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(Received for publication, September 20, 1995; and in revised form, January 17, 1996) From the
The vacuolar proton-translocating ATPase is the principal
energization mechanism that enables the yeast vacuole to perform most
of its physiological functions. We have undertaken an examination of
subunit-subunit interactions and assembly states of this enzyme. Yeast
two-hybrid data indicate that Vma1p and Vma2p interact with each other
and that Vma4p interacts with itself. Three-hybrid data indicate that
the Vma4p self-interaction is stabilized by both Vma1p and Vma2p.
Native gel electrophoresis reveals numerous partial complexes not
previously described. In addition to a large stable cytoplasmic complex
seen in wild-type,
The V-type proton-translocating ATPase is the cornerstone of the
yeast vacuole. This multisubunit membrane-bound enzyme converts the
energy of ATP into a proton electrochemical gradient that is essential
for the majority of vacuolar functions, including ion homeostasis,
accumulation of amino acids, and the correct targeting of vacuolar
resident proteins(1, 2, 3) . Enzymes of the
V-ATPase class are found throughout the biological world serving many
functions, oftentimes at different subcellular locations or with
tissue-dependent activities(4) . Many of the genes encoding
ATPase subunits, as well as genes necessary for vacuolar acidification,
have been identified(4, 5) . Null mutations in the VMA (vacuolar membrane ATPase) genes result in a conditional
phenotype characterized by several different traits. Growth of vma mutants is severely inhibited in media buffered to pH 7.0 or
higher; best growth is obtained in media buffered to pH
5.5(6) . These mutants are sensitive to high concentrations of
Ca
Biochemical analyses have begun to elucidate the
assembly and regulation of the enzyme(10, 11) . The
vacuolar ATPase is divided into two subcomplexes: a membrane-bound
V Our laboratory has traditionally
studied the biogenesis of the yeast vacuole and its role in cellular
metabolism. We became interested in the ATPase due to the substantial
influence of ATPase mutants on the targeting of proteins to the
vacuole(2, 3, 15) . Because of its central
role in most vacuolar functions, we have begun to explore the
structure/function relationship and regulation of the ATPase. We
initiated our studies using the two-hybrid method for examining
interactions between subunits(16) . To further explore
interactions we developed a native gel system for looking at
subcomplexes of the V
To produce
antiserum to Vma1p, Vma2p, and Vma8p, synthetic peptides were made
(Multiple Peptide Systems, San Diego, CA) based on the deduced amino
acid sequence of the VMA1, VMA2, and VMA8 genes. Peptides corresponding to amino acid residues 4-24
and 932-951 for Vma1p, 386-400 and 482-499 for Vma2p,
and 191-205 and 209-232 for Vma8p were separately
conjugated at their C termini to keyhole limpet hemocyanin. Standard
procedures were used to generate antiserum in New Zealand White
rabbits. Antiserum to Vma4p has been described previously(15) . The two-hybrid vectors, pGAD424 and pGBT9, which contain the GAL4 trans-activating domain and DNA binding domain,
respectively, were purchased from Clontech. The multiple cloning sites
(MCS) ( The three-hybrid plasmid was constructed by eliminating the
two-hybrid portion of pGAD424, which lies between two HindIII
sites, thus leaving the alcohol dehydrogenase promoter and terminator
intact. We cloned into the HindIII site an oligonucleotide
duplex of 5`-AGCTATGCCCAAGAAGAAGCGGAAGGTCGGATCCGAATTCACTAGTGTCGACA-3`
and 5`-AGCTTGTCGACACTAGTGAATTCGGATCCGACCTTCCGCTTCTTCTTGGGCAT-3` coding
for the SV40 nuclear localization sequence (NLS) and an MCS. The entire
promoter, NLS, MCS, and terminator cassette was then moved by
subcloning from flanking SphI sites into pRS423S, a derivative
of pRS423 (HIS3) which has had its MCS replaced with a single SphI site. The final construct was designated pMAT. The third
hybrid constructs were built by subcloning from pGAY1, pGAY2, and
pYES2VMA4 into the appropriate sites in pMAT, to create pMAT1, pMAT2,
and pMAT4. The HA epitope-tagged VMA7 construct was a gift
from T. Stevens and L. Graham (University of Oregon) and has been
described previously(33) .
For liquid assays, cells were grown to late logarithmic
growth phase (A
An interaction
between ATPase subunits would be detected as a
Figure 1:
Two-hybrid filter assay of interactions
between VMA genes. Full-length ORFs of VMA1
All signals from the
filter assays were distinctly above background, yet required from one
to 20 h to develop on the filter at 30 °C. The positive control, by
contrast, developed a strong signal within 30 min. Liquid assays for
Results from all possible three-hybrid crosses were somewhat
equivocal. In certain instances a three way interaction (i.e. including Vma1p, Vma2p, and Vma4p, each in a different fusion
vector) gave detectable signals on filter assays. However, these
signals were not detectable by liquid assay, and the interactions did
not show reciprocal relationships. The one clear result which did
emerge involved the Vma4p self-interaction (Fig. 2). In this
case we saw a significant and reproducible enhancement of the
Figure 2:
Three-hybrid liquid assay of interactions
between VMA genes. The combination of VMA genes in
the three-hybrid cross are indicated beneath the respective columns.
The N-terminal fusion is indicated on the bottom left. The
data are the product of four independent experiments (n = 4).
Figure 3:
ATPase complexes in wild-type yeast.
Extracts were prepared as described under ``Experimental
Procedures'' and run for 100 min on a 6% acrylamide native gel.
For second dimensions, strips of the native gel were soaked in Laemmli
buffer and wedged atop a 10.5% SDS-PAGE gel. Gels were transferred to
PVDF membranes and blotted as described. Roman numerals indicate the complexes described in the text. A,
immunoblot of single dimension native gel separation. The same lane is
shown four times, blotted with antiserum to the protein indicated
beneath the lane. The temporal order of blotting was Vma2p, Vma1p,
Vma4p, Vma8p. B, two-dimensional gels blotted with the same
antibodies as in A, but in the following order: 4p, 8p, 2p,
1p. Molecular masses in kilodaltons are given to the left of
each blot. The arrow at the right of the blot
indicates the position of the protein in the second dimension. The arrows on the blot indicate the position of complex II and
lower complexes and smears of the given subunit. Although the blots
were stripped between antibodies, some residual antibody from the
previous blot(s) does remain. Complex I is not detectable in wild-type
extracts.
The immunoblots were stripped
of antibodies and reprobed with antibodies to Vma2p, Vma4p and Vma8p,
shown in the second, third, and fourth lanes, respectively, of Fig. 3A. The actual order of blotting is 2p, 1p, 4p,
8p. Stripping of the blot is sometimes incomplete, but the second
dimensions for Vma2p, Vma4p, and Vma8p shown in Fig. 3B clarify the situation (order of probing: 4p, 8p, 2p, 1p). The
antibodies to Vma2p and Vma4p have strong nonspecific signals which
proved to be useful as reference points for comparison of strains. What
can clearly be seen is that all four proteins run in the higher band of
the first dimension, designated complex II, and also run at second,
separate locations lower in the first dimension. These data strongly
suggest that 1p, 2p, 4p, and 8p form a complex together. Vma4p also
runs at an anomalously high location and was designated complex III for
reasons given below. We have designated all the complexes from this
study with Roman numerals, counting down from the top of the native gel
as indicated in Fig. 3. Complex I does not appear in detectable
quantities in wild-type extracts and is therefore not indicated in Fig. 3. To confirm that complex II was dependent on all four
proteins, extracts from
Figure 4:
ATPase complexes in strains deleted for
Vma1p, Vma2p, Vma4p, and Vma8p. A, immunoblot of single
dimension native gel with extracts from
In
Figure 5:
Hedrick-Smith plot for calculation of
complex sizes. A set of the standard proteins indicated were run on
separate native gels, each of a different percent acrylamide. A, relative mobilities were measured and plotted as a function
of gel percent (inset) and the negative slopes plotted as a
function of molecular weight. B, extracts from wild-type,
Complex III, which contains Vma4p and perhaps other
unidentified subunits, migrates with a molecular mass of 96 ± 28
kDa. If this is exclusively Vma4p, then this would indicate a complex
of three subunits (predicted mass of 81 kDa). Although we cannot rule
out other subunits at this time, we have seen no effect on this complex
in other deletion strains tested (see below) and cannot detect the
presence of Vma1p and Vma2p in the complex, even though its stability
is dependent upon these subunits. The apparent weight of complex III
further supports our hypothesis that Vma4p forms a homomultimer
complex.
Figure 6:
ATPase complexes in a
We further examined extracts from
Figure 7:
ATPase
complexes in other deletion strains. Strains
To further examine complexes for the presence of
Vma7p, we expressed an HA epitope-tagged version of Vma7p in our
wild-type,
Figure 8:
Presence of HA epitope-tagged Vma7p in
ATPase complexes.
The assembly of multisubunit enzymes from numerous different
proteins is a fascinating and complex problem. We have begun to explore
the pathways of assembly for the V
Figure 9:
Model for cytoplasmic assembly of V
Based on our results, as well as those
from other laboratories, we propose that the initial V The pattern exhibited by Vma2p in the
native gel analysis is very complex. In addition to the complexes,
including Vma2p, which we discussed in this study, we can also identify
at least three species in the lower portion of the gel with strong
signals and additional weak signals at several places in the gel. We
believe that these are due to modifications of Vma2p which alter its
charge-to-mass ratio. It has been shown that Vma2p is phosphorylated in
both plant and animal systems(47, 48) , and there is
recent evidence that it is also phosphorylated in yeast. ( Recently, two
laboratories have cloned VMA8(34, 35) , the
product of which one laboratory has proposed is the homologue of the
F-type Another candidate for The interaction of
Vma4p with itself is supported by both our two-hybrid data and our
native gel analysis, as well as the observation from other laboratories
that this protein runs anomalously in glycerol gradients(12) .
Why Vma4p interacts with itself and if this is necessary for assembly
remain interesting questions, to which we have no answers at this time.
We cannot rule out the possibility that Vma4p interacts with proteins
which we have not yet examined and that these proteins make up a
substantial portion of complex III. The addition of Vma7p is
necessary for stabilization, but not formation, of the ATPase
subcomplexes (complex II and IV). We are currently making the
As has been demonstrated previously, Vma5p is not
necessary for the assembly of V Our current studies seek to characterize the components of complex
II, which we consider to be the definitive V
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10397-10404
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
vma3 and vma5 strains,
we see partial complexes in the vma4 and vma7 strains. All larger complexes are lost in the vma1,
vma2, and vma8 strains. We designate the
large complex seen in wild-type cells containing at least subunits
Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V
complex.
(
50 mM) in the media(7) . In vma strains that are also ade2, the reddish color
caused by accumulation of fluorescent amino-imidazole ribotide
conjugates in the vacuole is diminished, providing a convenient visual
screen for ATPase mutants (8) . Deacidification of the vacuolar
lumen, which is normally maintained at pH 6 in wild-type cells, can be
determined by direct fluorescent ratio measurements (9) and has
been used as a screen for vacuolar ph mutants(5) . Screens
utilizing these characteristics have revealed the genes catalogued in Table 1. Each of these genes is essential for vacuolar ATPase
activity and most are required for proper assembly of the complete
V-ATPase complex.
complex, which is responsible for the translocation of
protons, and a peripheral membrane V complex, which
contains the ATP-hydrolyzing subunits. In yeast, more extensive
biochemical studies have lagged behind the more readily achieved
genetic analyses. It has been shown that the ATPase can form the
soluble, cytoplasmic V complex independent of the vacuolar
membrane components (V) and that this complex requires at
least Vma1p, Vma2p, and Vma4p, but not Vma5p(12) . Studies on
the homologous complex from bovine clathrin-coated vesicles have shown
that the ATPase can be disassembled and reassembled in
vitro(13) . The homologues to Vma1p, Vma2p, Vma4p, Vma8p,
but not the Vma5p homologue, are essential for reassembly and
activity(14) . The subtleties of the interactions between these
core subunits, however, have not been examined. Furthermore, the role
of other peripheral subunits for which genes have been cloned from
yeast have yet to be determined. complex. From these studies we have
discovered a surprising number of cytoplasmic subcomplexes which we
predict will be assembly intermediates on the pathway to the complete
enzyme.
Reagents, Antibodies, Strains, and Plasmids
All
reagents were from Sigma unless otherwise stated. HEPES buffer was from
Research Organics, Inc. Media components were from Difco. All strains
were derived from the wild-type strain W303-1B (MAT
leu2 his3
ade2 trp1 ura3) with the exception of the vph1, stv1, and vph1stv1 strains(17, 18) , which were a gift from M.
Manolson (Hospital for Sick Children, Toronto). All W303-1B-based vma strains were a gift from N. Nelson (Tel Aviv
University), except for the vma4 strain, which was
prepared in W303-1B using the vma4::LEU2 disruption construct
described previously(15) , and confirmed by Western blot. The
two- and three-hybrid assays were conducted in strain SFY526 (MATa
ura3 his3 ade2 lys2 trp1 leu2 gal4 gal80
URA3::GAL1-lacZ)(19) .)in these vectors were modified by digestion with EcoRI and PstI to eliminate the existing sites, and
ligation with the complementing oligonucleotides
5`-AATTCGGATCCACTAGTTCGACCCGGGCTGCA-3` and
5`-GCCCGGGTCGAACTAAGTGGATCCG-3`, to create pA` and pB`, respectively.
This altered the reading frame of the sites, and added a unique SpeI site into each of the vectors. The VMA1vde ORF was obtained by polymerase chain reaction from plasmid
pVMA1vde, a gift from F. Gimble (Texas A & M University),
using the primers 5`-GAAAAGGATCCATGGCTGGTGC-3` and
5`-GCTCGAGATATCTTAATCGG-3`. This incorporates a BamHI site at
the 5` end and a XhoI site at the 3` end. Likewise, the VMA2 ORF was obtained from YPN1VAT2 (25) with primers
5`-AATATTGGATCCATGGTTTTGTC-3` and 5`-CAGCACTCGAGCTCTTAGATTAGAG-3`,
which incorporate the same two sites. These were cloned, individually,
into the BamHI and XhoI sites of plasmid pYES2 from
Invitrogen to generate pGAY1 and pGAY2, respectively. These are
galactose inducible and were used to demonstrate that the polymerase
chain reaction products could complement their respective deletion
strains. The BamHI-XhoI fragments were subcloned from
pGAY1 and pGAY2 into the BamHI and SalI sites of pA`
to create pA`1 and pA`2, respectively. Likewise, the fragments were
cloned into pB` to create pB`1 and pB`2. The VMA4 ORF and
terminator were cloned as a BamHI fragment from
pYES2VMA4(15) , which can complement the vma4 strain, and into pA` and pB` to make pA`4 and pB`4, respectively. Two- and Three-hybrid Assays
The two- and
three-hybrid constructs were transformed into strain SFY526 and plated
onto synthetic medium with glucose supplemented with appropriate amino
acids. Colonies were patched to fresh plates, grown, and transferred
onto Whatman filter paper #50 discs which had been layered onto
synthetic medium with glucose plates. The patches were allowed to grow
overnight and then lysed by two freeze/thaw cycles in liquid nitrogen.
The filters were then placed on additional filters soaked in Z-buffer
(100 mM sodium phosphate, pH 7.0, 10 mM KCl, 1 mM MgSO
, 0.3% 
-mercaptoethanol) with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (0.34
mg/ml) and incubated at 30 °C for 36 h. The filters were dried and
scanned.
1.0), and then lysed in
Z-buffer containing SDS (0.005%) and chloroform (5%). o-Nitrophenyl--D-galactopyranoside was added to
a final concentration of 0.4 mg/ml and the samples incubated at 30
°C for 30 min, then quenched with Na
CO
, 300
mM final concentration. Color development was measured by
absorbance at 420 nm and the activity calculated according to the
formula of Miller(20) . Multiple assays were conducted on
several independent transformations of each cross.Preparation of Extracts for Native Gel
Electrophoresis
Wild-type and deletion strains described above
were grown to late log in YPD (1% yeast extract, 2% bactopeptone, 2%
glucose) buffered to pH 5.5 with 50 mM MES and 50 mM MOPS buffers. The cells were pelleted and treated with 100 mM Tris-SO, pH 9.4, 10 mM dithiothreitol for 5
min at 30 °C. These were pelleted and resuspended in osmotically
supportive media containing 1% glucose and 1.5 mg of oxalyticase
(Enzogenetics)/A of cells/ml and incubated for
40 min at 30 °C. The resulting spheroplasts were pelleted gently
and washed once with supportive media lacking oxalyticase. The cells
were then pelleted and lysed on ice in native sample buffer (10 mM Tris acetate, pH 6.9, 5 mM potassium acetate, 1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride (Boehringer
Mannheim), 10% glycerol, 0.01% bromphenol blue, 0.01% xylene green) at
a concentration of 5 mg/ml total protein, based on an estimate of 200
µg of protein/A/ml of cells. These samples
were quickly aliquoted on ice, frozen in liquid nitrogen, and stored at
-80 °C until use. For native gels, the samples were thawed on
ice, centrifuged briefly to pellet particulate matter, and loaded
immediately.Native Gel Electrophoresis
Native gels employed a
continuous buffer system of HEPES and imidazole at pH 7.4. For
qualitative first dimensions and two dimensional gels, 6% acrylamide
(37.5:1 acryl:bis; Boehringer Mannheim) gels were used. For
Hedrick-Smith analyses, varying percentages from 6 to 8.25% were used.
Gels were prerun at a constant voltage of 150 V (25 V/cm) at 4 °C
for 2 h until the current was steady. Buffer was changed before sample
loading, and the gels were run for 100 min at 150 V. For qualitative
first dimensions and Hedrick-Smith analyses, the gels were
electroblotted onto Immobilon PVDF membrane (Millipore) overnight in
Tris/glycine transfer buffer at 60 mA constant current. For
two-dimensional gels, the first dimension was cut into strips and
soaked in Laemmli buffer for 1 h at 37 °C. These were loaded onto
the 3% stack of a 10.5% SDS-PAGE preparative gel and run at 100 V
constant until the dye front was at the bottom of the gel. These gels
were then transferred to PVDF as described above.Immunoblotting
The membranes were blocked with 5%
powdered milk (Blotto) in TTBS (20 mM Tris-HCl, pH 7.6, 137
mM NaCl, 0.1% Tween 20) for a time from 15 min to overnight.
Primary antibodies were typically used at a dilution of 1:5,000 or
1:10,000 in 1% Blotto TTBS and incubated at room temperature for
1-2 h. After three washes in TTBS, the membrane was blocked again
for at least 15 min and then incubated for 1 h with goat-anti-rabbit
antibodies conjugated to horseradish peroxidase (Cappel) at 1:10,000
dilution in 1% Blotto TTBS. After three washes in TTBS, bound
horseradish peroxidase was detected on film by chemiluminescent
reaction(21) .
Interactions Detected by Two-hybrid Assay
To
begin our examination of interactions between V subunits,
we cloned VMA1vde, VMA2, and VMA4 into the
two-hybrid vectors pA` and pB` and examined all possible crosses in
yeast strain SFY526, which contains the -galactosidase gene under
control of the GAL1-10 promoter integrated into the
genome. The VMA1 gene codes for a self-splicing
protein(54) , and the VMA1vde version has the VDE
coding region deleted from the ORF(55) .-galactosidase
activity on filters in the two-hybrid system (blue color). Fig. 1shows a filter assay for -galactosidase activity of
yeast patches containing all possible pairs of two-hybrid constructs.
For a positive control we crossed the SV40 large T-antigen and murine
p53 proteins (data not shown), whose interaction has been demonstrated
by independent methods. Signals from the VMA1 
VMA2 crosses are clearly evident in both possible combinations on the
filter, indicating that Vma1p and Vma2p may interact directly.
Surprisingly, we also discovered that Vma4p can interact with itself.
This result was not expected, since Vma4p has a proposed stoichiometry
of one per ATPase complex based on comparison with the V-ATPase complex
from bovine clathrin-coated vesicles(43) . We found no
interactions between VMA1 VMA4 and VMA2 VMA4 in the two-hybrid assay, although all three
proteins are necessary for formation of V complexes(12, 13) .
vde(1) , VMA2(2) ,
and VMA4(4) were cloned behind the GAL4 trans-activating domain (pA`) or the GAL4 DNA binding
domain (pB`) and transformed into the yeast two-hybrid detector strain
SFY526. The filter was soaked in
5-bromo-4-chloro-3-indolyl--D-galactopyranoside-containing
buffer for 30 h at 30 °C. The dark spots, indicating an
interaction between the respective VMA gene products, are blue in the original data.
-galactosidase activity gave low signals (1-2 Miller units)
from the VMA4 VMA4 interaction, and no
detectable signals from the VMA1 VMA2 crosses
were obtained. Again, the positive control gave a reproducible signal
of 80-100 Miller units. Western blots indicated that the
full-length fusion constructs were being stably expressed, albeit at
low levels (data not shown). We suspect that the Vma1p-Vma2p
interaction is transient and requires stabilization by other subunits
for a strong interaction.Three-hybrid Interactions
We conjectured that
two-hybrid interactions could be stabilized by the presence of a third
ATPase subunit targeted to the nucleus. Therefore, we constructed the
three-hybrid vector which fuses the SV40 nuclear localization sequence
to the N terminus of a protein so that it is targeted to the nucleus
for potential interaction with the two-hybrid fusion products. It has
been previously demonstrated that this signal is sufficient to target
passenger proteins to the yeast nucleus(44) . We envisioned
three possible interactions: 1) a bridge interaction, where two
proteins which did not show an interaction previously would now give a
positive signal in the -galactosidase assay; 2) an enhancement
interaction, where the third protein would stabilize the two-hybrid
products and increase the signal from the assay; and 3) a competitive
interaction, where the third protein would diminish a pre-existing
signal.-galactosidase activity in liquid assays when NLS-Vma1p or
NLS-Vma2p was included with the Vma4p fusion constructs. In contrast,
when the NLS-Vma4p fusion is present we see a competitive diminishment
of the self-interaction, further indicating that the interaction is
specific. The enhancement by NLS-Vma1p and NLS-Vma2p suggest that they
interact with Vma4p. Why Vma4p does not show an interaction with Vma1p
or Vma2p in a direct two-hybrid cross is uncertain, but we hypothesize
that the site of interaction in Vma1p and Vma2p may be masked by the
two-hybrid fusions, yet accessible in the third hybrid fusion
construct.
Two-dimensional Analysis of V
Vexed yet tantalized by our two- and
three-hybrid results, we developed a native gel analysis to identify
cytoplasmic complexes more directly, and by extension subunit
interactions. The first lane of Fig. 3A shows an
immunoblot of the first dimension from our wild-type strain of yeast,
probed with an antibody to Vma1p. Several bands are visible, one near
the top of the gel and an indeterminate smear further down. It must be
remembered that although native gels separate in part on molecular size
and shape, separation depends substantially upon the charge/mass ratio
and that therefore a higher band does not necessarily mean a larger
protein or complex. The top panel of Fig. 3B shows the second dimension probed with Vma1p. Here, two positions
of Vma1p can be distinguished from background. Positive identification
of two-dimensional spots was determined by comparison with a
two-dimensional analysis of a Complexesvma1 extract probed with
antibodies to Vma1p (data not shown).
vma1, vma2,
vma4, and vma8 strains were prepared and
resolved on two-dimensional gels. Fig. 4A shows the
first dimension from these strains. Complex II (Fig. 3) is
completely missing from all four deletion strains. The Vma4p-containing
complex III, which does not contain detectable amounts of either Vma1p
or Vma2p, also disappears from the vma1 and vma2 strains, but is clearly evident in the vma8 strain.
We have noticed that this complex is unstable in the vma1 and vma2 strains, visible only after long exposure
of the immunoblot. Analysis of our extracts by SDS-PAGE and immunoblot
shows that Vma4p is being degraded during spheroplasting and lysis,
rather than disassembling from complex III (data not shown). However,
steady state levels of Vma4p in vma1 and vma2 strains are similar to wild-type (data not shown). We have been
unable to detect Vma1p, Vma2p, Vma7p, or Vma8p in complex III, and this
complex is present and stable in vma7 or vma8 deletion strains (see below). We think complex III may be
multimerized Vma4p, which is somehow stabilized by Vma1p and Vma2p.
This corroborates our observation from the three-hybrid system, that
Vma4p can form a complex with itself, and that this complex is
stabilized by Vma1p and Vma2p.
vma1,
vma2, vma4, and vma8 strains.
Matching lanes are shown probed with antibodies to Vma1p, Vma2p, Vma4p,
and Vma8p. The order of blotting was 2p, 1p, 4p, 8p, resulting in some
residual signals in the vma4, Vma4p lane. B,
two-dimensional immunoblots of vma4 with Vma1p, Vma2p,
and Vma8p antibodies. The order of blotting was: 4p (not shown), 8p,
2p, 1p. Some residual signals remain on the Vma1p
blot.
vma4, complex II is
missing, but several new intermediate complexes have now appeared that
are detectable with antiserum to Vma1p and/or Vma2p. Two of these have
been designated complexes IV and V. Two-dimensional analysis reveals
that complex IV contains Vma1p, Vma2p, and Vma8p (Fig. 4B). Complex V contains Vma2p, but not Vma1p or
Vma8p. This seemed unusual, since complex V does not appear in either
deletion strain. Long exposures of our blots have revealed very small
amounts of this form of Vma2p in wild-type and deletion strains (data
not shown). This suggests that this form of Vma2p is rapidly altered in
a Vma4p-dependent manner, either by simple association or perhaps by
direct or indirect modification of Vma2p. In any event, Vma4p is
necessary for assembly of complex II, suggesting that complexes IV and
V are intermediates leading to complex II.Calculation of Molecular Weights
We wanted to know
how large these complexes are so that we might estimate the subunit
composition. We used the Hedrick-Smith method (45) to calculate
the molecular weights ( Fig. 5and Table 2). Native gels
ranging from 6 to 8.25% were run under identical conditions, and the
relative mobilities of the complexes were measured. Standard proteins
were run on the same gels, blotted, and stained with Amido Black. The
relative mobilities were plotted as a function of gel percent (Fig. 5A, inset), and the negative slopes of these
lines were plotted as a function of molecular weight (Fig. 5A). The relative mobilities of our complexes
were likewise plotted (Fig. 5B) and the molecular
weights calculated from the standard curve. The molecular weights
calculated from multiple experiments are compiled in Table 2.
Since Vma1p and Vma2p have proposed stoichiometries of three each per
complex, this would indicate that complex II (calculated molecular mass
of 576 ± 97 kDa) is large enough to be a 1p2p hexamer (predicted molecular mass of 375 kDa) with additional
subunits, including Vma4p and Vma8p. Complex IV (calculated mass 317
± 49 kDa) from the vma4 strain is too small to
encompass a 1p2p hexamer, but may be a
1p2p tetramer (predicted weight of 249 kDa)
with accessory subunits, including Vma8p. We have noticed a trend for
our calculated molecular masses to be higher than expected.
Calculations made on lower bands give no molecular mass closer than one
and a half times the predicted mass of the subunit examined. Further
experiments examining these lower molecular mass intermediates are
underway. These results strongly suggest that Vma4p is necessary for
assembly to a 1p2p4p8pXY complex
(where X and Y indicate other as yet unidentified
subunits).
vma3, and vma4 strains were run on the same
gels as in A, immunoblotted, and the relative mobilities
plotted as a function of gel percent. The average molecular weights of
multiple experiments are shown in Table 2.
V
We wished to know if complex II required a membrane
sector for assembly. To answer this question, we examined a
Is Unnecessary for Cytoplasmic
Assemblyvma3 strain, which lacks the V, in our
two-dimensional system. Fig. 6shows immunoblots of the first
dimension. Both complex II and the poorly resolved lower bands are
present. Hedrick-Smith analysis indicates that this complex II is the
same size as seen in the wild type. Two-dimensional analysis revealed
the same composition as in wild-type extracts (data not shown). In the
vma3, strain all of the complex II signals are stronger
than in wild-type, even though the total protein load was identical. We
believe this is due to loss of membrane-bound complexes from the
wild-type extracts, which are centrifuged to eliminate particulate
matter before being loaded onto the native gel. We also see strong
signals at the position of the gel-well from wild-type extracts,
suggesting that membrane-bound material does not enter the gel. Since
no complexes are membrane-bound in vma3, there is a
larger pool of cytoplasmic complexes detected in this strain. This
result also suggests that complex II does not contain any of the
membrane subunits.
vma3 strain lacking the V. Identical experiment as Fig. 3, but in a vma3 strain. Immunoblot of a
single dimension native gel probed with antibodies to Vma1p, Vma2p,
Vma4p, and Vma8p. Order of blotting: 2p, 1p, 4p,
8p.
vph1, stv1, and the
vph1stv1 double deletion strains. VPH1 and STV1 code for 96- and 102-kDa integral membrane
proteins, respectively, which are part of the membrane complex and are
believed to be responsible for targeting of the ATPase to specific
organellar membranes(17, 18) . In the absence of
either or both of these proteins, complex II is seen in the native
dimension (data not shown). Therefore, V is unnecessary for
assembly of complex II.Involvement of Other Peripheral Subunits
We have
examined several other peripheral subunits to see if they are necessary
for formation of the large cytoplasmic complex. Fig. 7shows
immunoblots in the first dimension for extracts from vma5 and vma7. Vma5p is unnecessary for formation of
complex II, although it has been demonstrated to be necessary for
assembly of V on the membrane. In the vma7 strain complex II vanishes, but is replaced by a weakly detectable
complex, complex I, higher in the gel, and a broad smear of Vma1p,
which runs immediately below this complex. Because of the weak signal
from complex I, we were unable to get complete data sets for
Hedrick-Smith analysis. However, two-dimensional analysis reveals that
it contains Vma1p, Vma2p, Vma4p, and Vma8p (data not shown). Hence, we
suspect that complex I is a loose hexamer which is stabilized by Vma7p
to form complex II.
vma5 and vma7 were run on a single dimension native gel and probed
with antibodies to Vma1p, Vma2p, Vma4p, and Vma8p. Order of blotting:
2p, 1p, 4p, 8p.
vma3 and vma4 strains. This
construct has been demonstrated to complement the Vma7p null mutation
and hence to be competent for properly assembly into
complexes(33) . Fig. 8shows immunoblots of extracts
from vma3 and vma4 strains expressing the
tagged Vma7p (order of blotting: HA, 4p, 1p, 8p, 2p). The signals from
wild type were similar to vma3, but weaker due to loss of
membrane-bound material (data not shown). The tagged protein can be
seen to run with both complex II and complex IV, as indicated by the
subsequent blot against Vma2p. Complex V is obscured by a nonspecific
band which was seen in extracts not expressing the HA construct. No
other bands were seen in the absence of the HA construct (data not
shown).
vma3 and vma4 strains
were transformed with pLG20 expressing an HA epitope-tagged version of
Vma7p and extracts prepared as described, run on native gels, and
blotted. Order of blotting: HA, 4p (not shown), 8p (not shown), 1p (not
shown), 2p.
sector of the vacuolar
proton-translocating ATPase. From our current results, we would propose
a tentative model for cytoplasmic assembly of the V complex
of the vacuolar ATPase (Fig. 9). The value of this model is that
it provides many testable hypotheses. While our results do not exclude
the possibility of interactions with other large proteins or protein
complexes (e.g. chaperones), we have applied Occam's
razor and assumed that only ATPase subunits are components of the
complexes we have observed.
complex. Roman numerals indicate complexes separated in
the native gels. The asterisk indicates the Vma4p-dependent
modified version of Vma2p seen unmodified as complex V in the
native gels. A plausible alternative to this model would have the
Vma4p-complex cycling with complex IV to render complex
I.
assembly event is an interaction between the Vma1p and Vma2p
proteins. Vma1p has been implicated as the ATP-hydrolyzing subunit of
the ATPase complex(23, 24) , while Vma2p is a
nucleotide-binding protein, which is believed to be involved in
regulation of the ATPase(25, 26) . Both subunits have
a stoichiometry of three per complex, based on comparison with the
bovine clathrin-coated vesicle ATPase(43) . From our two-hybrid
results, it appears that these two subunits can interact with each
other in the absence of other subunits. In addition, these subunits
show homology to F-type ATPase/synthase subunits(46) . The
Vma1p is homologous to the catalytic protein, whereas Vma2p is
homologous to the protein. From the crystal structure of the
F-type enzyme, these proteins can be seen in close proximity and appear
to interact(51) .)At least one modification may be dependent upon Vma4p,
since an unusual band appears in gel-resolved extracts from the
vma4 strain, with Vma2p as the only detectable component.
It may also be the case that other subunits, which we have not
explored, are associated with Vma2p in this band.
subunit(34) , although there is no significant
sequence homology between these proteins. From the results of our
native gel analysis, it is apparent that Vma8p is essential for the
assembly of Vma1p-Vma2p into larger complexes. It may be, therefore,
that Vma8p has some structural homology to . The issue remains
open to debate. is the Vma4p protein,
which has been suggested for this role in Neurospora
crassa(49) . From our native gel analysis, it is clear
that higher order complexes can form in the absence of this subunit.
Some F-type ATPases are capable of forming higher order complexes from
just and , but in general or 
is also necessary
for this assembly (reviewed in (50) ). Therefore, by comparison
with the F-type ATPases, Vma4p is an unlikely homologue. However,
V-type ATPases are not F-type ATPases, and we believe that a search for
one-to-one homologues is a faulty perspective on the situation. From
crystal structures it can be seen that the F-type subunit has a
coiled-coil as an essential structural motif (51) .
Coiled-coils are involved in protein-protein interactions, most notably
in the leucine zipper motif. Both Vma4p and Vma8p are predicted to have
high -helical content. We envision a scenario where the structural
role of is filled by two separate proteins, potentially Vma4p and
Vma8p, each of which contribute a coil, and both of which are necessary
for stable assembly of the complete Vma1p-Vma2p hexamer, which is
believed to surround the coiled-coil structure.vma4vma7 double deletion to examine if
complex IV has an unstable ``7-less'' counterpart. While
complex II is stable in extract for over 90 min on ice, complex I is
substantially diminished even after 30 min (data not shown, Fig. 8). Complex IV shows similar stability to complex II. The
Vma7p subunit is interesting, since it has been shown to be necessary
for assembly of both the V and the V complex,
and it was therefore proposed that Vma7p may be involved in the
attachment of V to V(33) . Vma7p may
serve as a multipurpose ``clamp'' to hold the V and V subcomplexes in stable conformations which may
then interact., although it has been shown
to be necessary for assembly of the complete complex in
yeast(12, 13) . Examination of other subunits is
currently under way. We suspect that Vma13p is unnecessary for the
formation of complex II, since deletion of its gene does not eliminate
the ability of the ATPase to assemble on the vacuolar
membrane(41) . Likewise Vma6p, which is a hydrophilic
peripheral protein tightly associated with the integral membrane
subunits and therefore regarded as a component of V, is
unlikely to be involved in the assembly of V(31) . complex. This
is the largest and most stable self-contained complex that can form
independent of the membrane. Other subunits, such as Vma5p, which are
soluble and cytoplasmic but not part of complex II, we would regard as
accessory factors necessary for assembly or activity of the complete
complex. We have also observed that our native gel system is extremely
sensitive to modifications caused by glucose deprivation, which has
recently been demonstrated to cause disassembly of the
ATPase(52) . We are initiating experiments to explore
modification of the V complex under stress conditions. We
are also pursuing experiments to demonstrate that these complexes are
not simply dead ends, but are bona fide intermediates in the
assembly pathway. We hope, ultimately, to establish the order of
interactions and regulation necessary for assembly and disassembly of
the V complex.
))
We thank Dr. Nathan Nelson for the gift of
vma strains and plasmids; Dr. Morris Manolson for the
gift of vph1, stv1, and
vph1stv1 strains; Dr. Fred Gimble for the VMA1vde plasmid; and Dr. Tom Stevens and Dr.
Laurie Graham for plasmid pLG20 and antisera to the HA epitope. We also
thank Dr. Sidney Scott and Kevin Morano for critical reading of the
manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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