Báo cáo Y học: Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-deficient strain of Saccharomyces cerevisiae and study of its complementation pdf

Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3 -deficient strain of Saccharomyces cerevisiae and study of its complementation Mikiko Ikeda 1 , Misato Hinohara 1 , Kimiko Umami 1 , Yuki Taguro 1 , Yoshio Okada 1 ,YohWada 2 , Yoichi Nakanishi 3 and Masayoshi Maeshima 3 1 Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan; 2 Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Japan; 3 Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6 ) was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase. Expression of the cDNAs in the strain revealed that four cDNAs from the six complemented the proton transport activity into the vacuole, visualized by fluor- escence microscopy. The vacuolar-membrane-enriched fractions from the four transformants showed cross- reactivity with antibodies against the subunits a and A of S. cerevisiae V-ATPase. Two translation products from the other two cDNAs were demonstrated not to be localized in vacuolar membranes, and thus could not complement the function of the VMA3-deficient strain. As the primary structures deduced from the former four cDNAs are similar but clearly different from those of the latter two, the latter two translation products may not be able to substitute for theVMA3 gene product. Keywords: proton transport; proteolipid subunit; V-ATPase; Acetabularia acetabulum; heterologous expression. The vacuolar H 1 -ATPase (V-ATPase) is ubiquitous both in prokaryotes and eukaryotes. This enzyme is composed of two domains, a large peripheral domain (V 1 ) and a membrane integral domain (V O ). The major component of the V O portioncommontoallV-ATPasesisthe N,N 0 -dicyclohexylcarbodiimide-binding 16-kDa subunit (proteolipid subunit). In higher plants, the V-ATPase has been well characterized biochemically and at the molecular level [1]. Its physiological roles in plant cells are to regulate cytoplasmic pH and ion levels, and to drive secondary active transport of various ions and metabolites such as Ca 21 , anions, amino acids and sugars into the vacuole. Plant V-ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits [1]. Among these subunits, the proteolipid subunit is present in six copies per holoenzyme [1], which forms a functional proton channel [2]. Acetabularia acetabulum, a giant unicellular marine alga, belongs to the Dasycladaceae family. We have already reported the presence of V-ATPase in this organism and demonstrated the proton-pumping activity in tonoplast- enriched vesicles and by immunoblot analysis [3]. Three subunits (A, B and the proteolipid subunit) form a small multigene family encoding V-ATPase; two different cDNAs coding the subunits A [4,5] and B [5,6], and six different cDNAs for the proteolipid subunit [7,8] have been isolated. In this study we focussed our attention on the function of the translation products of six cDNAs (AACEVAPD1 to AACEVAPD6 ) [7,8] for the proteolipid subunit of A. acetabulum V-ATPase. By heterologous expression in a VMA3-deficient strain of Saccharomyces cerevisiae and its complementation study, we confirmed that four cDNAs (AACEVAPD2, 4, 5 and 6 ) encode functional proteolipid subunits in the yeast V-ATPase complex, but two others (AACEVAPD1 and 3 ) do not. In addition to the results of immunoblot analyses, the relation between the functional complementation and the primary structures is also discussed. MATERIALS AND METHODS Yeast strains S. cerevisiae strains YN45 [MATa, ade2 –101, his3- D 200, leu2 D 1, lys2-801, trp1, ura3-52, D cup5(vma3)::LEU2, pep4::HIS3], YN11 [MATa, ade2-101, his3- D 200, leu2 D 1, lys2-801, trp1- D 63, ura3-52, D cup5 (vma3)::LEU2], YPH499 [MATa, ade2-101, his3- D 200, leu2 D 1, lys2-801, trp1- D 63, ura3-529], BJ5458 [MATa, ura3–52] [9], trp1, lys2–801, leu2 D 1, his3 D 200, pep4::HIS3, prb1 D 1.6R, can1, GAL] [10]. The YN11 strain was derived from YPH499 strain, and YN45 strain was prepared by the use of YN11 and BJ5458 strains. Preparation of AACEVAPD1 –6 5 0 RACE and 3 0 RACE products of the respective gene were used as a template for PCR to obtain the respective full Correspondence to M. Ikeda, Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Kuboki 111, Soja 719-1197, Japan (Received 23 April 2001, revised 21 September 2001, accepted 26 September 2001) Abbreviations: V-ATPase, vacuolar H 1 -ATPase; VMA3, gene coding for the proteolipid subunit of Saccharomyces cerevisiae V-ATPase; GraP DH, glyceraldehyde 3-phosphate dehydrogenase. Eur. J. Biochem. 268, 6097–6104 (2001) q FEBS 2001 length recombinant. About 100 ng of templates (pVC13/ pVC25 for AACEVAPD1, pVC39C/pVC39 for AAC- EVAPD2, pVC10/pVC10N for AACEVAPD3,pVC18/ pVC18N for AACEVAPD4, pVC51/pVC51N for AAC- EVAPD5 and pVC74/pVC74N for AACEVAPD6 ) [7,8] were subjected to PCR with primer sets of AP2 and adaptor 1 for AACEVAPD1, AP2 and adaptor 1 for AACEVAPD2, AP2 both at 5 0 and 3 0 ends for AACEVAPD3– 6, respectively. The temperature program consisted of 20 cycles of 94 8C for 1min, 608C for 2 min, and 72 8Cfor3min.For amplification, ExTaq DNA polymerase from TaKaRa (Kyoto, Japan) was used. The PCR products were separated by agarose gel electrophoresis, excised and purified over a Qiagen DNA extraction kit (Qiagen, Duesseldorf, Germany). The purified fragments were treated with a Klenow fragment and ligated into the Eco RV site of pBluescript SK (1) (pBS) (Stratagene, LaJolla, CA, USA). After transformation in Escherichia coli XL1-Blue, plasmid DNAs were prepared and subjected to DNA sequencing. The respective transformant without any misreading was innoculated in 30 mL of Luria –Bertani/ampicillin medium and the plasmid DNA was purified over a Qiagen-Tip100 column. Conversion of TAA to CAA and preparation of recombinants in yeast expression vector In the case of AACEVAPD1, 3 and 6, TAA is used as an Acetabularia-specific codon usage (translated as Gln). Conversion of TAA to CAA was performed by PCR as described below. AACEVAPD1 has two TAA codons in its open reading frame (ORF). Fragment 1 was amplified with AP2 and VC1*Q2 (5 0 -CACGAGCTCGGGTCTCATAACACCCATT TGAGC-3 0 ), fragment 2 with VC1*Q1 (5 0 -GCTCAAATG GGTGTTATGAGACCCGAGCTCGTG-3 0 ) and VC136*Q4 (5 0 -CCCACAAAAAGCTTGGGTTGTTGAGC-3 0 )and fragment 3 with VC136*Q3 (5 0 -GCTCAACAACCCAAG CTTTTTGTGGG-3 0 ) and AP2. The temperature program consisted of 20 cycles of 94 8C for 1 min, 55 8C for 1 min and 72 8C for 2 min. About 100 ng of template (AAC EVAPD1 ) and an ExTaq DNA polymerase were used for amplification. The amplified fragments 1, 2 and 3 were separated by agarose gel electrophoresis, excised from the gels and purified as described above. The purified fragments 1, 2 and 3 (<100 ng) were used as templates and subjected to further PCR with AP2. The temperature program consisted of 20 cycles of 94 8C for 1 min, 60 8C for 2 min, and 72 8C for 3 min. A fragment about 820 bp was specifically amplified, and treated in the same manner as described above. The purified fragment was subjected to Klenow repair and ligated into the Eco RV site of pBS. After transformation, plasmid DNAs were isolated and subjected to DNA sequencing. A transformant without any misreading was innoculated in 30 mL of Luria–Bertani/ampicillin medium and the plasmid DNA was purified over a Qiagen- Tip100 column. The purified plasmid DNA was digested with Sma I and Eco RI, and a fragment about 780 bp was treated in the same manner as described above. The purified fragment was subjected to Klenow repair and ligated into pKT10DATG [11], which was digested with Eco RI and treated with a Klenow fragment. After transformation in E. coli XL1-Blue, colonies were subjected to colony Southern hybridization. The nucleotide sequences of plasmid DNAs were confirmed by restriction mapping and by DNA sequencing. In the cases of AACEVAPD3 and 6, one TAA codon in their ORFs, thus should be converted to CAA. Fragment 1 was amplified with AP2 and VC136*Q4, and fragment 2 with VC136*Q3 and AP2 for both. The PCR conditions were the same as AACEVAPD1. Both fragments were digested with HindIII, and ligated by the use of a T4 DNA ligase. The ligated fragments were subjected to agarose gel electrophoresis, excised and purified over a Qiaex resin. The purified fragments were digested with Not I and ligated into a NotI-digested pBS. After transformation and mini- preparation, the transformants were subjected to DNA sequencing. The purified plasmid DNAs without any misreading were digested with Not I, treated with a Klenow fragment, and ligated into pKT10DATG and then selected as described above. AACEVAPD2, 4 and 5 have no TAA or TAG codon as Gln in the ORFs. AACEVAPD2 was digested with Eco RI and Sma I (846 bp), AACEVAPD4 with Sma I (809 bp) and AACEVAPD5 with Sma I (750 bp). They were separated by agarose gel electrophoresis, excised and purified over a Qiaex resin. After treatment with a Klenow fragment, they were ligated into pKT10DATG and selected as described above. Sequencing Nucleotide sequencing of double-stranded templates was performed with a Sequi-Therm Cycle Sequencing kit (Epicentre Tech., Chicago, IL, USA) and a Li-Cor dNA sequencer, Model 4000 L (Li-Cor, Lincoln, NE, USA), according to the manufacturer’s instructions. Expression of AACEVAPD1 –6 in yeast The constructs prepared as described above were introduced into S. cerevisiae YN45 strain by the LiOAc/PEG method [12] and grown in AHCW/Glc medium (0.17% yeast nitrogen base without amino acid, 0.5% ammonium sulfate, 1% casein hydrolysate, 0.002% adenine sulfate dihydrate, 0.002% tryptophan, 50 m M potassium phosphate, pH 5.5, 2% glucose). Accumulation of ade fluorescent dye in vacuole and observation by fluorescent microscopy The transformants of the S. cerevisiae YN45 strain were grown in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) at 30 8C, overnight to the exponential growth phase. A total of 1 mL of culture medium was transferred to a sterile 1.5-mL microtube and centrifuged at 2400 g for 5 min. The pellet was resuspended in 1 mL of sterile distilled water and mixed with a Vortex mixer. Cells were collected by centrifugation as described above. The pellet was resuspended in 1 mL of SCD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acid, 2% glucose) containing a low adenine concentration (4 mg : L 21 ) and tryptophan (20 mg : L 21 ). A total of 500 mL of the suspension was added to 5 mL of the above medium in a 50-mL tube which was shaken at 150 r.p.m./30 8C over- night. Cells were collected in a 1.5-mL microtube by 6098 M. Ikeda et al. (Eur. J. Biochem. 268) q FEBS 2001 centrifugation as described above. The pellet was resus- pended in 1 mL of aniline blue staining solution [1% aniline blue in NaCl/KCl/P i solution (0.8% NaCl, 0.02% KCl, 0.144% Na 2 HPO 4 , 0.024% KH 2 PO 4 , 2% glucose adjusted to pH 7.4 with 1 M NaOH)] and mixed with a Vortex mixer. After centrifugation, the supernatant was removed and the staining procedure was repeated twice (three times in total). After centrifugation, the pellet was resuspended in 1 mL of NaCl/KCl/P i , and centrifuged. The supernatant was removed by decantation, cells were resuspended using a pipette and an aliquot (3– 5 mL) was transferred onto a slide glass. After covering with a cover glass, ade fluorescence was observed under a fluorescent microscope with blue and green excitation, then cell walls were observed under UV excitation. YN45 strain as a negative control and YPH499 strain as a positive control were stained in the same manner as described above, except that 20 mg : L 21 uracil was added to the above SCD medium containing adenine and tryptophan. Membrane preparation from yeast To prepare crude microsomes, we harvested cells at the mid- exponential phase in YPD medium. Cells were treated with Zymolyase 20T (Seikagaku Kogyo Co., Tokyo, Japan) and further processed as described previously by Ueoka- Nakanishi et al. [13]. The crude microsomal fraction was subsequently subjected to a stepwise sucrose gradient centrifugation (15% and 35% sucrose) as described previously by Nakanishi et al. [14]. The interface between 15 and 35% sucrose solutions was collected and centrifuged at 150 000 g for 30 min. The pellet was resuspended in a stock buffer, frozen with liquid nitrogen and then stored at 280 8C until use as a vacuolar-membrane-enriched fraction. Protein preparation, SDS/PAGE and immunoblotting Proteolipid subunits were purified from the spheroplast suspensions and the membrane fractions by chloroform/ methanol extraction according to the method described previously by Umemoto et al. [15]. In the case of spheroplast suspension, SDS and dithiothreitol were added at the same concentrations as for preparing SDS/PAGE samples. SDS/PAGE on mini-gels and subsequent immunoblotting were carried out as described previously [16]. Binding of antibody was detected using ECL Western blotting detection reagents (Amersham Pharmacia Biotech). The antibody against 70-kDa (A) subunit of S. cerevisiae V-ATPase was a gift from R. Hirata of the Institute of Physical and Chemical Research (Wako, Japan), and the antibody against the 100-kDa (a) subunit of S. cerevisiae V-ATPase was purchased from Molecular Probes Inc., Eugene, OR, USA. RESULTS Functional expression of AACEVAPD2 , 4 , 5 and 6 in yeast VMA3 -deficient strain The cDNAs for the proteolipid subunit of A. acetabulum V-ATPase (AACEVAPD1– 6 ) encodes 164, 176, 164, 168, Fig. 1. Primary structures of A. acetabulum V-ATPase, proteolipid subunit isoforms. (A) Putative amino-acid sequences derived from AACEVAPD1 –6 were compared and divided into two groups. Hyphens in AACEVAPD 2–6 represent identical amino-acid residues to AACEVAPD1. Asterisks are identical amino acids when compared to Vma3p and Vma11p, and colons are gaps. Underlined sequences in Vma3p and Vma11p represent hydrophobic domains, I, II, III and IV in the descending order. (B) phylogenetic tree of the six isoforms of A. acetabulum, Vma3p, Vma11p and Vma16p according to the CLUSTALW program. q FEBS 2001 Expression of A. acetabulum V-ATPase subunit (Eur. J. Biochem. 268) 6099 167 and 168 amino-acid proteins [7,8]. The primary structures of the six proteins are shown in Fig. 1: they are divided into two groups, group 1 (AACEVAPD1 and 3 ) and group 2 (AACEVAPD2, 4, 5 and 6 ). Alignments of those primary structures to Vma3p (VMA3 gene product) and Vma11p (VMA11 gene product) are also depicted in Fig. 1. As summarized in Table 1, group 1 gave similar identities to Vma3p and Vma11p, while group 2 showed higher identity to Vma3p than Vma11p although the difference was slight. The respective gene was introduced into the S. cerevisiae strain YN45 (VMA3 coding for one of the proteolipid subunits of V-ATPase is deleted) to examine whether the translation product can be incorporated into the functional V-ATPase complex. S. cerevisiae strains with ade1 or ade2 mutations such as YPH499 accumulate purine intermediate metabolites in the vacuole, which polymerize in the compartment and form red pigments. These strains form red colonies on an agar plate, and the pigments accumulated in the vacuole fluoresce green under blue excitation and red under green excitation. When a vma mutation is introduced into those strains, no acidification of the vacuole occurs because of the lack of assembly of the V-ATPase complex. Therefore, vma mutants such as YN45 are not able to accumulate purine intermediates in the vacuole, form white colonies on an agar plate and no fluorescence in the vacuole is observed by microscopy. In the present experiment, AACEVAPD1 –6 were inserted between the yeast glyceral- dehyde 3-phosphate dehydrogenase promoter and termin- ator of a pKT10DATG yeast–E. coli shuttle vector that contained a 2-mm ori [11] (Fig. 2) after conversion of TAA (A. acetabulum-specific Gln codon) to CAA as described in Materials and methods. All the transformants were tested for accumulation of the purine intermediate metabolites in vacuole by fluorescence microscopy. The results are shown in Fig. 3; AACEVAPD2, 4, 5 and 6 clearly complemented the VMA3-deficient strain, while AACEVAPD1 and 3 did not, i.e., the translated products of the former genes were incorporated into the yeast V-ATPase complex and functioned as the proteolipid subunit which forms the H 1 channel forming V O sector. Intracellular distribution of the translated products of AACEVAPD1 –6 Functional expression was confirmed for AACEVAPD2, 4, 5 and 6 as described above. To examine the intracellular distribution of the translated products of AACEVAPD1–6 in yeast, we carried out SDS/PAGE and silver staining for the chloroform/methanol extracts of spheroplast suspensions and vacuolar-membrane-enriched fractions. The results Fig. 2. Constructs for expression of AACEVAPD1–6 in yeast. The whole reading frames of AACEVAPD1 –6 were inserted into the site between the yeast glyceraldehyde 3-phosphate dehydrogenase (GraP DH) promoter and terminator of a pKT10DATG yeast–E. coli shuttle vector. Table 1. Alignments of the A. acetabulum six proteolipid subunits and yeast three subunits (% identity). AACEVAPD1 AACEVAPD3 AACEVAPD2 AACEVAPD5 AACEVAPD4 AACEVAPD6 Vma3p Vma11p Vma16p AACEVAPD1 100 AACEVAPD3 97 100 AACEVAPD2 80 80 100 AACEVAPD5 80 80 94 100 AACEVAPD4 80 79 93 94 100 AACEVAPD6 80 80 93 95 94 100 Vma3p 50 49 54 54 55 54 100 Vma11p 50 50 51 52 51 52 52 100 Vma16p 32 32 28 29 30 29 30 28 100 6100 M. Ikeda et al. (Eur. J. Biochem. 268) q FEBS 2001 shown in Fig. 4 indicated that four genes were expressed in yeast (bands shown by arrows in Fig. 4B), the respective proteolipid subunit from AACEVAPD2, 4, 5 and 6 was located in vacuolar membranes but the translated products from AACEVAPD1 and 3 were not (Fig. 4A). The latter two translated products were supposed to be expressed in yeast (see Discussion), but may be degraded by proteases as the proteins were not integrated into vacuolar membranes as the yeast V-ATPase complex. Assembly of V-ATPase complex in vacuolar-membrane- enriched fraction of the respective transformant Western blot analysis was carried out to examine the assembly of V-ATPase complex in the vacuolar-membrane- enriched fraction of the respective transformant. The antibody against the subunit A in yeast V 1 portion and that against the subunit a in the yeast V O portion were used for this purpose (Fig. 5). Both subunits were detected in the membrane fractions of the four transformants (AAC EVAPD2, 4, 5 and 6 ), but were not detectable for the two transformants (AACEVAPD1 and 3 ). Data supported the functional assembly of the V-ATPase complex in the former, while no assembly occurred in the latter. DISCUSSION Yeast V-ATPase is the best characterized member of the V-type ATPase family. Biochemical and genetic screens have led to the identification of 14 genes; the majority designated VMA (for vacuolar membrane ATPase) encode subunits of the enzyme complex. At least eight genes encode proteins comprising the peripherally associated catalytic V 1 subcomplex, and the other six genes code for proteins forming the H 1 -translocating membrane V O subcomplex [17]. The V 1 domain is a 570-kDa peripheral complex composed of the subunits A –H with molecular masses of 14–70 kDa, and the V O domain is a 260-kDa integral complex composed of the subunits a (100 kDa), c (17 kDa), c 0 (17 kDa), c 00 (23 kDa) and d (36 kDa) [18]. The subunits c, c 0 and c 00 are designated the proteolipid subunit, and have Fig. 3. Accumulation of ade fluorescent dye in vacuoles of wild-type cells (A, strain YPH499), vma3 mutant cells (B, strain YN45) and the respective transformant of AACEVAPD1–6 (C– H). Fluor- escence images of cells with aniline blue are shown in the upper panels. Fluorescence images of cells with ade dye are depicted in the lower panels (Blue light excitation and 8 s exposure). Both images were observed at 1000 Â magnification. Central vacuoles are also seen as a bright area in cells by aniline blue staining. Fig. 4. Extraction of 16-kDa proteolipid from vacuole-membrane enriched fraction(s) (A) and spheroplast suspension (B) with organic solvent. (A) Vacuole-enriched membrane fractions (< 30 mg) from YPH499 (lane 1, wild-type), YN45 (lane 2:, VMA3-deficient strain) and transformants of AACEVAPD1–6 (lane 3–8) were washed with EDTA, extracted with chloroform/methanol solution, solubilized in SDS sampling buffer, and subjected to SDS/PAGE (15% gel). The gel was stained with silver. (B) An aliquot (0.5 mL) of spheroplast suspension was extracted with chloroform/methanol in the presence of SDS and dithiothreitol and the half was subjected to SDS/PAGE. White arrows indicate the proteolipid subunit incorporated into vacuolar membrane. q FEBS 2001 Expression of A. acetabulum V-ATPase subunit (Eur. J. Biochem. 268) 6101 been reported by Hirata et al. [19] to be the gene products of VMA3, VMA11 and VMA16 named Vma3p (160 amino acid with Glu137], Vma11p (164 amino acids with Glu145) and Vma16p (213 amino acids with Glu108), respectively. Umemoto et al. demonstrated that disruption of the VMA3 gene caused complete loss of the vacuolar membrane H 1 -ATPase activity and the occurrence of vacuolar acidification in vivo [20]. In addition, they found that the Vma3p was indispensable for the assembly of subunits A and B. Hirata et al. [19] investigated the functions of Vma11p and Vma16p in the S. cerevisiae V-ATPase complex, and reported that the two subunits c 0 and c 00 are essential for function and assembly of the V-ATPase complex into the vacuolar membrane. They also reported that VMA11p and VMA16p exist at lower levels than Vma3p in the vacuolar membranes, although the exact molar ratio of the three proteins could not be estimated. Forgac described in a recent minireview [21] that the V O domain consisted of six copies of the c/c 0 subunits and single copies of the other subunits a, c 00 and d. As described earlier, we have isolated six cDNAs encoding the putative proteolipid subunits of A. acetabulum V-ATPase [7,8]. The length of the cDNAs varies (AAC EVAPD1, 736 bp; AACEVAPD2, 825 bp, AACEVAPD3, 1032 bp; AACEVAPD4, 752 bp; AACEVAPD5, 722 bp; AACEVAPD6, 826 bp), and the sequences of their 5 0 and 3 0 untranslated regions are divergent. The putative gene products are proteins of 164 amino acids with Glu142, 176 with Glu156, 164 with Glu142, 168 with Glu148, 167 with Glu147 and 168 with Glu148 for AACEVAPD1–6, respectively. The Northern blot analysis showed a different expression ratio, AACEVAPD4 ¼ 6 3 . 2 ¼ 5 1 in descending order (data not shown). Judging from the primary structures in Fig. 1A, they are divided into two groups, group 1 and group 2 as described in the Results section. Are all the gene products in these six proteolipid- coding genes functional and all integrated into V O subcomplex of the vacuolar membrane H 1 -ATPase of A. acetabulum? In the present study, we carried out a complementation study with the vma3 mutant for the respective gene of A. acetabulum to answer this question. As a result, group 2 proteins complemented the function of the subunit c in the vma3 mutant, while group 1 protein could not in the mutant (Fig. 3). Further biochemical analyses also supported the finding in vivo; chloroform/ methanol extractable proteolipids (Fig. 4A) and Western blot analysis of the vacuolar-membrane-enriched fractions (Fig. 5). Among the transformants of the complementable four genes, the AACEVAPD2 product appears to be present in lower amounts in the vacuolar membrane compared to the wild-type and transformants AACEVAPD4, 5 and 6 (compare Figs 4 and 5). It may be due to the larger molecular size of the product, 176 amino acids with the longest N-terminal sequence among group 2 proteins (see Fig. 1). Functional assembly is possibly suppressed by the configuration of the product at the N-terminal region. Alignment data suggested that group 2 proteins are slightly more homologous to Vma3p than the group 1 protein Vma11p (Table 1). This could give a feasible explanation for the complementability of the group 2 proteins for the yeast vma3-mutant. A comparison of the structures, especially in the four hydrophobic domains (underlined sequences in Fig. 1A), showed that domains I and III are most divergent in group 1 and 2 proteins. The results we obtained indicate that these two domains may play a critical role in complementability. The genes AACEVAPD1 and 3 were constructed in another yeast expression vector, pYES2, which were tested for accumu- lation of ade fluorescent dye after galactose induction. When the genes were overexpressed by induction, accumulation of the dye in the vacuoles of both transformants was observed, and the intensity was weaker than that in the transformants of the other four genes constructed in pYES2 (data not shown). We did not take into consideration whether the A. acetabulum six proteolipid subunits function as Vma16p in yeast V-ATPase complex, as the primary structures deduced from the six genes have low identity (29–30%) to Vma16p. Also, Vma16p has a larger molecular size (213 amino acids) than any of the A. acetabulum gene products (164 –176 amino acids). Heterologous expression and complementation studies of the six genes in the yeast VMA11-deficient strain could give more information on that point, and studies by disruption of the VMA11 gene are now in progress. Isoforms of V-ATPase subunits have so far been reported in higher plants as reviewed by Sze et al. [1], three isoforms of the subunit a of mouse enzyme [22,23], four isoforms of the proteolipid subunit in Caenorhabditis elegans [24] and two isoforms of the subunit A in human osteoclastoma [25]. Isoforms in higher plants have been reported to be tissue- specific [26] or stress-induced, such as salinity stress [27]. Three isoforms of the subunit a in mouse were expressed in a tissue-specific manner [22], and the a3 isoform in mouse osteoclast was suggested to be a component of the plasma membrane V-ATPase, but the a1 isoform was localized in Fig. 5. Detection of subunits A (V 1 portion) and a (V O portion) of the yeast vacuolar membrane H 1 -ATPase in vacuolar-membrane- enriched fractions of the transformants of AACEVAPD1 –6. The vacuolar-membrane-enriched fractions were prepared as described in Materials and methods. Samples of the membrane fractions (< 17 mg protein) were subjected to SDS/PAGE (12.5% gel) stained with Coomassie Brilliant Blue (A) or transferred to nitrocellulose membranes and reacted with monoclonal anitibodies against the subunit A (B) and the subunit a (C), respectively. Lane 1, YPH499; lane 2, YN45; lane 3– 8, transformants of AACEVAPD1–6, respectively. 6102 M. Ikeda et al. (Eur. J. Biochem. 268) q FEBS 2001 the cytoplasmic endomembrane compartments of mouse osteoclast [23]. Among the four isoforms (VHA-1 to VHA-4) in C. elegans, the vha-3 gene was found to be expressed differently from the other proteolipid genes in a cell-specific manner [24]. van Hille et al. reported that one isoform (VA68-type) is ubiquitous, while the other isoform (HO68-type) is tissue-specific and located in the osteoclast plasma membrane [25]. Oka et al. [24] reported for the first time the presence of four proteolipid isoforms in a single organism. A. acet- abulum is also a single-cell organism, thus the enzymatic and physiological roles of the six proteolipid isoforms should be clarified. For this purpose, we believe, A. acetabulum is one of the best organisms, as V-ATPase is supposed to be localized in its vacuole, Golgi apparatus and lysozome. Tissue specificity and salinity stress can be excluded from physiological roles of isoforms as observed in higher plants V-ATPase. We have also isolated two different cDNAs coding for the subunits A and B of V-ATPase. The intracellular localization of these isoforms and the combination of the isoforms is yet to be determined. We are now trying to express fusion proteins of A. acetabulum proteolipid subunits and green fluorescent protein in A. acetabulum and tobacco cells. The stepwise approaches described above should help elucidate the function(s) of different subunit isoforms of the V-ATPase that are not yet understood. 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(2000) Tissue specificity of E subunit isoforms of plant vacuolar H 1 -ATPase and existence of isotype enzymes. J. Biol. Chem. 275, 6515–6522. 27. Lehr, A., Kirsch, M., Viereck, R., Schiemann, J. & Rausch, T. (1999) cDNA and genomic cloning of sugar beet V-type H 1 -ATPase subunit A and c isoforms: evidence for coordinate expression during plant development and cooridinate induction in response to high salinity. Plant Mol. Biol. 39, 463–475. 6104 M. Ikeda et al. (Eur. J. Biochem. 268) q FEBS 2001 . Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3 -deficient strain of Saccharomyces cerevisiae and study of its complementation Mikiko. the A. acetabulum six proteolipid subunits and yeast three subunits (% identity). AACEVAPD1 AACEVAPD3 AACEVAPD2 AACEVAPD5 AACEVAPD4 AACEVAPD6 Vma3p Vma11p