrested worm development at the L1 stage. These results suggest that luminal proton transport by V-ATPase with the VHA-6 isoform may be essential for the digestion of food or uptake through microvilli. In this regard, V-ATPase localized on the plasma membrane in the larval midgut of the tobacco hornworm, Manduca sexta, is important to generate a proton gradient (44).
In contrast to the intestinal expression during postembryonic development, VHA-6 was found predominantly in P2 cells in 4-cell embryos. The expression continued to a germ line cell lineage (P3, P4, Z2, and Z3 cells) until the comma stage. Introduction of vha-6 dsRNA abolished this embryonic expression (data not shown). Although Z2 and Z3 cells finally proliferate to germ cells (45), VHA-6 was not observed in the gonads or spermathecia of adult worms. It should be noted that the expression of VHA-5 and VHA-6 changed dramatically between the embryonic and larval stages. As described above, expression of VHA-6 was observed in germ line cells during the embryonic stage but was observed in intestinal cells in larvae and adults. This suggests the requirement of different acidic compartments during development.