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

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.

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

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

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

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

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 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

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

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

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

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

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