Journal of Experimental Botany, Vol. 53, No. 378, pp. 2273-2275,
November 1, 2002
© 2002 Oxford University Press
Functional expression of Acetabularia acetabulum vacuolar H+-pyrophosphatase in a yeast VMA3-deficient strain
Received 31 May 2002; Accepted 4 July 2002
2 Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja 719-1197, Japan
3 Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Abbreviations: V-PPase, vacuolar H+-pyrophosphatase; V-ATPase, vacuolar H+-ATPase; VMA3, gene coding for the proteolipid subunit of Saccharomyces cerevisiae V-ATPase.
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The function of the translation product of cDNA for Acetabularia vacuolar H+-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H+-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.
Key words: Key words: Acetabularia acetabulum, heterologous expression, proton transport, vacuolar H+-pyrophosphatase, yeast.
Vacuolar H+-pyrophosphatase (V-PPase) is a primary H+ pump that uses inorganic pyrophosphate (PPi) instead of ATP as an energy source. V-PPase acidifies vacuoles together with vacuolar H+-ATPase (V-ATPase) in the plant cell and actively exports protons from the cytosol in the bacterial plasma membrane (Baltscheffsky et al., 1999; Maeshima, 2000). V-PPase consists of a single polypeptide of about 80 kDa for land plants and algae (Maeshima 2000) or with smaller molecular masses in the photosynthetic bacterium, Rhodospirillum rubrum (Baltscheffsky et al., 1998), and the archaebacterium, Pyrobaculum aerophilum (Drozdowicz et al., 1999). The simplicity of the enzyme structure and its substrate is an advantage for the analysis of the structure–function relationship: catalytic domain (DVGADLVGKVE for mung bean V-PPase) (Takasu et al., 1997; Nakanishi et al., 2001), N-ethylmaleimide-binding cysteine residue (Cys634) and the N, N'-dicyclohexylcarbodiimide-binding residues (Glu305 and Asp504) (Zhen et al., 1997) both for Arabidopsis V-PPase.
V-PPase is distributed among higher plants, algae, protozoa, some eubacteria and archaebacteria (Drozdowicz and Rea, 2001). Acetabularia acetabulum is a unicellular marine alga and belongs to the Chlorophyta, which is thought to be one of the ancestral phyla of the plant kingdom. Regarding the molecular evolution of V-PPase, the Acetabularia V-PPase stands at the interface between the higher plants and the prokaryotes. The presence of V-PPase in its vacuole-rich fractions has already been reported (Ikeda et al., 1991). The primary structure of V-PPase was also elucidated by molecular cloning and its putative open reading frame may encode 721 or 751 amino acids (Ikeda et al., 1999). Sequence alignment revealed that Acetabularia V-PPase was about 45% identical with R. rubrum and Chara corallina (Nakanishi et al., 1999) V-PPase, respectively. The aim of this study was to clarify the real open reading frame of the gene and the function of the translation product of AcVP (Acetabularia V-PPase gene). By heterologous expression of AcVP in a VMA3-deficient strain of Saccharomyces cerevisiae, the acidification of yeast vacuoles was observed by fluorescence microscopy. Western blot analysis also supported the localization of the expressed protein in the vacuolar membrane.
S. cerevisiae strains BJ5459 [Mat
, ura3-52, trp1, lys2-801, leu2
1, his3200, pep4::HIS3, prb11.6R, can1, GAL>], YN45 [MAT, ade2-101, his3-200, leu21, lys2-801, trp1, ura3-52, cup5 (vma3)::LEU2, pep4::HIS3] and YPH499 [MAT, ade2-101, his3-200, leu21, lys2-801, trp1-63, ura3-52]. pKT10ATG (abbreviated as pKT) and pYES2 (abbreviated as pYES) were used as yeast expression vectors. As described in a previous paper (Ikeda et al., 1999), three cDNAs, pPP1, pPP2 and pPP3 were isolated by a reverse-transcription-PCR technique. pPP2 and pPP3 were used as a template for PCR to obtain the full length recombinant. About 100 ng of templates were subjected to PCR with primer sets of adaptor1 (AP1) and adaptor2 (AP2) in the same manner as described in a previous paper (Ikeda et al., 2001). The AcVP gene containing plasmid (pBluescript SK II (+), pBS) was purified over a Qiagen-Tip 100 column. In the case of AcVP, TAA may be used as an Acetabularia-specific codon (translated as Gln). Conversion of TAA to CAA was performed by PCR as described below. AcVP has one TAA codon in its open reading frame at 2958 nt. Fragment 1 was amplified with M13 forward primer and pp2984 (5'-AACCTGA AGTGTGTTTTTAGTTGATTGATCTAGAAAAGG-3') and fragment 2 with M13 reverse primer and pp2955 (5'-CCTTTTCTAGAT CAATCAACTAAAAACACACTTCAGGTT-3'). The temperature programme consisted of 20 cycles of 94 °C for 1 min, 50 °C for 2 min and 72 °C for 3 min. About 100 ng of template (AcVP/pBS) and an ExTaq DNA polymerase was used for amplification. Both fragments were separated by agarose gel electrophoresis, excised from the gels and purified over a Qiagen DNA extraction kit. Fragment 1 was digested with KpnI and XbaI and fragment 2 with XbaI and SacI. They were ligated with KpnI and SacI digested pBS. After transformation, plasmid DNAs were isolated and subjected to digestions with restriction enzymes before DNA sequencing. A transformant without any misreading was cultivated and plasmid DNA was isolated and purified as described above (AcVPTAA/pBS). Both AcVP and AcVPTAA was digested with SpeI and SacI. After Klenow repair, they were ligated into pKT and pYES, as described earlier (Ikeda et al., 2001). The constructs were introduced into S. cerevisiae YN45 strain by the LiOAc/PEG method and were grown in AHCW/Glc medium (0.17% yeast nitrogen base without amino acid, 0.5% ammonium sulphate, 1% casein hydrolysate, 0.002% adenine sulphate dihydrate, 0.002% tryptophan, 50 mM potassium phosphate, pH 5.5, and 2% glucose). The transformants (AcVP/pKT, AcVPTAA/pKT, AcVP/pYES, and AcVPTAA/pYES) were grown in YPD medium at 30 °C overnight until the exponential growth phase. The transformants in pKT were subjected to culture in SCD medium with low adenine concentration and processed as described previously (Ikeda et al. 2001). The transformants in pYES were innoculated in YPRaffinose medium (OD600, about 0.25) and grown to OD600 of around 1. Galactose was added at a final concentration of 2% and induction was conducted at 30 °C for 16 h. Cells were then subjected to culture in SCD medium with low adenine concentration and to observation by fluorescent microscopy. Membrane preparation from yeast was conducted in a similar manner except that cell disruption was done with glass-beads. SDS-PAGE and immunoblotting were performed as described previously (Ikeda et al., 2001). The antibody against mung bean V-PPase was used for immunoblotting.
As described previously (Ikeda et al., 1999), the AcVP gene encodes 721 amino acids, while the AcVPTAA gene 751 amino acids. In the present experiment, AcVP and AcVPTAA were inserted between the yeast glyceraldehyde 3-phosphate dehydrogenase promoter and terminator of a pKT10ATG yeast–E. coli shuttle vector that contained a 2 µm ori, and between the yeast GAL1 promoter and CYC1 terminator of pYES2. All the recombinants were transformed in S. cerevisiae BJ5459 strain, and expressions of rAcVP and rAcVPTAA were tested by SDS–PAGE and Western blot analysis. As shown in Fig. 1, the translated products were confirmed in total microsomal fractions of the AcVP/pYES and AcVPTAA/pYES transfromants. In the case of AcVP/pKT and AcVPTAA/pKT transformants, about one-fifth expressed proteins were observed (data not shown). From the molecular masses of the expressed proteins, the rAcVPTAA was judged to be native V-PPase in Acetabularia (Fig. 5 in Ikeda et al., 1991). Both the AcVPTAA transformants were tested for the accumulation of the purine intermediate metabolites in the vacuole by fluorescence microscopy. The results are shown in Fig. 2; the AcVPTAA/pYES transformant clearly complemented the VMA3-deficient strain, while AcVPTAA/pKT did not (data not shown, but was similar to Fig. 2B), i.e. the overexpressed translated product in the pYES transformant was incorporated into the vacuole and functioned as the proton pump. To examine the intracellular distribution of the translated products, Western blot analysis was carried out using the vacuolar-membrane-enriched fraction of the respective transformant. The results are shown in Fig. 3, indicating that the translated product in the AcVPTAA/pYES transformant was localized in the vacuole, while that in the AcVPTAA/pKT transformant was not integrated into the vacuole. There is a possibility that AcVP protein expressed in yeast is transported to the vacuolar membrane through a default pathway. However, the belief is that AcVP was precisely localized to the vacuolar membrane in yeast since both the stable activity and the full-size protein of AcVP was detected in the transformed yeast vacuole.
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From the studies of Arabidopsis V-PPase, plants contains not one, but two distinct subclasses: prototypical K+-activated AVP1-like (type I) V-PPases and K+-insensitive AVP2-like (type II) V-PPases (Drozdowicz et al., 2000). Acetabularia V-PPase belongs to type I as judged from the primary structure, but is insensitive to K+ (Ikeda et al., 1991). Nakanishi et al. (2001) examined the functional role of charged residues in the putative substrate-binding site (DVGADLVGKVE) and two conservative acidic regions of V-PPase by site-directed mutagensis in combination with a heterologous expression system in S. cerevisiae. They found that Lys-261 and Glu-263 of mung bean V-PPase are essential for the substrate-binding function, and Asp-253 and Glu-263 are essential for the Mg2+-binding function. Zhen et al. (1997) have proposed the DCCD-binding residues as Glu-305 and Asp-504 of Arabidopsis V-PPase by the method of site-directed mutagenesis. In pumpkin V-PPase, however, Maruyama et al. (1998) have identified the carbodiimide-reactive Glu residue (Glu-749) (Glu-751 of Arabidopsis V-PPase). In Acetabularia V-PPase, Glu-732 corresponding to Glu-751 of Arabidopsis V-PPase is present, when TAA is translated to Gln as described above. Zhen et al. (1997) also revealed the importance of Glu-427 in enzymatic activity. Since the Glu residue corresponding to Glu-427 of Arabidopsis V-PPase is conserved among V-PPase of all species including Acetabularia and Rhodospirillum, it has been speculated that Glu-427 may be the first H+-carrying residue of V-PPase to transport H+ from the cytosol into the vacuole. The direct observation of vacuole acidification presented in this report will help with the identification of H+-carrying residue(s) of V-PPase, and these studies are now in progress.
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Acknowledgement |
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This work was partly supported by a Grant-in-Aid for Scientific Research (C) (Nos 09640780, 14540600) from the Japanese Society for the Promotion of Science.
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