From the
The vacuolar H
Vacuolar proton-translocating ATPases
(H
Studies of the assembly of the yeast
vacuolar H
The
wild-type yeast strain SF838-1D (genotype: MAT
In the experiment shown in ,
yeast cells were grown overnight, converted to spheroplasts, and
labeled with Tran
In the absence of the DSP
cross-linker, a similar collection of subunits, with the exception of
the 42-kDa subunit, could be immunoprecipitated from wild-type cells by
the 8B1 antibody. The 42-kDa subunit appeared to be lost during
immunoprecipitation without cross-linker even though it could be
isolated with the complex by other means(14) . Addition of
cross-linker allowed consistent immunoprecipitation of the 42-kDa
subunit with the wild-type complex and allowed more reliable
comparisons between different experiments. In the presence of DSP,
several proteins in addition to the 100-, 36-, and 17-kDa subunits of
the ATPase are co-precipitated consistently by the 10D7 antibody. These
included a band at 75 kDa and a faint, diffuse band at 19 kDa that were
co-precipitated previously in the absence of cross-linker(14) ,
as well as a band at 200 kDa and several at 40-60 kDa.
Precipitation of these bands was characteristic of the 10D7 antibody,
but the amount of protein in additional bands did not appear to change
under conditions for disassembly and reassembly as the 100-, 36-, and
17-kDa proteins did.
Vacuolar vesicles were prepared as described(20) , except
that spheroplasts were washed once with 1.2 M sorbitol, then
incubated 15 min at 30 °C in either YEPD containing 1.2 M sorbitol or YEP containing 1.2 M sorbitol. After the
incubation, the spheroplasts were pelleted by centrifugation, then
lysed immediately. ATPase activities of isolated vesicles were measured
at 37 °C using the coupled enzyme assay of Lotscher et
al.(21) , and a Lowry (22) assay was used to
quantitate protein. In order to assess the amount of ATPase activity
specific for V-type ATPases, vacuolar vesicles were preincubated with
100 nM concanamycin A (Wako Chemicals) for 20 min on ice, and
the difference between the ATPase activities of vesicles with and
without preincubation with inhibitor was taken as the vacuolar ATPase
activity. (Concanamycin A is a highly specific inhibitor of V-type
ATPases(23) .)
To determine whether the state of assembly of the yeast
vacuolar H
The
dissociation of the peripheral V
The presence of relatively
stable, assembled V
All of the labeled subunits are present in complexes reassembled
during the chase period, so new synthesis of subunits is probably not
necessary for reassembly. Nonetheless, it is still possible that other
proteins involved in disassembly or reassembly must be newly
synthesized. To address this possibility, we included 100 µg/ml
cycloheximide in the chase medium (Fig. 2, A and B, lanes 6-8) and examined the effects on the
immunoprecipitated complexes. (This level of cycloheximide was shown
previously to rapidly inhibit yeast protein synthesis(27) .)
Comparison of lanes3 and 6 in Fig. 2shows that cycloheximide does not appear to have a direct
effect on the assembled vacuolar H
In order to obtain a quantitative estimate of the extent
of disassembly and reassembly of the vacuolar H
Disassembly and reassembly of the yeast
vacuolar H
In Fig. 6, the
kinetics and extent of disassembly under several different conditions
are compared. Immunoprecipitation data were quantitated using a
phosphorimager, and the ratio of the 100- and 69-kDa subunits
immunoprecipitated by the 8B1 antibody is shown as a measure of
assembly and disassembly. The topcurve represents
spheroplasts chased in glucose, and these data demonstrate that a small
amount of assembly of the 100-kDa subunit with the 69-kDa subunit
appears to continue during the chase period. However, incubation of the
labeled spheroplasts in the absence of any carbon source or in the
presence of galactose (2%) or glycerol/ethanol (3%/2%) induced a rapid
dissociation of the 100-kDa subunit from the immune complexes
precipitated by the 8B1 antibody. The 17- and 36-kDa subunits were lost
at a similar rate under these conditions (data not shown). The
disassembly may have been slightly slower in galactose than in the
other carbon sources, but for each condition, almost all of the
disassembly was complete by the 20-min chase point. (The apparent
increase in the 100/69-kDa ratio at the 20-min time point in medium
lacking glucose was not seen in other experiments.) It is also notable
that the final extent of disassembly is similar under each of the
different conditions. Complexes immunoprecipitated with the 8B1
antibody after a 40-min chase period in media that cause disassembly (i.e. no carbon source, galactose, and glycerol/ethanol)
contain only 34 ± 2% as much 100-kDa subunit, 33 ± 6% as
much 36-kDa subunit, and 25 ± 6% as much 17-kDa subunit as those
immunoprecipitated after a chase period in 2% glucose.
The presence of the peripheral subunits in the supernatant
from cells grown overnight in raffinose could result from a reduction
in the amount of V
The results reported here have clear implications for the
structure of the yeast vacuolar H
Previous work has supported a model for
V-type ATPases in which the V
The most significant new structural insight from these
results is that the free V
Although there has been
extensive research into the cellular responses to changes in carbon
sources and the mechanisms for sensing glucose, a unified picture is
only beginning to emerge(30, 31, 32) . Growth in
glucose, the preferred carbon source for rapid fermentative growth,
results in transcriptional repression of many of the gene products
involved in utilization of other carbon sources(32) . Upon a
shift from glucose to a less-preferred carbon source, cells adapt by
reversing the process of glucose repression and in some cases actively
inducing genes required for metabolism of the available
sugar(32) . Both raffinose and glucose are ultimately used in
fermentation, but raffinose mediates weak glucose repression and
glucose repression is relieved during growth on galactose.
Glycerol/ethanol is a non-fermentable carbon source, and thus requires
a still different set of proteins for metabolism. Despite the variety
of eventual outcomes of shifting to these different carbon sources, the
immediate disassembly of the vacuolar H
The disassembly and reassembly
processes identified here could potentially provide a mechanism for
regulating acidification of the yeast vacuole or other acidic
organelles. If membrane attachment is required for ATPase activity in vivo, as it is in vitro, then removing the
catalytic subunit, along with the other peripheral subunits, from the
vacuolar membrane, could both reduce proton pumping and prevent
unproductive ATP hydrolysis. Recently, it has been shown that rapid
changes in transepithelial voltage seen at specific developmental
stages of tobacco hornworm larvae were caused by a specific loss of the
V
Yeast spheroplasts
were labeled with Tran
Yeast spheroplasts were labeled with Tran
I thank David Turner for the use of his microscope,
Jianzhong Liu for assistance with photography, and Christine Tachibana
for a critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase of the yeast Saccharomyces cerevisiae is composed of a complex of
peripheral subunits (the V
sector) attached to an integral
membrane complex (the V
sector). In the experiments
described here, attachment of the V
to the V
sector was assessed in wild-type cells under a variety of growth
conditions. Depriving the yeast cells of glucose, even for as little as
5 min, caused dissociation of approximately 70% of the assembled enzyme
complexes into separate V
and V
subcomplexes.
Restoration of glucose induced rapid and efficient reassembly of the
enzyme from the previously synthesized subcomplexes. Indirect
immunofluorescence microscopy and subcellular fractionation revealed
detachment of the peripheral subunits from the vacuolar membrane in the
absence of glucose, followed by reattachment in the presence of
glucose. Rapid dissociation of vacuolar H
-ATPases
could also be triggered by shifting cells into a variety of other
carbon sources, and reassembly could be generated by addition of
glucose. Disassembly and reassembly of vacuolar
H
-ATPases in vivo may be a means of
regulating organelle acidification in response to extracellular
conditions, or a mechanism for assembling alternate complexes of
vacuolar H
-ATPases in different intracellular
compartments.
-ATPases)
(
)are multisubunit
complexes, found in all eukaryotic cells, that acidify one or more
intracellular compartments(1) . Vacuolar
H
-ATPases from plants, animals, and fungi are very
similar. All consist of a V
sector, which is a complex of
peripheral membrane proteins containing the ATP-binding sites, and a
V
sector, comprising a complex of integral membrane
proteins containing the proton pore(2, 3, 4) .
The vacuolar H
-ATPases share sequence and structural
similarities with the F
F
-ATPases of
mitochondria, chloroplasts, and bacteria, and these similarities are
believed to reflect an evolutionary relationship(5) . However,
unlike the F
F
-ATPases, which can be separated
into a soluble F
complex that is capable of ATP
hydrolysis(6) , and a membrane-bound F
complex that
appears to function as a proton channel(7) , a variety of
biochemical studies indicate that the V
sector of the
vacuolar H
-ATPases does not retain its ATPase activity
when detached from the
membrane(8, 9, 10, 11, 12) , and
the V
complex is not an open proton
pore(12, 13) .
-ATPase have shown that mutants lacking one
subunit of the enzyme complex can still assemble the V
complex in the cytoplasm under conditions where the V
complex is not assembled, and the V
complex can be
assembled and transported to the vacuole in the absence of an assembled
V
complex(14, 15, 16, 17) .
Independent V
and V
complexes were also seen in
wild-type yeast cells, but it was not clear whether these complexes
were assembly intermediates, products of breakdown of the vacuolar
H
-ATPase, or artifacts of the biochemical
isolation(14) . This paper describes the reversible dissociation
of the wild-type V
complex from the V
complex in vivo. Both the disassembly and subsequent reassembly of the
complex occur rapidly and efficiently in response to changes in growth
conditions, suggesting that these processes could play a role in
regulation of vacuolar H
-ATPase activity or in
cellular distribution of the active enzyme.
Materials and Strains
Zymolyase 100T was
purchased from Seikagaku America, Inc. TranS-label was
purchased from ICN. Dithiobis(succinimidylpropionate) (DSP) was
obtained from Pierce.
C-Labeled molecular mass markers
(high range) were obtained from Life Technologies, Inc. All secondary
antibodies used for indirect immunofluorescence microscopy
(fluorescein-conjugated and unconjugated) were obtained from Organon
Teknika-Cappel. All other reagents were purchased from Sigma.
, ade6,
leu2-3, leu2-112, ura3-52, pep4-3, gal2)
was used in all experiments. Yeast media were prepared as described
previously(18, 19) .
Immunoprecipitations
Immunoprecipitations were
carried out under nondenaturing conditions as described(14) ,
with the following modifications. Cells were converted to spheroplasts
in supplemented minimal medium lacking methionine that contained 2%
glucose and 1.2 M sorbitol (SD-Met, 1.2 M sorbitol)
by incubation for 20 min with 0.1 unit of zymolyase/10 cells. For each immunoprecipitation, 0.5
10
spheroplasts were suspended in 150 µl of SD-Met, 1.2 M sorbitol, shaken 20 min at 30 °C, and then incubated for the
indicated labeling time with 50 µCi of Tran
S-label. At
the end of the incubation, unlabeled methionine and cysteine were added
to a final concentration of 0.33 mg/ml each, and the cells were
pelleted by centrifugation. For samples with a subsequent chase, the
pellets were resuspended in the appropriate medium to the same density
as for labeling and shaken at 30 °C for varied times. After the
labeling and chase times were completed, spheroplast pellets were
solubilized in phosphate-buffered saline (PBS; 137 mM NaCl,
2.6 mM KCl, 12 mM sodium phosphate, pH 7.2)
containing 1% (w/v) C
E
and a protease
inhibitor mixture (final concentrations: 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 2 µg/ml chymostatin, 5 µg/ml aprotinin). DSP was
added as 25 mM solution in dimethyl sulfoxide, to a final
concentration of 0.67 mM. After 60 min, an equal volume of
quenching buffer (50 mM Tris/HCl, pH 7.5, 10% glycerol, 1
mM EDTA) containing a 2-fold concentration of the protease
inhibitor mixture was added to the samples to quench the cross-linker,
and after another 15 min, samples were presorbed with Protein
A-Sepharose CL-4B, as described. Antibody incubations were carried out
as described(14) , except that 5 µl (12.5 µg) of
purified 8B1 antibody (9) and 495 µl of PBS containing 5
mg/ml bovine serum albumin were added instead of a cultured supernatant
containing this antibody. Immunoprecipitated proteins were washed,
solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography as described(14) . In some cases
immunoprecipitation results were quantitated by exposing the gels
overnight to the phosphor-containing screen and analyzing on a Fuji
BAS100 phosphorimager.
S-label as described above except that 2%
raffinose was used as the sole carbon source instead of 2% glucose.
Unlabeled methionine and cysteine were added to initiate the chase
period, but raffinose (to give a total concentration of 4%) and glucose
(to give a final concentration of 2%) were then added directly to the
labeling medium rather than spinning down the spheroplasts and
resuspending them in fresh medium.
Indirect Immunofluorescence Microscopy
Cells were
grown to mid-log phase in YEPD (1% yeast extract, 2% peptone, 2%
dextrose medium; Ref. 18), then diluted to approximately 1
10
cells/ml in YEPD or YEP (1% yeast extract, 2% peptone
medium without dextrose) and incubated at 30 °C. After the
indicated time of incubation, cells were fixed for 30 min in the same
medium, then pelleted and fixed overnight(20) . Monoclonal
antibodies 13D11 and 10D7 (15) were used at a 1:10 dilution and
undiluted, respectively, and the signal was amplified as
described(20) .
Fractionation of Spheroplast Lysates and Isolation of
Vacuolar Vesicles
Cells grown overnight at 30 °C to early to
mid-log phase in YEPD, YEP containing 2% raffinose, or YEP containing
3% glycerol, 2% ethanol were converted to spheroplasts and washed as
described above, except that all incubations were carried out in the
presence of the appropriate carbon source. Spheroplasts from
approximately 2.5 10
cells were resuspended in 1 ml
of the appropriate incubation medium containing 1.2 M sorbitol
and incubated for 20 min at 30 °C. In some samples the incubation
was continued for another 20 min in the presence of added 2% glucose to
assess reassembly; otherwise, the cells were pelleted after the initial
20-min incubation then resuspended in 1 ml of PBS containing the same
protease inhibitor mixture used for immunoprecipitations. DSP was added
to a final concentration of 0.6 mM, and the mixture was
incubated on ice for 60 min. The cross-linker was quenched by addition
of 1 ml of quenching buffer, followed by incubation for 20 min on ice.
After addition of another 3 ml of PBS containing the protease inhibitor
mixture, the spheroplast lysate was homogenized in a Dounce homogenizer
with 10 strokes of the A-pestle. The lysate was then centrifuged for 60
min at 35,000 rpm (147,000
g at r
) in an SW50.1 rotor. Supernatant and pellet
fractions were analyzed by Western blotting as described(14) .
-ATPase is altered in response to changes in
extracellular conditions, yeast spheroplasts were biosynthetically
labeled with Tran
S-label for 45 min and then shifted to
different media containing unlabeled methionine for a 15-min
``chase'' time prior to immunoprecipitation. Fig. 1A shows that after labeling for 45 min, all of the
previously identified subunits of the yeast vacuolar
H
-ATPase can be co-precipitated with monoclonal
antibody 8B1 against the 69-kDa catalytic subunit of the enzyme (lane1), and there is little change in the
distribution of subunits immunoprecipitated after a subsequent 15-min
chase period in the same medium (lane2). This
indicates that assembly of the yeast vacuolar H
-ATPase
is almost completed during the 45-min labeling time, and that the
assembled complexes are stable through the subsequent 15-min chase
period. Very similar complexes were obtained from cells chased in
medium buffered to pH 5 (lane5), buffered to pH 7.5 (lane3), or containing 50 mM CaCl
(lane4). These conditions were chosen because
yeast mutants lacking vacuolar H
-ATPase activity fail
to grow in medium buffered to pH 7.5 or containing 100 mM
CaCl
but are able to grow on medium buffered to pH 5,
suggesting that cellular requirements for the vacuolar
H
-ATPase differ under these different growth
conditions (19, 24, 25). However, these results indicate that the
different media do not grossly affect assembled vacuolar
H
-ATPase complexes. In contrast, assembly of the yeast
vacuolar H
-ATPase was significantly affected by
transferring the labeled cells to medium lacking glucose for 15 min (lane6). Complexes co-precipitated from cells chased
in medium without glucose showed greatly reduced levels of the 100-,
42-, 36-, and 17-kDa subunits. (Although there appears to be some
reduction in the level of the 32-kDa subunit in Fig. 1A,
this was not seen as consistently. The level of the 32-kDa subunit in
the experiments shown in Fig. 2A and 5A was
more typical.) The 60-, 32-, and 27-kDa proteins that remain associated
with the 69-kDa subunit after incubation in medium lacking glucose have
been implicated as members of the peripheral V
sector of
the vacuolar H
-ATPase, along with the 42-kDa subunit
(reviewed in Ref. 4). The 100- and 17-kDa subunits have been shown to
be integral membrane proteins(15, 16, 17) , and
the 36-kDa subunit appears to be closely associated with these subunits
even though it is not an integral membrane subunit (14, 26).
Figure 1:
Disassembly of the yeast vacuolar
H-ATPase. Yeast cells were converted to spheroplasts
and labeled with Tran[
S]-label for 45 min as
described under ``Experimental Procedures.'' Labeled
spheroplasts were then lysed immediately (lane1) or
chased for 15 min in SD-Met (lane2), YEPD buffered
to pH 7.5 (lane 3), YEPD containing 100 mM CaCl
(lane 4), YEPD buffered to pH 5 (lane 5), or YEP buffered to pH 5 (lane 6) before
lysis. Chase media contained 1.2 M sorbitol. Monoclonal
antibodies 8B1, against a V
subunit (A), and 10D7,
against a V
subunit (B), were used to isolate
complexes containing vacuolar H
-ATPase subunits, and
immunoprecipitated proteins were visualized by autoradiography.
Positions of
C-labeled molecular mass markers are
indicated on the left (from top: 200, 97, 68, 43, 29, 18.4,
and 14.3 kDa), and previously identified subunits of the yeast vacuolar
H
-ATPase are indicated by arrows on the right.
Figure 2:
Disassembly and reassembly of the vacuolar
H-ATPase do not require new protein synthesis. Yeast
cells were converted to spheroplasts and labeled with
Tran
S-label for 45 min as described under
``Experimental Procedures.'' Labeled spheroplasts were then
lysed immediately (lane1), or chased for 20 min in
YEPD buffered to pH 5 (lane2), 40 min in YEPD
buffered to pH 5 (lanes 3 and 6), 20 min in YEP
buffered to pH 5 (lanes4 and 7), or 20 min
in YEP buffered to pH 5, followed by an additional 20 min with 2%
glucose added (lanes5 and 8). In lanes
6-8, the chase medium contained 100 µg/ml cycloheximide.
All of the chase media contained 1.2 M sorbitol. Monoclonal
antibodies 8B1 (A) and 10D7 (B) were used, and
immunoprecipitated proteins were visualized by autoradiography.
Molecular mass markers and H
-ATPase subunits are
identified as in Fig. 1.
Fig. 1B shows the complexes immunoprecipitated from
the same biosynthetically labeled spheroplasts by the 10D7 monoclonal
antibody, which recognizes an epitope on the 100-kDa subunit that
appears to be accessible only when the V subunits are not
bound(15) . This antibody has been used to localize (15) and immunoprecipitate (14) V
complexes
under conditions where the peripheral V
complex cannot bind
to the membrane. The 10D7 antibody immunoprecipitates a complex
containing the 100-, 36-, and 17-kDa subunits, in addition to several
other proteins that have not been characterized, from wild-type cells
suggesting that the cells contain a pool of assembled V
complexes that are not associated with the V
subunits (Fig. 1B, lanes1-5). After the
chase period in no glucose medium (lane6), there is
an increase in the amount of 100-, 36-, and 17-kDa subunits
immunoprecipitated by the 10D7 antibody. The results in Fig. 1indicate that: 1) incubation in medium lacking glucose has
caused dissociation of the peripheral V
sector of the yeast
vacuolar H
-ATPase from the integral membrane V
complex, and 2) after dissociation, the subunits of each sector,
with the exception of the 42-kDa subunit, remain assembled.
sector would be expected
to reduce the ATPase activity of vacuolar membranes. To assess this
directly, vacuolar membrane vesicles were isolated from spheroplasts
incubated in the presence or absence of glucose immediately before
lysis. Vesicles prepared from spheroplasts incubated in medium
containing 2% glucose contained 3.9 units/mg of vacuolar ATPase
activity (where 1 unit = 1 µmol of ATP hydrolyzed/min and
vacuolar ATPase activity is taken as the ATPase activity inhibited by
100 nM concanamycin A). The spheroplasts incubated in the
absence of glucose yielded membranes containing only 1.5 units/mg
vacuolar ATPase activity. This suggests that incubation of spheroplasts
in the absence of glucose results in a loss of 61.5% of the vacuolar
ATPase activity from the membranes.
and V
sectors in the cells
incubated in medium lacking glucose (Fig. 1) suggested that the
disassembled complexes might remain competent for reassembly. To
investigate whether disassembly of the vacuolar
H
-ATPase is a reversible process, we incubated labeled
spheroplasts first with medium lacking glucose for 20 min and then
added back glucose for 20 min and examined the immunoprecipitated
complexes. As shown in Fig. 2(A and B, lane4), the assembled vacuolar
H
-ATPase complexes were extensively dissociated after
20 min in medium without glucose. However, if glucose was then added
back to the cells for 20 min (Fig. 2, A and B, lane5), the complexes obtained were almost
indistinguishable from those immunoprecipitated from cells incubated
with glucose throughout the chase period (Fig. 2, A and B, lanes2 and 3). Neither
disassembly nor reassembly required incubation of the spheroplasts at
pH 5. Disassembly occurred in unbuffered medium lacking glucose, and
reassembly occurred when glucose was added back to unbuffered medium.
-ATPase complexes,
and lanes7 and 8 demonstrate that both
disassembly and reassembly can occur in the absence of new protein
synthesis.
-ATPase
in the presence and absence of glucose, the distribution of the 100-,
36-, and 17-kDa subunits between the complexes immunoprecipitated by
the 8B1 antibody (assembled V
V
complexes) and
the complexes immunoprecipitated by the 10D7 antibody (V
subunits only) was measured using a phosphorimager. The results
are shown in . In cells chased for 20 min in the presence
of 2% glucose, 75-80% of the total quantity of these V
subunits precipitated by both antibodies was co-precipitated with
the V
subunits, suggesting that most of the V
subunits are found as part of fully assembled ATPase complexes.
However, when the cells were subjected to a 20-min chase in the absence
of glucose, only 22-33% of the subunits were immunoprecipitated
by the 8B1 antibody and and 67-78% were precipitated by the 10D7
antibody, indicating that the three subunits are found predominantly in
V
complexes not containing V
subunits.
Readdition of glucose restored the distribution seen before cells were
deprived of glucose.
-ATPase were also visualized by indirect
immunofluorescence microscopy, and the results are shown in Fig. 3and Fig. 4. Yeast vacuoles appear as depressions when
cells are visualized under Nomarski optics ( Fig. 3and Fig. 4, A, C, and E; Ref. 20).
Indirect immunofluorescence microscopy with the 13D11 antibody, which
recognizes the 60-kDa peripheral subunit of the enzyme, shows
colocalization of the V
sector with the vacuolar membrane
in yeast cells incubated in the presence of 2% glucose (Fig. 3B). After the cells have been deprived of glucose
for 20 min, the same antibody shows diffuse staining of the cytoplasm,
in addition to some staining of the vacuolar membrane (Fig. 3D). Restoration of glucose to the cells results
in loss of cytoplasmic staining and restoration of bright vacuolar
membrane staining (Fig. 3F). These results are entirely
consistent with the results from immunoprecipitations described in Fig. 1and Fig. 2. Further evidence of disassembly and
reassembly of the vacuolar H
-ATPase complex was
obtained from indirect immunofluorescence microscopy using the 10D7
monoclonal antibody (Fig. 4, B, D, and F). This antibody was previously shown to give little or no
staining of wild-type cells grown in the presence of 2%
glucose(15) , and in this experiment, there appears to be very
little staining of cells incubated with glucose throughout (Fig. 4B) or cells deprived of glucose that subsequently
had the glucose restored (Fig. 4F). However, the
antibody did recognize the vacuolar membrane in cells deprived of
glucose (Fig. 4D), suggesting that the epitope on the
100-kDa subunit is available in these cells. Previous experiments on
mutants that fail to assemble the peripheral subunits onto the vacuolar
membrane gave similar results(15) , indicating that exposure of
the 10D7 antigen reflects disassembly of the peripheral membrane
subunits from the integral membrane complex. The microscopy results in Fig. 3and 4 also eliminate any possibility that disassembly and
reassembly occurs only in yeast spheroplasts, since whole cells were
incubated in the presence and absence of glucose and fixed before they
were converted to spheroplasts in these experiments.
Figure 3:
Indirect immunofluorescence microscopy of
the 60-kDa subunit in yeast cells treated under varied conditions.
Cells were incubated in YEPD for 40 min (A and B),
YEP for 20 min (C and D), or YEP for 20 min, followed
by an additional 20 min with 2% glucose present (E and F) as described under ``Experimental Procedures.''
Cells were fixed and then stained with monoclonal antibody 13D11 to
visualize the V sector. Identical fields were viewed under
Nomarski optics (A, C, and E) or fluorescein
fluorescence optics (B, D, and F).
Figure 4:
Indirect immunofluorescence microscopy of
the 100-kDa subunit in yeast cells treated under varied conditions.
Cells were prepared as in Fig. 3, but were stained with monoclonal
antibody 10D7 to visualize the V sector when it is not
complexed with the V
subunits. Identical fields were viewed
under Nomarski optics (A, C, and E) or
fluorescein fluorescence optics (B, D, and F).
The reversible
dissociation of the yeast vacuolar H-ATPase in medium
lacking any carbon source suggested that there might also be rapid
changes in the ATPase structure when cells were shifted between
different carbon sources. Yeast cells grown and biosynthetically
labeled for 50 min in 2% glucose were shifted to three different carbon
sources for a 20-min chase period, and the results are shown in Fig. 5. Incubation of the labeled complexes in 2% raffinose (lane4), 2% galactose (lane6),
and 3% glycerol, 2% ethanol (lane8) resulted in
disassembly of the vacuolar H
-ATPase. Under all of
these conditions, the 100-, 42-, 36-, and 17-kDa subunits appear to be
lost from the complexes immunoprecipitated by the 8B1 antibody, and
increased amounts of the 100-, 36-, and 17-kDa subunits appear in the
V
sectors precipitated by the 10D7 antibody. Readdition of
glucose (to a final concentration of 2%) for 20 min could once again
stimulate reassembly of the vacuolar H
-ATPase from
previously synthesized V
and V
sectors in all
of these carbon sources (Fig. 5, lanes5, 7, and 9).
Figure 5:
Disassembly and reassembly of the yeast
vacuolar H-ATPase can occur in a variety of carbon
sources. Yeast cells were converted to spheroplasts and labeled with
Tran[
S]-label for 50 min in medium containing 2%
glucose. Labeled spheroplasts were lysed immediately (lane1) or chased for 20 min in YEPD (lanes2 and 3), YEP containing 2% raffinose (lanes4 and 5), YEP containing 2% galactose (lanes6 and 7), or YEP containing 3% glycerol/2% ethanol (lanes8 and 9). All chase media contained
1.2 M sorbitol. Glucose (final concentration 2%) was added to
the samples shown in lanes3, 5, 7,
and 9 after the initial 20-min incubation, and the incubation
was continued for another 20 min. Monoclonal antibodies 8B1 (A) and 10D7 (B) were used in the
immunoprecipitations, and precipitated proteins were visualized by
autoradiography. Molecular mass markers and H
-ATPase
subunits are indicated as in Fig. 1.
The kinetics of disassembly of the
vacuolar H-ATPase were examined by varying the chase
time in medium lacking glucose. At the shortest chase times examined (2
min at 30 °C without glucose), the immunoprecipitated complexes
were almost identical to those seen after 20 min in medium without
glucose (data not shown). Similarly, the kinetics of reassembly were
examined by immunoprecipitation of complexes at various times after
restoration of glucose to labeled spheroplasts that had been deprived
of glucose for 20 min. Reassembly also appeared to be complete after a
2-min incubation in glucose (data not shown). Although processing of
the spheroplasts may have added as much as 2 min to the time between
removal or addition of glucose and solubilization, both disassembly
from glucose deprivation and reassembly with glucose readdition are
essentially complete in less than 5 min.
Figure 6:
Kinetics of vacuolar
H-ATPase disassembly in different carbon sources.
Spheroplasts were labeled for 45 min in medium containing 2% glucose (t = 0 for all samples), then shifted to YEPD (
),
YEP (
), YEP containing 2% galactose (
), or YEP containing
3% glycerol/2% ethanol (▾), all containing 1.2 M sorbitol, for an additional chase period at 30 °C. Aliquots
were withdrawn after 5, 20, and 40 min of chase, and fully or partially
assembled complexes of the vacuolar H
-ATPase were
immunoprecipitated with the 8B1 antibody as described under
``Experimental Procedures.'' The amount of radioactivity
present in the 100-kDa and 69-kDa bands after SDS-polyacrylamide gel
electrophoresis was quantitated using a phosphorimager, and the ratio
of the two bands is expressed as a function of chase
time.
The
microscopy results shown in Fig. 3and Fig. 4indicate that
disassembly and reassembly in the absence of glucose are not restricted
to the vacuolar H-ATPase complexes synthesized within
the 45-min labeling time of the immunoprecipitation experiments. This
conclusion was further supported by Western blot analysis of the
distribution of the 69- and 60-kDa subunits between supernatant and
pellet fractions obtained from spheroplasts that had been incubated
under varied conditions, lysed, and cross-linked with DSP as described
under ``Experimental Procedures'' (Fig. 7). The
peripheral subunits were found predominantly in the pellets from
spheroplasts incubated in glucose before lysis (samples1 and 3), even if the spheroplasts had been subjected to a
period of glucose deprivation prior to glucose readdition (sample3). However, if the spheroplasts were incubated in medium
lacking glucose immediately before lysis (sample2),
the peripheral subunits were found predominantly in the supernatant
fraction.
Figure 7:
Membrane
attachment of peripheral subunits of the yeast vacuolar
H-ATPase in cells incubated under varied conditions.
Cells were grown overnight in YEPD (samples 1-3), YEP
containing 2% raffinose (samples4 and 5),
and YEP containing 3% glycerol/2% ethanol (sample6),
then converted to spheroplasts. Spheroplasts were incubated for 20 min
in YEPD (sample1), 20 min in YEP (sample2), 20 min in YEP followed by 20 min with glucose added
to 2% (sample3), 20 min in YEP containing 2%
raffinose (sample4), 20 min in YEP containing 2%
raffinose followed by 20 min with 2% glucose added (sample5), and 20 min in YEP containing 3% glycerol/2% ethanol.
All incubations of spheroplasts contained 1.2 M sorbitol.
Spheroplasts were lysed, cross-linked, and fractionated into
supernatant (S) and pellet (P) fractions as described
under ``Experimental Procedures.'' The fractions were
solubilized, subjected to SDS-polyacrylamide gel electrophoresis, and
analyzed by Western blotting with monoclonal antibodies 8B1 (against
the 69-kDa subunit) and 13D11 (against the 60-kDa subunit.
Approximately 2.5
10
cells from each treatment were
fractionated into supernatant and pellet fractions, and 1/50 of the
total volume obtained from each fraction was loaded to obtain the blot
shown.
All of the experiments described so far examine changes in
assembly of vacuolar H-ATPase complexes that had
initially been allowed to assemble in glucose-containing medium.
However, the disassembly of the ATPase in a variety of different carbon
sources suggests that assembly of the enzyme during growth on these
carbon sources could also be affected. To address this, yeast cells
were grown overnight in media containing 2% raffinose or 3% glycerol,
2% ethanol as their sole carbon source, and the distribution of the 69-
and 60-kDa subunits between membrane (pellet) and supernatant fractions
was determined by Western blot analysis (Fig. 7). In cells grown
overnight in 2% raffinose (sample 4), the 69- and 60-kDa
peripheral subunits appear to be predominantly in the supernatant
fraction, indicating that they are either not assembled or not retained
on the membrane efficiently. In cells grown overnight in
glycerol/ethanol, approximately 50% of the peripheral subunits are
found in the supernatant fraction (sample 6), suggesting that
assembly or retention of the subunits on the membrane is less efficient
than in glucose-grown cells but more efficient than in raffinose-grown
cells.
sector relative to the V
subunits so that there are no longer enough membrane-binding
sites for the V
subunits. However, when spheroplasts
derived from cells grown in raffinose are treated for 20 min in
glucose, the 69- and 60-kDa subunits are transferred almost
quantitatively to the pellet fraction (Fig. 7, sample5). This suggests that there are existing V
subunits or complexes present in the raffinose-grown cells that
become available to bind V
subunits after a relatively
brief incubation in glucose. This possibility was further explored by
biosynthetically labeling vacuolar H
-ATPase complexes
during growth on raffinose, and then examining the assembly of the
100-kDa subunit into V
V
complexes during a
chase period in raffinose or glucose. Spheroplasts from cells grown
overnight in 2% raffinose were labeled for 45 min with
[
S]methionine in the presence of 2% raffinose,
then an excess of unlabeled methionine was added along with additional
raffinose or glucose. shows the percentage of the total
100-, 36-, and 17-kDa subunits immunoprecipitated by the 8B1 antibody
as part of V
V
complexes, as quantitated by
phosphorimager analysis. There is very little change in the percentage
of the V
subunits coprecipitated by the 69-kDa subunit (8B1
antibody) during a 15-min chase in raffinose, suggesting that only
about one-third of these subunits can assemble into complete
V
V
complexes in raffinose, while the rest are
in free V
complexes. In contrast, addition of glucose for
15 min causes a significant redistribution of these subunits from free
V
complexes to fully assembled V
V
complexes. Although the 54-60% assembly of the V
subunits into V
V
complexes is still lower
than the 75-80% assembly shown in after incubation
in glucose, it is clear that the free V
complexes present
in raffinose-grown cells must be able to be incorporated into fully
assembled complexes when the proper signal is received.
-ATPase and also
suggest intriguing possibilities for enzyme regulation or
redistribution of vacuolar H
-ATPases in response to
extracellular conditions.
and V
sectors may
be structurally independent but are functionally
interdependent(8, 9, 10, 11, 12, 13, 14) .
Independent V
and V
complexes can clearly form
in mutants lacking one subunit(14) , and a number of
investigators have reported the presence of free V
and
V
sectors in wild-type yeast cells as well as in other cell
types(13, 14, 17, 28) . One possible
explanation for these results is that fully assembled complexes are
disassembled during biochemical purification. Although it is difficult
to completely eliminate this possibility, the data presented here
suggest that dissociation after lysis cannot account for all of the
free V
and V
complexes. Clear differences in
subunit distribution with different treatments are seen by a number of
different methods, including immunoprecipitation under nondenaturing
conditions, subcellular fractionation, and immunofluorescence
microscopy, and in some cases, these differences have been supported by
demonstrating functional differences in ATPase activity of isolated
vacuoles. In order to minimize dissociation of the complex during
purification, a cross-linking reagent was also included in both the
immunoprecipitation and cell fractionation experiments. In the presence
of the cross-linker, both the subunit composition of the
immunoprecipitated complexes and the distribution of the V
subunits between soluble and membrane fractions resemble those
obtained previously in the absence of cross-linker(14) , with
the exception of the 42-kDa subunit, which could only be
co-precipitated in the presence of cross-linker. However, the amount of
the V
subunits coprecipitated with the V
subunits and the distribution of subunits in experiments like
that shown in Fig. 7were found to be more consistent between
different experiments when the cross-linker was included. In the
absence of cross-linker, there were some experiments when very little
of the V
subunits were coprecipitated by the 8B1 antibody
and some experiments where almost all of the peripheral subunits were
found in the supernatants from cell lysates, regardless of whether the
cells had been incubated in glucose. This lack of consistency may well
have been caused by variable loss of the 42-kDa subunit in the absence
of cross-linking, because experiments on the clathrin-coated vesicle
ATPase have indicated that although V
V
complexes can form in the absence of the comparable 40-kDa
subunit, these complexes are unstable(29) . By approaching the
distribution of the V
and V
subunits with a
number of different, independent methods and limiting the possibility
for dissociation of complexes after lysis, the data presented here
provide strong support for indications that the V
and
V
sectors of the V-type ATPases can exist separately in
intact cells.
and V
subcomplexes
may, in fact, be in a dynamic equilibrium with fully assembled
complexes. Under conditions that promote ATPase disassembly, the 69-,
60-, 32-, and 27-kDa V
subunits are removed as a complex to
the cytosol, while the 100-, 36-, and 17-kDa V
subunits
remain assembled in the membrane. The 42-kDa subunit appears to be lost
from both sectors, in accord with previous genetic (14) and
biochemical data (29). More remarkably, yeast cells can reassemble
intact vacuolar H
-ATPase complexes using previously
synthesized subunits in response to changes in extracellular
conditions. Therefore, following disassembly in the absence of glucose,
the integral V
sector, the peripheral V
sector,
and the 42-kDa subunit must all remain competent for assembly. Both the
rapid kinetics of diassembly and reassembly and the occurrence of both
processes in the presence of cycloheximide indicate that any enzymatic
activity necessary for signaling dissociation and reassociation of the
complex must be contained within the assembled vacuolar
H
-ATPase itself or in other proteins synthesized
during the initial 45-50-min labeling time. The free V
and V
subcomplexes described in yeast cells (14, 17) and bovine clathrin-coated vesicles (13, 28) may be intermediates in an ongoing process of
assembly and disassembly of the collection of V-ATPases in these cells.
In a more dynamic structural picture of the V-type ATPases, it becomes
extremely important to understand the features holding the V
and V
together and how these structural features
might respond to extracellular signals.
-ATPase seen
upon a shift to any of these carbon sources is very similar to that
seen in the absence of any carbon source. Readdition of glucose to each
of these carbon sources elicited a similar, rapid reassociation of the
V
and V
sectors, as well. A large number of
transcriptional and post-translational changes also occur when cells
are shifted from respiratory or gluconeogenic growth to rapid
fermentative growth(30, 32) . The changes in assembly of
the vacuolar H
-ATPase when cells are shifted from
glucose are rapid and can occur in the presence of cycloheximide,
suggesting a post-translational mechanism. However, cells grown
overnight in ethanol/glycerol or raffinose also showed reduced levels
of ATPase assembly, suggesting both long term and rapid effects of
carbon source on ATPase assembly.
subunits from the insect gut plasma membrane V-ATPase,
which resulted in inactivation of ATPase activity and proton
pumping(33) . It is entirely possible that the same structural
changes that induce disassembly and reassembly in the yeast system also
cause the dissociation of the insect vacuolar
H
-ATPase, and it is intriguing to speculate whether
the same intracellular signal could operate in both systems. It is also
interesting that the yeast plasma membrane H
-ATPase is
activated post-translationally by addition of glucose to cells in
gluconeogenic or respiratory growth and by intracellular acidification
(one consequence of growth in a rapidly fermenting sugar such as
glucose)(30, 34) , and that activation is paralleled by
changes in phosphorylation of the enzyme(35) . Activation of the
plasma membrane H
-ATPase when growth in glucose is
initiated has been invoked as a mechanism of regulating cytoplasmic
pH(34) , and an increased number of assembled vacuolar
H
-ATPases induced by addition of glucose could also
assist in raising cytoplasmic pH. In addition, if cells contained
excess V
complexes, or multiple forms of the V
complex, as results in yeast and in other systems have
indicated(13, 36, 37, 38) , the
reassembly process could potentially redistribute the V
complexes to different organelles or change the distribution of
the catalytic subunits among biochemically distinct complexes.
Characterization of the molecular triggers of disassembly and
reassembly should provide new insights into the structural interactions
within the vacuolar H
-ATPase as well as the mechanisms
for regulation of organelle acidification.
Table: Percentages of 100-, 36-, and 17-kDa subunits
immunoprecipitated as part of VV
versus V
complexes following different treatments
S-label for 50 min and then
incubated for an additional chase period in the presence or absence of
glucose, as indicated. Assembled and partially assembled complexes of
the vacuolar H
-ATPase were immunoprecipitated using
the 8B1 and 10D7 antibodies and analyzed by SDS-polyacrylamide gel
electrophoresis as described under ``Experimental
Procedures.'' The total amount of radioactivity in each of the
100-, 36-, and 17-kDa subunit bands precipitated by the two antibodies
was quantitated using a PhosphorImager. The percentage of each subunit
co-precipitated as part of fully assembled V
V
complexes was calculated by comparing the quantity
immunoprecipitated by the 8B1 antibody to the total immunoprecipitated
by both antibodies. The percentage immunoprecipitated by the 10D7
antibody (not shown) represents the portion of the subunits present in
free V
complexes not assembled with V
subunits.
Table: Glucose induces assembly of
VV
complexes in cells grown in raffinose
S-label in
medium containing 2% raffinose for 45 min and then incubated for a
chase period of 5 or 15 min after addition 2% raffinose or 2% glucose.
Assembled and partially assembled complexes of the vacuolar
H
-ATPase were immunoprecipitated using the 8B1 and
10D7 antibodies and analyzed by SDS-polyacrylamide gel electrophoresis
as described under ``Experimental Procedures.'' The
percentage of each subunit co-precipitated by the 8B1 antibody as part
of fully assembled V
V
complexes was calculated
as described in Table I.
-ATPase, proton-translocating ATPase; YEPD, yeast
extract-peptone-2% dextrose medium; YEP, 1% yeast extract-2% peptone
medium without dextrose; SD-Met, supplemented minimal medium lacking
methionine; DSP, dithiobis(succinimidylpropionate); PBS,
phosphate-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.