Expression of the V-ATPase proteolipid subunit of Acetabulariaand study of its complementation Mikiko Ikeda1, Misato Hinohara1, Kimiko Umami1, Yuki Taguro1, Yoshio Okada1, Yoh Wada2, Yoi
Trang 1Expression of the V-ATPase proteolipid subunit of Acetabularia
and study of its complementation
Mikiko Ikeda1, Misato Hinohara1, Kimiko Umami1, Yuki Taguro1, Yoshio Okada1, Yoh Wada2,
Yoichi Nakanishi3and Masayoshi Maeshima3
1 Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan;2Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Japan;3Laboratory 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 H1-ATPase (V-ATPase) is ubiquitous both in
prokaryotes and eukaryotes This enzyme is composed of
two domains, a large peripheral domain (V1) and a
membrane integral domain (VO) The major component of
the VO portion common to all V-ATPases is the
N,N0-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 Ca21,
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
M A T E R I A L S A N D M E T H O D S
Yeast strains
S cerevisiae strains YN45 [MATa, ade2 – 101, his3-D200, leu2D1, lys2-801, trp1, ura3-52, Dcup5(vma3)::LEU2, pep4::HIS3], YN11 [MATa, ade2-101, his3-D200, leu2D1, lys2-801, trp1-D63, ura3-52, Dcup5 (vma3)::LEU2], YPH499 [MATa, ade2-101, his3-D200, leu2D1, lys2-801, trp1-D63, ura3-529], BJ5458 [MATa, ura3– 52] [9], trp1, lys2 – 801, leu2D1, his3D200, pep4::HIS3, prb1D1.6R, 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 ofAACEVAPD1 – 6
50RACE and 30RACE 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 H1-ATPase; VMA3, gene coding
for the proteolipid subunit of Saccharomyces cerevisiae V-ATPase;
GraP DH, glyceraldehyde 3-phosphate dehydrogenase.
Trang 2length 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 50and 30ends for AACEVAPD3 – 6, respectively
The temperature program consisted of 20 cycles of 94 8C for
1 min, 60 8C for 2 min, and 72 8C for 3 min 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 (50-CACGAGCTCGGGTCTCATAACACCCATT
TGAGC-30), fragment 2 with VC1*Q1 (50-GCTCAAATG
GGTGTTATGAGACCCGAGCTCGTG-30) and VC136*Q4
(50-CCCACAAAAAGCTTGGGTTGTTGAGC-30) and
fragment 3 with VC136*Q3 (50-GCTCAACAACCCAAG
CTTTTTGTGGG-30) 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 ofAACEVAPD1 – 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 mM potassium phosphate, pH 5.5, 2% glucose)
Accumulation ofade 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:L21) and tryptophan (20 mg:L21) 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
Trang 3centrifugation as described above The pellet was
resus-pended in 1 mL of aniline blue staining solution [1% aniline
blue in NaCl/KCl/Pi solution (0.8% NaCl, 0.02% KCl,
0.144% Na2HPO4, 0.024% KH2PO4, 2% glucose adjusted to
pH 7.4 with 1M 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/Pi, 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:L21uracil 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
R E S U L T S
Functional expression ofAACEVAPD2, 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 program.
Trang 4167 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 H1channel forming VOsector
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.
Trang 5shown 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 V1 portion and
that against the subunit a in the yeast VOportion 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
D I S C U S S I O N
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 V1 subcomplex, and the other six genes code for proteins forming the H1-translocating membrane VO subcomplex [17] The V1 domain is a 570-kDa peripheral complex composed of the subunits A – H with molecular masses of
14 – 70 kDa, and the VO domain is a 260-kDa integral complex composed of the subunits a (100 kDa), c (17 kDa),
c0(17 kDa), c00(23 kDa) and d (36 kDa) [18] The subunits
c, c0and c00are 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 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.
Trang 6been 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
H1-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 c0 and c00are
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 VO
domain consisted of six copies of the c/c0subunits and single
copies of the other subunits a, c00and 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 50and 30
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 VO subcomplex of the vacuolar membrane H1-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 H1-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.
Trang 7the 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
A C K N O W L E D G E M E N T S
We are grateful to Prof Dr Yasuhiro Anraku of Teikyo Science
University for intensive discussions Thanks are extended to Dr Ryogo
Hirata of the Institute of Physical and Chemical Research (Riken) for
the generous gift of the antibody and for instructions.
R E F E R E N C E S
1 Sze, H., Ward, J.M & Lai, S (1992) Vacuolar H 1 -translocating
ATPase from plants: structure, function and isoforms J Bioenerg.
Biomembr 24, 371 – 381.
2 Gogarten, J.P., Kibak, H., Dittrich, P., Taiz, L., Bowman, E.J.,
Bowman, B.J., Manolson, M.F., Poole, R.J., Date, T., Oshima, T.,
Konishi, J., Denda, K & Yoshida, M (1989) Evolution of the
vacuolar H1-ATPase: implication for the origin of eukaryotes.
Proc Natl Acad Sci USA 86, 6661 – 6665.
3 Ikeda, M., Satoh, S., Maeshima, M., Mukohata, Y & Moritani, C.
(1991) A vacuolar ATPase and pyrophosphatase in Acetabularia
acetabulum Biochim Biophys Acta 1070, 77 – 82.
4 Konishi, K., Moritani, C., Rahman, Md H., Kadowaki, H.,
Oesterhelt, D & Ikeda, M (1995) Molecular cloning of cDNAs
(D50528, D50529) encoding Acetabularia acetabulum V type
ATPase, A subunit Plant Physiol 109, 337.
5 Ikeda, M., Konishi, K., Kadowaki, H & Moritani, C (1996)
Molecular cloning of cDNAs (D50529 – D50531) encoding
Acetabularia acetabulum V type ATPase, A and B subunits.
Plant Physiol 111, 651.
6 Ikeda, M., Kadowaki, H., Rahman, Md H & Ohmori, S (1995)
Molecular cloning of cDNAs (D50530, D50531) encoding
Acetabularia acetabulum V type ATPase, B subunit Plant Physiol.
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