From the
The vacuolar H
Proton pumps are among the most important primary pumps in
biological systems. They provide the universal high energy intermediate
(called the protonmotive force) that can drive numerous secondary
uptake processes
(1, 2) . Vacuolar
H
In
contrast to the subunit structure of the catalytic sector that is
generally defined, that of the membrane sector of V-ATPase is largely
unknown. So far, four different gene products were clearly shown to
comprise the yeast V-ATPase membrane
sector
(17, 18, 19, 20, 21) . The
proteolipid is a pivotal subunit of the membrane sector and is most
likely involved in the proton translocating activity of the
enzyme
(17, 18, 22, 23) . This gene
product of VMA3 encodes a highly hydrophobic protein that
binds dicyclohexylcarbodiimide. The integrity of this and other
subunits of the enzymes is judged by their presence in the purified
V-ATPase and, more importantly, by their necessity for the activity
and/or assembly of the enzyme. This is checked by disrupting genes
encoding specific V-ATPase subunits in yeast, resulting in a phenotype
that cannot grow at high pH or high or low calcium concentrations in
the medium
(18, 22, 23, 24) . In
addition, a polypeptide can be defined as a subunit of a multisubunit
protein complex only if it is present in stoichiometric amounts of at
least one unit/enzyme. The other subunits that were defined as part of
the membrane sector of the enzymes are Ac39 and Ac115. Ac39 is a single
gene product (VMA6) that is hydrophilic in nature but is
copurified with the membrane sector
(19, 25) . It is not
released by cold inactivation from the membranes. Two genes encode
Ac115 (VPH1 and STV1), which contains several
potential transmembrane helices
(20, 21) . The inclusion
of these two gene products as genuine subunits of the enzymes is
somewhat controversial because inactivation of each of these genes did
not give the phenotype resulted from inactivating the genes encoding
all the other subunits
(20, 21) . However, when the two
genes were simultaneously inactivated, the desired phenotype was
obtained
(21) . Since a homologous cDNA was discovered in
mammalian cells
(26) , it may be appropriate to consider Ac115 as
a genuine subunit of the enzyme. However, we do not think that it
participates directly in the mechanism of action of V-ATPases. The
membrane sector of V-ATPase may contain additional hydrophobic subunits
that participate in the mechanism of proton conduction across the
membrane
(12, 27, 28, 29, 30, 31, 32) .
Alternatively, the proteolipid by itself may conduct the protons by a
completely unknown mechanism.
In looking for additional subunits of
the membrane sector, we isolated the V-ATPase from yeast vacuoles. We
detected one polypeptide with an apparent molecular mass of 16 kDa on
SDS gels that appears to have a larger copy number than the other
subunits of the V-ATPase. The polypeptide was subjected to proteolytic
cleavage and amino acid sequencing. The resulting amino acid sequences
identified a DNA sequence in the GenBank without an assigned reading
frame. Interruption of this gene (VMA10) yielded the
Hydrophobic membrane proteins are frequently hard to detect
in SDS gels stained with Coomassie Blue. The main obstacle to the
isolation of large quantities of such hydrophobic polypeptides is the
presence of phospholipids and other hydrophobic substances in the
preparation that hamper attempts to concentrate the sample.
Concentrating the sample usually results in the loss of resolution on
the SDS gel. Therefore, we devised a method for isolating these
polypeptides in large quantities (41). The method included the
isolation and purification of the membrane protein and precipitation of
the proteins by alcohol (see ``Experimental Procedures'' and
Fig. 1
). The resulting pellet was dissolved in a buffered
solution containing 1% SDS and 0.1% mercaptoethanol and was subjected
to a sucrose gradient centrifugation in a solution containing 0.1% SDS
and 0.01% mercaptoethanol. This treatment resulted in the dissociation
of polypeptides from the membrane and the displacement of phospholipids
by SDS. Therefore, many phospholipids ran as mixed micelles with SDS
and appeared at the bottom of the gradient (see Fig. 1, lane1). Polypeptides that were not highly hydrophobic and at
low molecular weight ran at the top of the gradients (Fig. 1,
lanes5-9). After analyzing the fractions on
SDS gels, similar fractions can be combined and reprecipitated with
ethanol and rerun on a sucrose gradient as before. Repetition of these
gradients will yield a much purer preparation of low molecular weight
subunits than in the original preparation. As shown in Fig. 1, we
identified several subunits of the enzyme by specific antibodies that
were available in our laboratory. Consequently, we were able to
identify new proteins. Fig. 1shows that a prominent protein band
with an apparent mass of about 16 kDa was present on the gel right
above subunit F. This protein was transferred to Immobilon and
subjected to trypsin treatment. After separation of the polypeptides by
HPLC, the polypeptides were sequenced. In addition, another preparation
of this polypeptide was cleaved by V8 protease as described
previously
(39, 41) . Sequencing of one of the
polypeptides resulting from the V8 cleavage gave the amino acid
sequence: FEQKNAGGV. Sequencing two of the polypeptides resulting from
the trypsin cleavage gave the amino acid sequences NGIATLLQAEK and
KAEAGVQGELAEIK.
A search in the GenBank revealed that the amino acid
sequences can be correctly translated from sequences within chromosome
VIII that were published in the GenBank in May 1994 (U00062) and as a
publication in October 1994
(43) . However, there was no
assignment for an open reading frame in the published sequence. The
translated reading frame identified by the alignment with the above
amino acid sequences was situated between the reading frames encoding a
protein related to aldehyde dehydrogenase (on the opposite DNA strand)
and an open reading frame encoding a protein exhibiting weak homology
to the product of HIT1 gene (on the same DNA strand). Since the
aforementioned amino acid sequences exhibited 100% identity with the
translated DNA fragment from chromosome VIII, we decided to examine
whether this DNA fragment encoded the protein that we isolated as a
potential subunit of the membrane sector of V-ATPase. Two
oligonucleotides were synthesized using the flanking regions of the
suspected reading frame as the following: ACC ACA GAA TTC CGC
CAT ACT TGC AAA TTG CCA CAC C and TGA AAA GCA TGC TAC TAT TCT
GCA AAT ACT ATT ATA. The oligonucleotides contained the respective
EcoRI and SphI restriction sites for cloning at the
same sites in the YPN2 plasmid (see Fig. 2and Ref. 22). To
eliminate the possibility that two identical genes are present in the
yeast genome, we performed a Southern blot analysis probed with a
Since the first isolation of V-ATPase from yeast vacuoles
(38) the number of reported subunits of this enzyme has
increased
(8, 10, 41) . This resulted primarily
from advancement in techniques used for the purification of the enzyme
and more importantly by identification of genes encoding subunits of
the enzyme utilizing the specific phenotype resulting from their
inactivation
(22, 24) . Recently the gene VMA7 was shown to encode subunit F of the catalytic sector of yeast
V-ATPase
(10, 11) . We believe that all subunits in the
catalytic sector have now been identified and that it is composed of
six subunits, denoted as subunits A to F in the order of decreasing
molecular mass from 69 to 14 kDa. The composition of the membrane
sector is far from being settled. So far, only the proteolipid (Vma3p)
withstands the criterion for a genuine membrane sector subunit that may
be directly involved in proton conduction across the
membrane
(23) . Since this protein is highly hydrophobic and
contains only one charged group inside the membrane, it is likely that
other membrane proteins may be involved in proton conduction across the
membrane sector of V-ATPase. In addition, it was demonstrated that the
membrane sector of V-ATPase provides the template for the assembly of
the enzyme
(19, 22) . The assembly of the catalytic
sector is totally dependent on the proper assembly of the membrane
sector, and the sequence of events in the assembly of V-ATPase is that
the membrane sector is assembled first followed by assembly of the
catalytic sector. These observations lead us to propose that more
subunits will be found to comprise the membrane sector of
V-ATPase
(2, 3) .
In all preparations of purified
yeast V-ATPase, we observed a prominent band of approximately 16 kDa.
Since dicyclohexylcarbodiimide labeling coincides with this prominent
band, we assumed that the band resulted from the presence of the
proteolipid at this position. However, analysis with epitope-tagged
proteolipid led us to conclude that the Coomassie Brilliant
Blue-stained 16 kDa band did not result from the stained proteolipid
(not shown). Therefore, we isolated this protein band, subjected it to
a proteolytic cleavage and obtained the amino acid sequences of the
resulting polypeptides. A search of the GenBank data base with these
amino acid sequences identified a potential gene in chromosome VIII
that was not previously delineated as an open reading frame. Since we
knew that this gene was expressed and the gene product was present in
the purified V-ATPase, we looked for the possibility that this gene
contained an intron and the initiator methionine was situated somewhere
upstream of the reading frame. We were successful in isolating from a
cDNA library a full-length clone corresponding to the VMA10 gene containing an initiator methionine that was 162 nucleotides
upstream from the nucleotides encoding the second amino acid (serine).
The intron starts with the canonical signal of GT and ends with the
expected nucleotides AG. Introns are quite rare in yeast, and most of
them, as does VMA10, start immediately after the initiator
methionine (42).
The integrity of Vma10p as a V-ATPase subunit was
checked by all of the available techniques for this purpose. Disruption
of the VMA10 gene yielded a mutant with identical phenotype to
all the other null mutations in V-ATPase
subunits
(10, 11, 24, 41) . This
phenotype could be complemented by transformation with the plasmid
carrying a VMA10 gene. The gene product denoted M16 for
membrane protein had an apparent molecular mass of 16 kDa. We would
like to suggest that the nomenclature of the other membrane proteins
contain the prefix M. This would change Ac39 and Ac115 to M115 and M39.
The calculated molecular mass of M16 (Vma10p) was about 13 kDa, and the
slower migration on the gel may be a result of its high content of
charged amino acids. Even though the predicted amino acid sequence M16
was highly hydrophilic, it behaved like a genuine membrane-associated
protein. It was not released from vacuoles by cold inactivation, and
the null mutation in this gene prevented not only the assembly of the
catalytic sector subunit but also the membrane sector subunit. It is
noteworthy that even though the null mutation in the VMA10 gene drastically reduced the assembled amounts of M39 and Vma12p,
it did not affect the assembly of the proteolipid into the membrane
(not shown). This observation endorses our previous notion that the
proteolipid serves as a template for the assembly of remaining subunits
in the membrane sector and thereby for the assembly of the catalytic
sector
(22) . The other criteria that indicate that M16 was a
genuine subunit of the membrane sector was its copurification with the
enzyme and its presence in stoichiometric amounts with the other
subunits of the enzyme. Preliminary results of quantitative amino acid
analysis revealed that M16 is present at least as three copies/enzyme
(not shown). As with all the other subunits of this enzyme, the precise
function of M16 is not known. However, the homology between M16 and
subunit b of F-ATPases may suggest similar function in energy
coupling and/or assembly of the catalytic sector onto the membrane
sector. Very recently, we cloned a cDNA encoding M16 in bovine adrenal
medulla.
The discovery of M16 as a subunit of the membrane
sector does not lessen the necessity to look for additional hydrophobic
protein(s) that may function alongside the proteolipid in proton
conduction across the membrane. Indeed, in preparations of V-ATPases
from mammalian and yeast sources, such a protein candidate was
identified as a protein band that migrates on SDS gels at about 20
kDa
(12, 27, 28, 29, 30, 31, 32) .
We are actively looking for the genes or cDNAs encoding this
polypeptide, which, when found, we suggest to name it VMA9.
With the recent discovery of numerous genes encoding subunits and
factors that influence the assembly of V-ATPase, this enzyme has become
an attractive model for the study of biogenesis and the assembly of
complexed membrane proteins.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
-ATPase (V-ATPase) functions as a
primary proton pump that generates an electrochemical gradient of
protons across the membranes of several internal organelles. It is
composed of distinct catalytic and membrane sectors, each containing
several subunits. We identified a protein (M16) that copurifies with
the V-ATPase complex from Saccharomyces cerevisiae and
appears to be present at multiple copies/enzyme. Amino acid sequencing
of its proteolytic products yielded three nonoverlapping peptide
sequences matching an unidentified reading frame located on chromosome
VIII. Sequence analysis of cDNA encoding M16 revealed that the gene
encoding this protein (VMA10) is interrupted by a
162-nucleotide intron that begins after the ATG codon of the initiator
methionine. The cDNA encodes an hydrophilic protein of 12,713 Da with a
basic isoelectric point of pH 9. A
vma10::URA3 null
mutant exhibited growth characteristics typical of other
vma disruptant mutants in genes encoding subunits of
V-ATPase. The null mutant does not grow on medium buffered at pH 7.5.
It fails to accumulate quinacrine into its vacuole, and subunits of the
catalytic sector are not assembled onto the vacuolar membrane in the
absence of M16. A cold inactivation experiment demonstrated that M16 is
a subunit of the membrane sector of V-ATPase. M16 exhibits a
significant sequence homology with subunit b of F-ATPase
membrane sector.
-ATPase (V-ATPase)
(
)
is one of
these proton pumps, and it is present in every known eukaryotic cell.
Its main function is to energize the vacuolar system of eukaryotic
cells and to provide the protonmotive force necessary for many
biochemical and physiological processes. This enzyme is composed of two
distinct structures, the catalytic and the membrane sectors, and each
is composed of several
subunits
(1, 2, 3, 4, 5, 6, 7) .
Recently it became apparent that the initial number of subunits
implicated in the structure and function of V-ATPase was minimal.
Biochemical studies have suggested that yeast V-ATPase is composed of
at least 10 subunits ranging in molecular mass from 17 to 100
kDa
(8, 9) . However, recent studies suggest that the
number of subunits that make V-ATPase is much greater. In addition,
several gene products may also function in different steps in the
biogenesis and assembly of the enzyme or are necessary for its proper
activity (10-15). The family of vacuolar
H
-ATPases is related to the F-ATPases that are present
in the chloroplasts and mitochondria of eukaryotic cells and in most,
if not all, eubacteria
(1, 2) . The catalytic sector of
F-ATPases contains five different subunits. Initial biochemical studies
of V-ATPase catalytic sector suggested that it contains five different
subunits as well. These subunits were denoted as subunits A to E in
order of decreasing molecular weight from 6 to 28 kDa
(3) . Very
recently it was demonstrated that the enzyme's catalytic sector
from insects and Saccharomyces cerevisiae contains an
additional sixth subunit
(10, 11, 16) . The gene
(VMA7) encoding this subunit in yeast was cloned and
sequenced, and its product (Vma7p) was denoted as subunit F of the
catalytic sector. Even though we do not expect to find more subunits
for the catalytic sector of V-ATPase, we do anticipate finding more
gene products that may regulate the catalytic sector by inhibiting or
promoting the ATPase and proton pumping activities of the enzyme.
vma10::URA3 mutant that exhibited a similar phenotype to
the other null mutants of yeast V-ATPase.
Materials
Restriction enzymes were purchased
from Boehringer Mannheim or New England Biolabs. Peroxidase-conjugated
protein A and antibodies were obtained from Sigma. Radioactive
chemicals and the ECL antibody detection system were from Amersham
Corp. Amplitaq DNA polymerase for PCR amplification was
purchased from Perkin Elmer-Cetus Instruments.
Methods
Published procedures were used for
transformation of yeast cells by lithium acetate treatment
(33) ,
recombinant DNA methods
(34) , purification of yeast genomic
DNA
(35) , protein determination
(36) , assaying ATPase and
proton uptake activities
(37) , and screening
libraries
(34) . A 5`-STRETCH yeast cDNA library in gt11 was
purchased from Clontech. The library was screened using a
P-labeled DNA fragment obtained by PCR from yeast genomic
DNA. Positive plaques were isolated, and the cDNA inserts were
amplified by PCR using
gt11 primers. The resulting DNA fragments
were cloned in pCRII plasmid (Invitrogen). Both DNA strands of the
isolated cDNA were sequenced using oligonucleotide primers. Western
blots were performed according to the protocol of the ECL antibody
detection system. Samples were denatured by SDS sample buffer (0.1
M Tris, pH 6.8, 2% SDS, 2% 2-mercaptoethanol, 0.05% bromphenol
blue, and 10% glycerol) and electrophoresed on 12% polyacrylamide
minigels (Bio-Rad) as described previously
(27) . Following
electrotransfer at 0.5 A for 15 min, the nitrocellulose filters were
blocked for 1 h in a solution containing 100 mM NaCl, 100
mM sodium phosphate, pH 7.5, 0.1% Tween 20, and 5% nonfat dry
milk. The blocked filters were incubated with antibodies for 1 h at
room temperature at dilution of 1-1000 in a similar solution
containing 1% dry milk. Following four washes in the same solution,
peroxidase-conjugated secondary antibody or protein A was added to the
filters. After incubation for 1 h and four washes with the same
solution, the nitrocellulose filters were subjected to the ECL
amplification procedure. The filters were exposed to Kodak X-Omat AR
film for 5-30 s.
Construction of Epitope-tagged Vma10p
A DNA
fragment encoding the amino acid sequence: YPYDVPDYAS (tag), which is
influenza hemagglutinine epitope (HA), was introduced by PCR into the
carboxyl terminus of the reading frame of VMA10 gene.
Following cloning into EcoRI and SphI sites of the
YPN2 plasmid
(22) , the correct insertion was verified by DNA
sequencing. Transformation of vma10 null mutants with the
plasmid containing the tagged gene resulted in complementation of the
null phenotype. Monoclonal antibody against the epitope tag (12CA5
mouse cell line) was purchased from BAbCO and used at a dilution of
1:1000.
Yeast Strains and Analysis of Mutants
S.
cerevisiae strain W303-1B was used for this study. Wild-type
and mutants were grown in YPD medium containing 1% yeast extract, 2%
Bacto-peptone, and 2% dextrose. The medium was buffered by 50
mM Mes and 50 mM Mops, and the pH was adjusted as
specified by NaOH
(24) . Agar plates were prepared by the
addition of 2% agar to the YPD buffer medium at the given pH. After
transformation by the lithium acetate method
(33) , yeast cells
were grown on minimal plates containing 0.67% yeast nitrogen base, 2%
dextrose, 0.1% casamino acids, 2% agar, and the appropriate nutritional
requirements. For measurements of quinacrine uptake, 1 ml of yeast
culture (A = 0.8) was sedimented,
resuspended in 0.1 ml of YPD containing 100 mM Hepes, pH 7.6,
and 200 µM quinacrine. After incubation at 30 °C for
10 min, the cells were washed 3 times with ice-cold solution containing
100 mM Hepes, pH 7.6, and 2% dextrose. The cells were
resuspended in 0.1 ml of the same buffer, mixed with equal volume of 1%
low melting point agarose, mounted on glass slides, covered with
coverslips, and observed for fluorescence or Nomarski within 10 min.
For preparation of vacuoles, cells were grown in YPD medium adjusted to
pH 5.5 by HCl and harvested at cell density of about A
= 0.8. Vacuolar membranes were prepared according to
Uchida et al.(38) , except that the homogenization
buffer contained no magnesium and the vacuoles were washed only once
with the EDTA buffer. ATP-dependent proton uptake activity was assayed
by following the absorbance changes of acridine orange at 490-540
nm as described previously
(37) . The 1-ml reaction mixture
contained 20 mM Mops-Tris, pH 7, 150 mM KCl, and 15
µM acridine orange. Iso-lated yeast vacuoles containing
5-20 µg of protein were added to the reaction mixture
followed by 10 µl of 0.1 M MgATP. The reaction was
terminated by the addition of 1 µl of 1 mM carbonyl
cyanide p-trifluoromethoxyphenylhydrazone.
Purification of Yeast V-ATPase and Isolation of
Proteolitic Cleaved Polypeptides
Yeast cells were grown in 10
20-liter fermentors in YPD medium, and vacuoles were isolated as was
described above. V-ATPase (about 0.5 mg of protein) was purified from
the isolated vacuoles as described
previously
(22, 38, 39) . Glycerol gradient
fractions containing the purified V-ATPase were precipitated by the
addition of 3 volumes of cold ethanol, incubated for 15 min at
-20 °C, and centrifuged at 20,000 g for 20
min. The resulting pellet was dissolved in a 1 ml of solution
containing 20 mM Mops-NaOH, pH 7.5, 1% SDS, and 0.1%
mercaptoethanol. Two 0.5-ml samples were applied onto sucrose gradients
(7-35%) in a buffer containing 10 mM Mops-NaOH, pH 7.5,
0.1% SDS, and 0.01% mercaptoethanol. The gradients were centrifuged at
15 °C in a SW60 rotor at 57,000 rpm for 15 h. Ten fractions were
collected from each tube and analyzed for subunits content by
electrophoresis on 12.5% SDS-polyacrylamide gel. Similar gels were
electrotransferred onto nitrocellulose filters that were decorated by
the subunit-specific antibodies (Fig. 1). In this way the
Coomassie Blue-stained bands were identified as the specified subunits
in Fig. 1.
Figure 1:
Fractionation of
purified V-ATPase on sucrose gradient containing SDS. The purified
V-ATPase preparation was processed as described under
``Experimental Procedures'' and applied on top of 3.5-ml
sucrose gradients (30%-7%) containing 10 mM Mops-NaOH, pH 7.5,
0.1% SDS, and 0.01% 2-mercaptoethanol. After 15 h of centrifugation at
57,000 rpm in a SW60 rotor, 10 fractions were collected from the bottom
and analyzed by SDS-PAGE and immunodecoration with subunit specific
antibodies. Top left, SDS gel of the purified V-ATPase stained
with Coomassie Blue. The identified subunits are indicated; M115 and M39 are the subunits that were previously denoted as
Ac115 and Ac39, respectively. The prominent protein band between subunits E and F is M16. Top
right, Coomassie Blue-stained SDS gel of fractions collected from
the sucrose gradient. Immunodecoration with antibodies raised against
V-ATPase subunits revealed that additional stained bands located
between subunits A and E are degradation products of subunit A. The
prominent band with apparent M = 16,000 did
not cross-react with any of the antibodies used. Bottom right,
identification of V-ATPase subunits in the gradient fractions by
antibodies raised against individual subunits. Five gels, identical to
the stained gel, were electrotransferred to nitrocellulose filters and
decorated with the specified subunit-specific antibodies. The position
of the individual subunits was matched using prestained molecular
weight markers.
To obtain internal amino acid sequence of Vma10p,
fractions 6 and 7 were dissociated in sample buffer containing 0.1
M Tris-Cl, pH 6.8, 2% SDS, 2% mercaptoethanol, 0.05%
bromphenol blue, and 10% glycerol. The dissociated sample was
electrophoresed using Mini-PROTEAN II apparatus (Bio-Rad) on two
15-well 12.5% polyacrylamide gels. One of the gels was briefly stained
by Coomassie Blue, destained, and washed with distilled water 3 times,
5 min each time. The band at 16 kDa (above subunit F) was excised and
briefly (about 5 min) lyophilized. A 10-well minigel of 15%
polyacrylamide was polymerized and incubated overnight at room
temperature. About 5 µl of a solution containing 0.1 M
Tris-Cl, pH 6.8, 10% glycerol, bromphenol blue, 0.05% SDS, and 0.5
µg of V8 protease (Boehringer Mannheim) was applied into each well
and electrophoresed until the stain entered the top of the stacking
gel. Then, the slices containing Vma10p were applied into the wells.
Electrophoresis was performed at 30 V for 40 min and then at 200 V for
an additional 25 min. The proteins were electrobloted onto an Immobilon
filter (Millipore) according to Matsudaira
(40) . The stained
bands were excised and subjected to amino acid sequencing by a
gas-phase Applied Biosystems sequenator. The second gel was
electrotransferred onto Immobilon filters, the filters were stained
with Amido Black (naphthol blue black from Fluka), and the 16-kDa
protein band was excised and subjected to trypsin cleavage as described
previously. Polypeptides were separated by reverse phase HPLC and
sequenced.
P-labeled DNA fragment corresponding to the proposed
reading frame of the gene. Southern analysis (not shown) indicated the
presence of a single gene in the yeast genome. A separate set of
oligonucleotides was designed to disrupt the gene (Fig. 2). The
URA3 gene was introduced into the construct by PCR, and the
resulting DNA fragment was used for transforming W-303-1b yeast
cells. Yeast colonies that grew on minimal plates without uracil were
subjected to further analysis. Colonies that did not grow on YPD plates
buffered at pH 7.5 were further analyzed by PCR for the correct
disruption of the gene. As shown in Fig. 3, the null mutant
vma10::URA3 failed to grow at pH 7.5 like any other
mutant in which a gene encoding V-ATPase subunit had been interrupted.
These results suggested that, indeed, the proposed open reading frame
is the gene VMA10 that encodes the protein Vma10p.
Figure 2:
Construction of a disrupted allele of the
VMA10 gene. A novel HANNH method of gene disruption was
utilized for the generation of vma10 null mutation (H.
Nelson and N. Nelson submitted for publication). Two oligonucleotides
marked in the figure as A and B (for their sequence,
see ``Results'') were used for PCR amplification of
the VMA10 gene and its flanking regions from yeast chromosomal
DNA. The second set of primers (C and D) was designed
where the 3` part of the oligonucleotides matches internal sequences of
VMA10 and 5` parts of oligonucleotides were derived from the
sequences of the URA3 gene. The combinations of primers A + C and B + D were used for
amplification of VMA10-flanking regions from the first PCR
product with the URA3 gene. The final construct was created in
two more PCR steps. First the 5`-flanking region of VMA10 was
fused to a URA3 gene, and then the 3`-flanking region of
VMA10 was added by amplification with primers A and
B. The product of the last PCR contains the URA3 gene
in place of VMA10 coding sequence and was used for
transformation to create the
vma10::URA3 strain. The
toppart of the picture shows the position of the
initiator methionine (M) and intron (region from M to
the start of VMA10) as revealed by cDNA
sequencing.
Figure 3:
The phenotype of mutants containing a
disrupted VMA10 gene. The VMA10 gene was interrupted
as described under ``Experimental Procedures'' and in the
legend to Fig. 2. Panel A, chromosomal DNAs from wild-type
cells W303B and one transformed colony that grew on minimal medium
lacking uracil, were amplified by PCR using primers A and B (see Fig.
2). PCR products were analyzed by electrophoresis in 1% agarose gel.
Lane 1, molecular weight standards from top to bottom were as
follows: 23, 9.4, 6.6, 4.4, 2.3, 2, 1.4, 1.1, 0.9, and 0.6 kilobases;
lane 2; wild-type strain; lane 3;
vma10::URA3 mutant. Panel B, growth on YPD
plates buffered to the indicated pH. 1, W303B; 2,
vma10::URA3; 3,
vma10::URA3 transformed
with the YPN2 plasmid; 4,
vma10::URA3 transformed with
YPN2 plasmid bearing the VMA10 gene. Each colony in the figure
represents an independent transformant.
However,
we could not identify the initiator methionine within the proposed
reading frame in the published DNA sequence. We therefore concluded
that this gene was probably interrupted by an intron and its initiator
methionine was upstream of the first amino acid that could be read from
translation of the DNA. To prove this point, a yeast cDNA library was
screened using a P-labeled DNA fragment encoding the
proposed reading frame. The resulting positive plaques were isolated,
and the cDNAs were isolated from the
gt11 by PCR and cloned by the
TA procedure into pCRII plasmid. Sequencing of the cDNA clearly showed
that, as shown in Fig. 4, there was an upstream initiator
methionine and the reading frame was interrupted by an intron of 162
nucleotides. The mRNA started at nucleotide 243 (Fig. 4) and
ended following the polyadenylation signal AATAAA that begins at
nucleotide 827. Consequently, the VMA10 gene was expressed as
a spliced mRNA. Search in GenBank revealed that Vma10p exhibits 24%
identity and about 40% similarity with subunits b of F-ATPases
from Escherichia coli or Vibrio alginolyticus (accession number X16050). Amino acid sequence alignment also
indicate possible relation to NtpF gene product from the gene
cluster for Na
-translocating ATPase from
Enterococcus hirae(44) . BESTFIT program showed 29%
identity and 46% similarity between Vma10p and NtpF.
Figure 4:
Nucleotide and derived amino acid
sequences of VMA10. The position of the initiator methionine
was determined by sequencing of the cDNA isolated from the gt11
library. The amino acid sequences of the peptides are
underlined. The cDNA nucleotide sequence has been submitted to
GenBank with accession number U21240. The intron starts at nucleotide
260 and ends at nucleotide 421.
To
further analyze the phenotype of vma10 null mutation, we
followed the quinacrine accumulation both in wild-type and null mutant
cells. As shown in Fig. 5the null mutants failed to accumulate
quinacrine into their vacuoles. In addition, vacuoles isolated from the
null mutant exhibited no ATP-dependent proton uptake activity
(Fig. 6). It was demonstrated in our and other
laboratories
(19, 20, 21, 22, 23, 24) that a null mutation in one of the V-ATPase subunits
prevents the assembly of the other subunits in the catalytic sector of
V-ATPase. Therefore, we checked the assembly of the different V-ATPase
subunits into the vacuole membranes. As shown in Fig. 7, the null
mutation
vma10::URA3 prevented the assembly of subunits A
(Vma1p), C (Vma5p), E (Vma4p), and M39 (Vma6p), the latter of which is
a subunit of the membrane sector of the enzyme. In contrast, the null
mutation
vma10::URA3 cells synthesized similar amounts of
these subunits as the wild-type cells (Fig. 4, cell lysate). It
is particularly interesting that the membrane sector subunit M39 is
also not present in the isolated vacuoles of the mutant cells. This
observation is in line with other observations showing that mutations
in the catalytic sector subunits did not prevent the assembly of the
membrane sector subunits. However, mutations that deleted one of the
membrane sector subunits influenced the assembly of the other membrane
sector subunits
(19, 20, 21, 22) . Vma12p
is a vacuolar membrane protein that does not copurify with the
V-ATPase
(14) . Null mutation in the gene (VMA12)
encoding this protein prevented the assembly of the enzyme. The lack of
Vma10p in its corresponding null mutant drastically reduced the amount
of Vma12p in the vacuolar membranes.
Figure 5:
The mutant with a disrupted VMA10 gene does not accumulate quinacrine into its vacuole. Accumulation
of quinacrine was measured as described under ``Experimental
Procedures.'' Nomarski and fluorescence images are shown.
Top, fluorescence picture of vma10 null mutant and
the wild-type (WT). Bottom, vma10 null
mutant cells as seen using Nomarski optics.
Figure 6:
The isolated vacuoles of
vma10::URA3 null mutant lack ATP-dependent proton uptake
activity. About 20 µg of isolated vacuolar membranes were used for
measuring ATP-dependent acidification of membrane vesicles by following
a decrease in absorption at 490-540 nm, as described in detail
under ``Experimental Procedures.'' Where indicated, 1
µmol of MgATP or 1 nmol of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP) were added
to the 1 ml reaction mixture. Trace1, wild-type
membrane vesicles (W303B); trace2,
vma10::URA3 mutant membrane
vesicles.
Figure 7:
Subunits of the catalytic sector are not
assembled onto the vacuolar membrane in the absence of
Vma10p. Crude cell lysates from wild-type cells and
the
vma10 null mutant were prepared as described
previously (10). Vacuoles were prepared from 5 liters of the
corresponding yeast culture grown in YPD, pH 5.5 (38). Fifty µg of
cell lysate protein or 5 µg of purified membranes were loaded/lane
of 12% SDS-polyacrylamide gel and, after separation, transferred to
nitrocellulose membrane. The presence of V-ATPase subunits was detected
by affinity-purified antibodies raised against indicated gene products.
Lane 1, wild-type cells; lane 2,
vma10 null mutant.
Cold inactivation of V-ATPases
resulted in inactivation of the enzyme and dissociation of the
catalytic sector from the
membrane
(10, 27, 28, 39) . To assess
whether Vma10p is a membrane-associated protein, we performed cold
inactivation with the isolated vacuoles from wild-type cells that
carried an epitope-tagged Vma10p. The gene encoding the tagged Vma10p
complemented the corresponding null mutation, and it grew well on a
medium buffered at pH 7.5. Vacuoles were isolated from these cells and
subjected to cold inactivation in the presence and absence of
MgATP
(10, 22, 39) . As shown in Fig. 8, in
the absence of MgATP, no loss of V-ATPase subunits from the vacuoles
was observed. In contrast, in the presence of MgATP, a major loss of
subunits A (Vma1p) and C (Vma5p) into the supernatant was observed. M39
(Vma6p) was also released to the supernatant but to a much lesser
extent than the catalytic sector subunits. Fig. 8also shows that
cold inactivation in the presence of MgATP failed to release Vma10p
from the membrane. These experiments support our conclusion that
VMA10 encodes a V-ATPase subunit that is associated with the
membrane sector of the enzyme.
Figure 8:
Vma10p is not released from
vacuolar membrane by cold inactivation. The
vma10::URA3 strain was transformed with YPN2 plasmid carrying VMA10 gene containing an epitope tag at its carboxyl terminus. The
resulting transformant was grown in 20 liters of YPD medium, and
vacuolar membrane vesicles were isolated. Membranes (0.75 mg) were
diluted into 3 ml of buffer containing 20 mM Mops-Tris, pH
7.0, 250 mM NaCl, and 1 mM dithiothreitol, and then
divided into three parts. MgATP was added to one of them to give 5
mM final concentration. After 2 h of incubation on ice, the
sample containing MgATP and one of the samples lacking MgATP were
centrifuged at 150,000
g for 25 min. Both
supernatants, as well as a sample that was not centrifuged, were
concentrated by trichloroacetic acid precipitation and dissolved in 0.1
ml of SDS dissociation buffer. Ten µl of each sample were
electrophoresed in 12% polyacrylamide gel, and after transfer to
nitrocellulose, membranes were decorated with antibodies against the
indicated subunit. Lane 1, starting material of purified
vacuoles; lane 2, supernatant after cold treatment without
MgATP; lane 3, supernatant after cold treatment in the
presence of MgATP.
(
)
This finding together with the
homology of M16 to bacterial subunit b of F-ATPase, suggests
that all V-ATPases including those of archaebacteria contain equivalent
subunits to M16.
/EMBL Data Bank with accession number(s) U21240.
-ATPase; PCR, polymerase chain reaction; Mes,
4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic
acid; HPLC, high performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.