From the
The isolated Saccharomyces cerevisiae vacuolar
proton-translocating ATPase (V-ATPase) is composed of at least 10
subunits. We have identified VMA8, the gene encoding the
32-kDa subunit of the V-ATPase, by 100% match between the sequences of
tryptic peptides and the predicted protein sequence of ORF11. The VMA8 gene contains a 768-base pair open reading frame encoding
a 256-amino acid protein with a predicted molecular mass of 29,176 Da.
Disruption of VMA8 resulted in a mutant exhibiting
pH-sensitive growth, slowed growth under all conditions, and an
inability to grow on nonfermentable carbon sources. Vacuolar membranes
isolated from vma8
The Saccharomyces cerevisiae vacuolar
H
The
V-ATPase is similar in structure and subunit composition to the well
characterized F
Biochemical analysis
has identified at least ten subunits of the yeast V-ATPase ranging in
molecular mass from 14 to 100 kDa. The V
Mutations
in a number of vacuolar membrane ATPase (VMA) genes (with the
exception of VPH1 encoding the 100-kDa subunit; Refs. 9 and
15) result in a characteristic set of growth phenotypes in yeast. Cells
in which a VMA gene has been disrupted exhibit increased
sensitivity to calcium and the inability to grow on nonfermentable
carbon sources or in media buffered to neutral pH. In addition,
vacuolar membranes isolated from these vma mutants lack ATPase
activity. Besides the VMA genes that encode V-ATPase subunits,
other proteins have been identified that are not associated with the
final V-ATPase complex but are required for its assembly (Vma12p,
Vma21p, and Vma22p; Refs. 16 and 17).
Complete assembly of the V-ATPase requires the
presence of every subunit except the 54-kDa subunit encoded by the VMA13 gene (11). Cells lacking any V
This paper describes the cloning of VMA8 and the
characterization of the encoded 32-kDa polypeptide (Vma8p). Vma8p is a
subunit of the V-ATPase and a component of the V
A disruption allele was constructed by
replacement of the VMA8 open reading frame with the LEU2 gene (Fig. 2B). Replacement of VMA8 with vma8::LEU2 at the genomic locus resulted in a mutant
displaying growth phenotypes characteristic of other vma mutants. Cells that carried vma8::LEU2 (KHY104) exhibited
pH-sensitive growth, the ability to grow in media buffered to pH 5.0
but not to 7.5. The vma8::LEU2 cells also showed increased
doubling time in all growth media tested and were incapable of growing
on glycerol, a nonfermentable carbon source, even in media buffered to
pH 5.0. Vacuolar membranes isolated from vma8
A search of the protein data
bases revealed several proteins that demonstrated homology to the yeast
32-kDa protein (Fig. 3). The highest homology was shared with a
hypothetical 28.8-kDa protein from Caenorhabditis elegans identified as a putative open reading frame in the genome (GenBank
accession number Z27080). The yeast and C. elegans predicted
proteins shared 52% identity and 68% similarity as calculated over the
entire amino acid sequence. The function of the C. elegans 28-kDa protein is unknown, but its high identity to a yeast
V-ATPase subunit suggests that it plays a similar role in C.
elegans. As pointed out by Nelson et al.(26) ,
yeast Vma8p shares 55% identity with a bovine 34-kDa predicted V-ATPase
polypeptide subunit. The yeast 32-kDa protein also shared homology (27%
identity and 48% similarity) with the NtpD protein subunit of Enterococcus hirae vacuolar type
Na
In
the absence of Vma8p, the 69-, 60-, 54-, 42-, and 27-kDa proteins were
not associated with the vacuolar membrane in a vma8
Vacuolar membranes isolated from vma8
The V-ATPase isolated from yeast is composed of at least ten
proteins, ranging in molecular mass from 14 to 100 kDa. A large
collection of genes has been identified that encode subunits or
nonsubunit proteins required for the assembly of a functional V-ATPase (). Loss of any one of these proteins, except Vph1p (8) or its isoform Stv1p(15) , results in a mutant cell
exhibiting a pH-sensitive growth phenotype. A number of uncharacterized vma mutants exist in our laboratory representing mutations in
genes possibly encoding additional subunits or assembly factors.
We
have cloned and characterized the VMA8 gene encoding the yeast
32-kDa V-ATPase subunit. Peptide sequence generated from the 32-kDa
polypeptide associated with the yeast V-ATPase (Vma8p) positively
identified the S. cerevisiae ORF11 as an additional VMA gene. Disruption of the VMA8 gene in yeast results in the
complete loss of V-ATPase activity. The absence of Vma8p did not affect
the assembly of the V
The yeast Vma8p exhibits sequence similarity to
several predicted amino acid sequences available through the GenBank
data base, including a putative protein from C. elegans described as similar to a ``membrane-associated ATPase
Recently published work by Nelson et al.(26) described the cloning and sequencing of a bovine
cDNA-encoding subunit D, a 34-kDa protein that copurifies with the
catalytic complex of chromaffin granules. Nelson et al.(26) identified the yeast ORF11 as encoding a putative
protein possessing 55% amino acid sequence identity to subunit D of the
bovine V-ATPase. These workers coincidentally designated ORF11 as VMA8, the gene encoding the yeast equivalent to the bovine
V-ATPase subunit D. A data base search by these workers for proteins
with sequences similar to the bovine subunit D also identified several
protein sequences including the C. elegans open reading frame,
hypothesized to encode a protein related to an ``ATPase
A comparison between the
subunits of the E. hirae Na
An intact
Na
The physical interaction between Vma8p, the vacuolar
membrane, and other V-ATPase subunits was examined by KNO
We thank Margaret Ho, Cynthia Bauerle, and Margaret
Lindorfer for assistance with tryptic peptide fragment sequencing and
analysis. We also thank Eric Whitters, Nia Bryant, and Dewaine Jackson
for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
yeast cells exhibited no V-ATPase
activity. Immunoblot analysis of vma8
cells revealed
normal levels of both V
and V
subunits. Whereas
the V
subunits failed to associate with the vacuolar
membrane in vma8
cells, the V
polypeptides
were transported to and stable in the vacuolar membrane. Density
gradient fractionation revealed that Vma8p associated only with the
fully assembled V-ATPase and did not associate with a separate lower
density V
subcomplex fraction. Finally, Vma8p was unable to
assemble onto the vacuolar membranes in the absence of other V
subunits.
-translocating ATPase (V-ATPase)
(
)belongs to a family of multisubunit
proton-translocating V-type ATPases present in all eukaryotic cells and
various bacteria. Members of the V-type ATPase family are necessary for
the acidification of several organelles including endosomes, Golgi,
secretory vesicles, vacuoles, and lysosomes (1). The proton gradient
generated by the ATP hydrolyzing activity of the yeast V-ATPase is
utilized by other transporters to drive the accumulation of ions, amino
acids, and metabolites into the vacuole(2, 3) .
F
-ATPase of the mitochondrial
inner membrane(4) . The V-ATPase is composed of a V
catalytic sector of peripherally associated proteins facing the
cytosol that are assembled on the V
sector of integral
proteins forming the proton-translocating pore. Unlike the
F
F
-ATPase, the V-ATPase is only capable of
hydrolyzing ATP coupled to the translocation of protons into the
vacuole resulting in vacuolar acidification.
sector appears to
be composed of multiple copies of a 17-kDa proteolipid forming the
proton pore in addition to the 36- and 100-kDa
subunits(5, 6, 7, 8, 9) . The
V
sector contains polypeptides of 69, 60, 54, 42, 32, and
27 kDa(10, 11, 12, 13) . The 14-kDa
subunit is unique because it behaves biochemically as a V
subunit, yet cells lacking this protein fail to transport V
subunits to the vacuolar membrane(14) . The 69- and 60-kDa
proteins are the catalytic subunits of the V
sector,
analogous to the
and
subunits of the
F
F
-type ATPase complex. The remaining subunits,
however, share no significant sequence similarity to the
,
,
and
F
F
-type ATPase subunits.
(
)Loss of
any of these nonsubunit proteins also results in a Vma
phenotype.
subunit (e.g. 60-kDa subunit encoded by VMA2) fail to
assemble the remaining V
subunits onto the vacuolar
membrane, but these unassembled proteins remain stable in the
cytoplasm(9) . However, these mutants have been found to
assemble the V
polypeptides and transport them to the
vacuole(7) . In contrast, disruption of a gene encoding a
V
subunit, such as the 17-kDa proteolipid (VMA3)
integral membrane protein, results in a significant decrease in the
steady-state level of the remaining V
subunits, reflecting
increased turnover of the unassembled subunits (7).
(
)Although, the loss of a V
subunit has no
effect on the steady-state level of the V
subunits, these
peripheral polypeptides are unable to assemble onto the vacuolar
membrane.
complex.
Loss of Vma8p from the cell prevents assembly of the remaining V
subunits onto the vacuolar membrane. Vma8p shares sequence
identity with V-ATPase subunits from a variety of organisms but lacks
similarity to any F
F
-ATPase subunits,
suggesting that Vma8p is unique to the V-ATPases.
Strains and Culture Conditions
Yeast strains
used in this study are listed in . The wild-type strain is
SF838-1D. vma2 (SF838-1DV1) is isogenic to
SF838-1D except vma2::LEU2; vma8
(KHY104)
is isogenic to SF838-1D except vma8::LEU2. Yeast were
grown in YEPD buffered to either pH 5.0 or 7.5 as described by
Yamashiro et al. (18) or in S.D. (0.67% yeast nitrogen
base, 2% dextrose) supplemented with the appropriate amino acids.
Cloning VMA8
Oligonucleotides synthesized
complementary to the 5`-flanking (5`-TTGCCTTTGCCCGTGCCTTA-3`) and
3`-flanking (5`-GCTACCATTGGCGTGGTCTC-3`) sequences of VMA8 were used to amplify by PCR from genomic DNA a 2-kb DNA fragment
containing the VMA8 gene. The PCR fragment was digested with
the enzymes SstI and BglII to generate a 1837-bp
restriction fragment containing VMA8, which was then cloned
into the BamHI/SacI sites of the vector pBluescript
KS+ (Stratagene, La Jolla, CA) to generate the plasmid pKH3201.
Construction of Disruption Strain
The plasmid
pKH3201 was digested with the restriction enzyme BbsI,
removing a 700-bp internal VMA8 fragment representing 92% of
the open reading frame. A 2.2-kb HpaI LEU2 fragment
was subcloned into the blunted BbsI sites of pKH3201 creating vma8::LEU2 (pKH3202). A subclone in which the LEU2 open reading frame was cloned in the opposite orientation to the VMA8 reading frame was chosen for the construction of a null
allele. The genomic locus of VMA8 was disrupted by the method
of Rothstein(19) . Yeast haploid strain, SF838-1D, was
transformed using the lithium acetate method (20) with a 3.3-kb SacI/PstI linear fragment containing vma8::LEU2. Leucine prototrophic transformants were screened
by PCR amplification to confirm integration into the correct genomic
locus using oligonucleotide primers complementary to genomic sequence
flanking the VMA8 open reading frame
(5`-GCATCTGTAGTACATAGGTTCCTAACA-3` and
5`-GGCTTACATATTTTTGAAAAGGGTCTT-3`). Correct integration of vma8::LEU2 into the VMA8 genomic locus resulted in
the amplification of a 2.3-kb fragment versus a 860-bp
wild-type fragment.
Protein Preparation, SDS-PAGE, and Western Blot
Analysis
Whole cell extracts and vacuolar membranes were
prepared from wild-type, vma2, and vma8
cells for SDS-polyacrylamide gel electrophoresis and immunoblot
analysis as described previously(14) .
Proteolipid Extraction
Vacuolar membranes from
wild-type and vma8 cells were extracted with
chloroform/methanol as described by Ho et al.(11) .
Samples were incubated for 30 min at 37 °C in 10 mM Tris,
pH 7.4, containing 1 mM EDTA, 8 M urea, 5% SDS, and
5%
-mercaptoethanol. Proteins were separated on a 15%
SDS-polyacrylamide gel and detected by silver staining the gel.
Epitope Tagging of Vma8p
A 1.9-kb SacI/HindIII fragment containing the VMA8 gene from pKH3201 was subcloned into the centromeric vector pRS316 (21) to create pKH3203. An oligonucleotide duplex encoding a
single epitope (YPYDVPDYA) of the influenza virus hemagglutinin protein
(HA; Ref. 22) flanked by BglII and BamHI staggered
ends was ligated into the BclI restriction site of VMA8 to generate pKH3204 (VMA8::HA). The HA epitope was
introduced 8 amino acids from the 3`-terminal stop codon, introducing
an additional 1509 Da to the molecular mass of Vma8p. A NdeI
restriction enzyme site engineered near the 5` termini of the HA
epitope allowed confirmation of the presence of the HA tag. Sequencing
of double-stranded template prepared from pKH3204, using the
oligonucleotides 5`-GCATCTGTAGTACATAGGTTCCTAACA-3` and
5`-GGCTTACATATTTTTGAAAAGGGTCTT-3`, confirmed that the HA tag was in
frame with the VMA8 open reading frame. Whole cell extracts
were prepared from vma8 cells (KHY104) carrying pKH3204
and screened by immunoblot analysis using HA monoclonal antibody 12CA5
(Babco, Inc., Berkeley, CA) for expression of the HA tag.
Purification of the Vacuolar ATPase
Complex
Vacuolar membranes were isolated from wild-type
(SF838-1D) and vma8 (KHY104) yeast cells
transformed with either pKH3203 (VMA8) or pKH3204 (VMA8::HA) as described previously(23) . Vacuolar
membranes from wild-type or vma8
cells expressing
Vma8p-HA were washed with 10 mM Tris, pH 7.4, 1 mM EDTA, solubilized with 2% zwitterionic detergent ZW3-14
(Calbiochem, San Diego, CA), and separated by centrifugation (20 h at
110,000
g) through a 12-35% glycerol density
gradient(24, 25) . Fractions were collected in
750-µl aliquots, and a portion of each fraction (100 µl) was
assayed immediately for both ATPase and dipeptidyl aminopeptidase B
activity(23) . Proteins in the gradient fractions were
precipitated with 10% trichloroacetic acid, and samples were prepared
for SDS-PAGE as described(24) . Immunoblot analysis of the
gradient fractions was performed by probing with antibodies specific
for the 100-, 69-, and 36-kDa V-ATPase subunits and Vma8p-HA. Proteins
were visualized by chemiluminescence (DuPont NEN) after incubation with
horseradish peroxidase-conjugated secondary antibodies (Amersham
Corp.).
Alkaline Carbonate Extraction of Vacuolar
Membranes
Vacuolar membranes from vma8 cells
carrying pKH3204 (VMA8::HA) were incubated with 100 mM Na
CO
, pH 11.5, or 5 M urea as
described previously(7) . Pellet and supernatant fractions were
analyzed using antibodies specific for the 100-, 69-, 60-, and 36-kDa
V-ATPase subunits or Vma8p-HA.
Nitrate Extraction of Vacuolar Membranes
Vacuolar
membranes from vma8 cells carrying pKH3204 (VMA8::HA) were treated with potassium nitrate in the presence
or absence of 5 mM MgCl
ATP as described
previously(24) . Pellet and supernatant fractions from 5 µg
of treated vacuolar membranes were separated by SDS-PAGE, transferred
to nitrocellulose, and probed with antibodies specific for 100-, 69-,
and 36-kDa V-ATPase subunits and Vma8p-HA.
Other Methods
DNA sequencing was performed at the
Molecular Genetics Instrumentation Facility, University of Georgia.
Protein alignment was generated using the MegAlign sequence analysis
program (DNASTAR, Inc.).
Identification of the Gene Encoding the 32-kDa V-ATPase
Subunit
The vacuolar ATPase was isolated by glycerol density
gradient separation of detergent-solubilized vacuolar membranes.
Proteins present in the glycerol gradient fraction possessing the
highest ATPase activity were examined by SDS-PAGE. Coomassie staining
of this V-ATPase fraction revealed at least 10 major proteins, enriched
from the total proteins present in the vacuolar membranes (Fig. 1, right panel). Most of the proteins present in
this peak fraction have been identified by various immunological,
biochemical, and genetic approaches as subunits of the yeast V-ATPase,
with the exception of the 32-kDa protein.
Figure 1:
Identification of the 32-kDa subunit of
the vacuolar ATPase. Coomassie-stained gels of vacuoles and purified
V-ATPase are shown. 10 µg of wild-type vacuolar membrane protein
and 3 µg of glycerol gradient isolated V-ATPase were separated on a
8-15% SDS-polyacrylamide gradient gel, and the proteins were
visualized by staining with Coomassie Brilliant Blue R-250. Apparent
molecular masses of V-ATPase subunits are shown (in kDa). The arrow indicates the 32-kDa product of the VMA8 gene. The asterisk denotes an unidentified protein
band.
The SDS-PAGE-separated
proteins of the peak V-ATPase fraction were transferred to
nitrocellulose, and the individual proteins were visualized by staining
with Ponceau S(7) . The band with apparent molecular mass of
32-kDa was excised and subjected to tryptic digestion, the fragments
were separated by microbore high performance liquid chromatography, and
several well resolved peptides were subject to microsequencing. The
sequence of the peptide fragments matched 100% with the sequence
available in the GenBank data base (accession number L10830, identified
in S. cerevisiae as ORF11 on chromosome V) described as a
yeast hypothetical 29.2-kDa protein (Fig. 2A). The open
reading frame ORF11, with the predicted amino acid sequence matching
the sequence generated from the 32-kDa protein, was located in the AFG1-CYC7 intergenic region of chromosome V. The
sequence contained a 768-bp open reading frame encoding a protein of
256 amino acids with a predicted molecular mass of 29,176 Da (Fig. 2A). We have designated this open reading frame as VMA8, the gene encoding a 32-kDa vacuolar membrane ATPase
subunit. During the preparation of this manuscript, the VMA8 sequence was reported by Nelson et al.(26) . In
that preliminary communication, Nelson et al. described only
that vma8 cells exhibited pH-sensitive growth.
Figure 2:
Predicted protein sequence of VMA8 and construction of gene disruption. A, translation of VMA8 open reading frame. The sequence of tryptic peptide
fragments generated from isolated 32-kDa V-ATPase subunit is shown underlinedabove the Vma8p sequence. The arrow indicates the site of insertion of the HA epitope tag. B,
physical map and construction of the gene disruption. The solid
line indicates the genomic sequence. The shaded box indicates the VMA8 open reading frame. The solid
triangle indicates the approximate site of insertion of the
sequence encoding the single HA epitope into the BclI enzyme
restriction site. The black box indicates the 2.2-kb HpaI LEU2 fragment used to replace the BbsI
fragment within the coding region of VMA8. The half arrows labeled 1 and 2 represent oligonucleotide
primers used to PCR amplify the VMA8 fragment. The figure is
not drawn to scale.
Cloning and Disruption of VMA8
Utilizing DNA
sequence information available through the data base, oligonucleotides
were synthesized complementary to the genomic sequence surrounding the VMA8 open reading frame. PCR amplification and subsequent
subcloning generated a 1837-bp fragment containing the VMA8 gene (Fig. 2B), which was sufficient for
complementation of a genomic disruption strain. The sequence of the
open reading frame of the PCR-generated VMA8 was confirmed by
double-stranded sequencing.
cells
completely lacked ATPase activity (<1% wild-type activity).
Transformation of vma8
disruption strain with a
centromeric plasmid-borne VMA8 PCR-derived gene fragment
(pKH3203) resulted in complete complementation of the Vma
phenotypes as determined by restoration of wild-type growth and
V-ATPase activity (data not shown).
-translocating ATPase (27) and with the
subunit of the archebacterium Sulfolobus acidocaldarius ATPase
(23% identity and 47% similarity, Ref. 28). The suggestion that ORF11
encoded the 32-kDa yeast V-ATPase subunit was first proposed by Takase et al. in their work describing the sequence of the entire ntp operon encoding the subunits of the E. hirae Na
-translocating ATPase(27) .
Figure 3:
Alignment of Vma8p with homologous protein
sequences. Clustal alignment of the predicted open reading frame from S. cerevisiae Vma8p V-ATPase subunit (Sc Vam8p) with
the predicted protein sequences from the 28.8-kDa C. elegans hypothetical protein (Ce 28kDa), the E. hirae Na-ATPase NtpD protein (Eh NtpD), and
the S. acidocaldarius ATPase
subunit (Sa
gamma). The sequences that are identical to Vma8p are boxed.
Fate of V-ATPase Subunits in vma8
In
order to determine the effect of the loss of Vma8p on the remaining
V-ATPase subunits, we examined the steady-state levels of proteins in
whole cell extracts and in isolated vacuolar membranes prepared from
wild-type (SF838-1D) and vma8 Cells
cells (KHY104). The
samples were separated by SDS-PAGE, and the relative protein levels in
wild-type and vma8
cells were compared by immunoblot
analysis using antibodies specific to each of the subunits. The
steady-state protein levels of the 100-, 69-, 60-, 54-, 42-, 36-, and
27-kDa subunits present in whole cell extracts prepared from vma8
cells were indistinguishable from the levels of
these proteins present in wild-type cells suggesting that the loss of
Vma8p had no effect on the synthesis or stability of the subunits
examined (Fig. 4A, left panel).
Figure 4:
Comparison of V-ATPase subunit levels in
whole cell extracts and vacuolar membranes prepared from wild-type and vma8 cells. A, detection of V-ATPase subunits by
immunoblot analysis. 30 µg of whole cell extract protein or 4
µg of vacuolar membrane protein were loaded per lane and then
separated by electrophoresis, and immunoblots were probed with
antibodies specific for the individual subunits, as described under
``Experimental Procedures.'' B, detection of the
17-kDa proteolipid V-ATPase subunit. Proteins extracted from 10 µg
of vacuolar membranes with chloroform/methanol as described under
``Experimental Procedures'' were separated by SDS-PAGE on a
15% acrylamide gel and detected by silver
staining.
In contrast
to whole cell extracts, vacuolar membranes isolated from vma8 cells (KHY104) contained only two of the seven subunits that could
be detected by immunoblot analysis using available antibodies. Only the
100- and 36-kDa subunits could be detected in vacuolar membranes from vma8
cells, but all subunits could be detected in the
vacuolar membranes prepared from wild-type cells (Fig. 4A, right panel). Additionally, the
17-kDa vacuolar ATPase proteolipid subunit was present at wild-type
levels in the vacuolar membranes of the vma8
mutant as
determined by silver-stained chloroform/methanol-extracted proteins (Fig. 4B). The results indicate that the three
well-characterized polypeptides of the V
sector are all
present in the vacuolar membranes of vma8
cells.
mutant (Fig. 4, right panel), even though they are
present in the cell at wild-type levels. Because these proteins are
identified as subunits of the ATP-hydrolyzing V
sector of
the vacuolar ATPase, the loss of Vma8p must prevent the association of
these V
subunits with the V
polypeptides on the
vacuolar membrane. Therefore, these data are consistent with the
suggestion that Vma8p functions as a subunit of the V
subcomplex or is required for its assembly.
Vma8p Is a Subunit of the V-ATPase
To
localize Vma8p in the cell and determine if it associates with the
V-ATPase, an epitope-tagged version of the protein was generated (see
``Experimental Procedures''). The sequence corresponding to
the HA epitope (YPYDVPDYA) was inserted between amino acid Ala and Asp
. VMA8::HA complemented the growth
phenotypes of the vma8
yeast cells. Vacuolar membranes
were isolated from the disruption strain, vma8
, carrying
either VMA8 or VMA8::HA, and the ATPase activities of
the vacuolar membranes were compared. The specific activity of vacuolar
membranes from vma8
cells expressing Vma8p-HA was 1.4
µmol of ATP hydrolyzed per min/mg of protein, compared with the
specific activity of vma8
expressing Vma8p, which was 1.8
µmol ATP hydrolyzed per min/mg of protein. The V-ATPase containing
the epitope-tagged Vma8p possessed approximately 78% of the
ATP-hydrolyzing activity of cells carrying the untagged protein and
indicated that the introduction of the HA epitope at the 3` terminus of
Vma8p did not appear to significantly alter its function in the cell.
cells expressing
Vma8p-HA were solubilized and separated by centrifugation through a
12-35% glycerol density gradient. Fractions were assayed for both
ATPase activity and dipeptidyl aminopeptidase B activity, and
immunoblot analysis was performed with antibodies specific to V-ATPase
subunits (Fig. 5). The peak ATPase activity was found in
fractions 7-9 of the 16 gradient fractions collected, and
dipeptidyl aminopeptidase B activity was highest in fraction 4.
Dipeptidyl aminopeptidase B is a 120-kDa integral membrane protein
component of vacuolar membranes (29) and is fractionated away
from the V-ATPase under these conditions as indicated by a separate and
distinct dipeptidyl aminopeptidase B activity peak (Fig. 5).
Figure 5:
Detection of Vma8p in solubilized vacuolar
membranes fractionated on a glycerol density gradient. Vacuolar
membranes were isolated from vma8 cells carrying plasmid
pKH3204. The proteins were solubilized in 2% ZW3-14 and separated
by centrifugation through a 12-35% glycerol density gradient.
Protein samples from each fraction were separated by SDS-PAGE and
probed with antibodies specific for the 100-, 69-, and 36-kDa V-ATPase
subunits and the HA epitope. The arrows indicate fractions
possessing peak ATPase and dipeptidyl aminopeptidase B (DPAP
B) activity.
The glycerol gradient fractions of solubilized vma8 vacuolar membranes expressing Vma8p-HA were probed with antibodies
against two V
subunits, the 100- and 36-kDa proteins, and
the V
69-kDa catalytic subunit. Immunoblot analysis
revealed all three of the V-ATPase subunits colocalized to gradient
fractions 7-9, and this corresponded to the fractions possessing
the highest ATPase activity, indicating that these fractions contained
assembled and functional V-ATPase. Glycerol gradient fractions probed
for the epitope-tagged Vma8p showed the protein cofractionating with
the active V-ATPase fractions, indicating that Vma8p is a subunit of
the vacuolar ATPase. The V
100- and 36-kDa subunits
typically showed a biphasic distribution in the glycerol density
gradients, results consistent with those previously
reported(7, 24) . The high density peak displayed
significant cofractionation between the 36- and 100-kDa proteins and
ATPase activity and a second, less dense protein peak possibly
representing isolated V
complex without the associated
V
subunits.
Fate of Vma8p When V
The assembly of a functional V-ATPase is dependent
on the presence of all the subunits of the complex, with the exception
of the 54-kDa subunit (VMA13; Ref. 11). In mutants lacking a
subunit of the VAssembly Is
Blocked
complex, such as the 60-kDa protein
encoded by the VMA2 gene, the V
complex was
assembled and present on the vacuolar membrane, but the V
subunits remained unassembled and in the cytoplasm of the cell.
To determine whether Vma8p behaved like other V
subunits,
whole cell extracts and vacuolar membranes were prepared from wild-type
and vma2
mutant cells expressing Vma8p-HA. Immunoblot
analysis of the protein samples was performed by probing with
antibodies specific to the 100- and 36-kDa V
subunits, the 69-kDa catalytic V
subunit, and
Vma8p-HA. Both the V
and V
subunits
were present in vma2
mutant cells at wild-type levels as
shown in the left panels of Fig. 6.
Figure 6:
Partitioning of vacuolar ATPase subunits
in vma2 cells. Whole cell extracts and vacuolar membrane
proteins prepared from wild-type and vma2
cells
expressing Vma8p-HA were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with antibodies against 100-, 69-, and
36-kDa V-ATPase subunits and Vma8p. 4 µg of vacuolar membrane
proteins or 30 µg of whole cell proteins were loaded per
lane.
As expected, loss
of the 60-kDa V protein (encoded by VMA2)
prevented the assembly of the V
complex and thus the
V-ATPase complex, as confirmed by the absence of the 69-kDa protein on
the vacuolar membranes of the vma2
cells. The level of
the Vma8p-HA detected in whole cell extracts of vma2
cells was identical to that present in wild-type cells expressing
Vma8p-HA. The loss of Vma2p had a very dramatic effect on the level of
Vma8p associated with the vacuolar membranes without effecting the
level present in the cell, as shown by comparing the bottom panels of Fig. 6(labeled Vma8p). A faint signal was
detected in the vacuolar membranes of vma2
cells
expressing Vma8p-HA but was present at a significantly diminished level
compared with Vma8p associated with wild-type vacuolar membranes.
Therefore, the behavior of Vma8p in vma2
cells was
consistent with the assignment of Vma8p as a subunit of the V
sector.
Vma8p Is a Peripheral Membrane Component of the
V
To determine the nature of the
association of Vma8p with membranes, vacuolar membranes were treated
with alkaline carbonate or urea. Treated membranes were separated from
soluble proteins by centrifugation at high speed (100,000 Sector
g). Immunoblot analysis of the pellet and supernatant
fractions was performed by probing with antibodies specific to the 100-
and 36-kDa V
subunits, the 69- and 60-kDa V
subunits, and Vma8p-HA (Fig. 7). For vacuolar membranes
treated with buffer only, all of the V-ATPase subunits examined were
associated with the membrane pellet fraction. A significant fraction of
the Vma8p was removed from the vacuolar membrane by treatment with
alkaline carbonate or urea. In contrast, the treatment of vacuolar
membranes with either alkaline carbonate or 5 M urea did not
alter the distribution of the integral 100-kDa V
subunit(4, 8) . The protein band below the 100-kDa
(identified by an asterisk in Fig. 7) represents the
75-kDa degradation product that remains associated with the membrane
fraction (9). In these same samples, the 36-kDa protein was very
efficiently released from the vacuolar membrane by these treatments,
confirming previously published results characterizing the unique
biochemical behavior of this peripheral V
subunit(7) . Finally, the 69- and 60-kDa V
proteins, forming the catalytic core of the V-ATPase, were
largely released into the supernatant fraction following treatment with
alkaline carbonate or urea. Like the hydrophilic 60-kDa V-ATPase
subunit, Vma8p was not completely extracted from the membrane fraction.
Figure 7:
Alkaline sodium carbonate or urea
treatment of vacuolar membranes from cells expressing Vma8p-HA.
Vacuolar membranes were incubated with 10 mM Tris, pH 7.4, 1
mM EDTA (TE Buffer), 100 mM NaCO
, or 5 M urea as described
under ``Experimental Procedures.'' The pellet (P)
and supernatant (S) fractions from 5 µg of vacuolar
membranes were separated by SDS-PAGE and analyzed by probing
immunoblots with antibodies specific to the 100-, 69-, 60-, and 36-kDa
V-ATPase subunits and Vma8p-HA. The asterisk indicates the
100-kDa degradation product.
The possible association of Vma8p with the V sector was
further examined through the use of the chaotropic reagent
KNO
. Treatment of vacuolar membranes with relatively low
concentrations of KNO
(100 mM) has been shown to
release the 69-, 60-, and 42-kDa V
subunits from the
membrane in a MgATP-dependent
manner(24, 30, 31) . Vacuolar membranes were
isolated from cells expressing Vma8p-HA and subjected to KNO
treatment in both the presence and the absence of MgATP. The
membrane samples were treated with buffer alone, 100 mM KNO
, or 100 mM KNO
plus 5 mM MgATP and centrifuged at high speed generating a membrane pellet
and supernatant fraction. Immunoblot analysis of the samples was
performed using antibodies against the 100- and 36-kDa V
subunits, the 69-kDa V
subunit, and Vma8p-HA. Buffer
or 100 mM KNO
treatment alone resulted in the
release of only trace amounts of any V-ATPase polypeptides into the
supernatant (Fig. 8). Treatment with KNO
plus MgATP
resulted in the release of the 69-kDa protein and Vma8p-HA from the
membrane. Neither the 100- or 36-kDa V
proteins were
removed from the vacuolar membranes under these conditions.
Figure 8:
Nitrate extraction of vacuolar membranes
from cells expressing Vma8p-HA. Vacuolar membranes were incubated with
buffer, 100 mM KNO, or 100 mM KNO
, plus 5 mM MgCl
ATP. Pellet (P) and supernatant (S) fractions from 5 µg of
vacuolar membranes were separated by SDS-PAGE and analyzed by probing
immunoblots with antibodies specific to the 100-, 69-, and 36-kDa
V-ATPase subunits and Vma8p-HA.
sector as determined by the presence
of the 100-, 36-, and 17-kDa proteins on vacuolar membranes of vma8
cells, but the V
subunits could not
assemble in the absence of Vma8p. HA-tagged Vma8p copurified with the
V-ATPase activity and behaved as a subunit of the V
sector
of the complex. Therefore, we conclude that the VMA8 gene
encodes the yeast 32-kDa V-ATPase subunit, which is required for
V-ATPase function.
chain'' (GenBank accession number Z27080) and a 25-kDa
subunit (encoded by atpG) of the S. acidocaldarius ATPase (Ref. 28; see Fig. 3). The designation of
for
the S. acidocaldarius subunit refers only to its
position in the ATPase operon, after
and
. No evidence
exists that either the C. elegans putative protein or the S. acidocaldarius subunit are functionally related to the well
studied F
F
-ATPase
subunit of Escherichia coli responsible for coupling proton translocation
to ATP synthesis(32) . No significant identity exists between
the predicted amino acid sequences of the yeast Vma8p and the
subunit of the yeast F
F
-ATPase (Atp3p; Ref.
33), suggesting that the 32-kDa subunit is unique to the vacuolar-type
ATPases.
subunit.'' Nelson et al. proposed that Vma8p, because it
has high identity to the bovine subunit D and to the C. elegans
-related subunit, is the yeast V-ATPase subunit equivalent to
the
subunit of the F
F
-type ATPase. In an
apparent contradiction, Nelson et al. also point out that no
significant sequence homology exists between subunit D and the
subunit of the F
F
-ATPase. No evidence exists,
despite the reasoning offered by these workers, that the function of
Vma8p in the V-ATPase complex is similar to the gamma subunit of the
F
F
-type ATPase.
-translocating
ATPase, encoded by the ntp gene cluster, and the well
characterized yeast V-ATPase identified several analogous proteins
shared by the two ATPase complexes(27) . The yeast 32-kDa
protein (Vma8p) encoded by VMA8 had been identified as a
protein similar to the NtpD subunit of the Na
-ATPase
present in the membranes of E. hirae (27). The subunit
composition of the E. hirae Na
-ATPase is very
similar to the yeast V-ATPase, suggesting that it is a V-type ATPase.
Interestingly, two subunits encoded by the E. hirae Na
-ATPase operon, NtpF (14-kDa) and NtpH (7-kDa),
lack corresponding sub-units characterized in yeast, suggesting that
additional yeast V-ATPase subunits may exist.
-ATPase catalytic sector can be isolated by mild
EDTA treatment of E. hirae cell membranes(34) .
Interestingly, the three major proteins comprising this intact
bacterial V
subcomplex are the 69- (NtpA), 52- (NtpB), and
29-kDa (NtpD) subunits of the E. hirae Na
-ATPase. The two largest proteins from this
bacterial complex are analogous to the 69- and 60-kDa yeast V
subunits. As already described, the NtpD protein shares high
similarity to the yeast 32-kDa protein characterized in this work. The
ability to isolate a V
subcomplex suggests that a strong
physical association exists between these peripheral subunits (69-,
52-, and 29-kDa), stronger than the association to the V
sector.
treatment. Proteins that were released only after ATP-dependent
treatment in the presence of 100 mM KNO
reflected
a modified membrane association possibly brought about by a
conformational change related to either ATP binding or ATP hydrolysis.
Vma8p, like the 69-kDa subunit, was sensitive to this ATP-dependent
release from the vacuolar membrane, but the 36- and 100-kDa V
subunits were insensitive to the ATP-induced conformational
change. The specific ability of ATP to affect the release of V
subunits reflects directly on the role of the V
subunits in forming the ATP hydrolyzing complex. Conversely, the
lack of stimulation of release of the V
subunits by ATP
directly reflects the role of the V
subunits, not in
hydrolyzing ATP, but in the formation of the proton pore through the
membrane. The results provided by the study of the vacuolar-type ATPase
from E. hirae suggest a similar and important role for Vma8p
in forming the catalytic core of the yeast vacuolar ATPase together
with the 69- and 60-kDa subunits.
Table: S. cerevisiae strains and plasmids
Table: 0p4in
Unpublished
data.(119)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.