Microtubules Are Involved in Glucose-dependent
Dissociation of the Yeast Vacuolar [H+]-ATPase in
Vivo*
Ting
Xu and
Michael
Forgac
From the Department of Physiology, Tufts University School of
Medicine, Boston, Massachusetts 02111
Received for publication, January 23, 2001, and in revised form, April 25, 2001
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ABSTRACT |
The vacuolar [H+]-ATPases
(V-ATPases) are composed of a peripheral V1 domain and a
membrane-embedded V0 domain. Reversible dissociation of the
V1 and V0 domains has been observed in both yeast and insects and has been suggested to represent a general regulatory mechanism for controlling V-ATPase activity in
vivo. In yeast, dissociation of the V-ATPase is triggered by
glucose depletion, but the signaling pathways that connect V-ATPase
dissociation and glucose metabolism have not been identified. We have
found that nocodazole, an agent that disrupts microtubules, partially blocked dissociation of the V-ATPase in response to glucose depletion in yeast. By contrast, latrunculin, an agent that disrupts actin filaments, had no effect on glucose-dependent dissociation
of the V-ATPase complex. Neither nocodazole nor latrunculin blocked reassembly of the V-ATPase upon re-addition of glucose to the medium.
The effect of nocodazole appears to be specifically through disruption
of microtubules since glucose-dependent dissociation of the
V-ATPase was not blocked by nocodazole in yeast strains bearing a
mutation in tubulin that renders it resistant to nocodazole. Because
nocodazole has been shown to arrest cells in the G2 phase of the cell cycle, it was of interest to determine whether nocodazole exerted its effect on dissociation of the V-ATPase through cell cycle
arrest. Glucose-dependent dissociation of the V-ATPase was examined in four yeast strains bearing temperature-sensitive mutations that arrest cells in different stages of the cell cycle. Because dissociation of the V-ATPase occurred normally at both the permissive and restrictive temperatures in these mutants, the results suggest that
in vivo dissociation is not dependent upon cell cycle phase.
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INTRODUCTION |
Vacuolar [H+]-ATPases
(V-ATPases)1 are
ATP-dependent proton pumps that are located in both
intracellular compartments and the plasma membrane (1-11). They
function to couple the energy of ATP hydrolysis to the active transport
of protons from the cytoplasm of the cell to either the lumen of
intracellular organelles or the extracellular environment.
Acidification of intracellular compartments is important for a variety
of basic cellular processes, including membrane traffic, zymogen
activation, protein breakdown, and the coupled transport of small
molecules such as neurotransmitters (1-8). Proton transport across the
plasma membrane has been shown to function in pH homeostasis (9), renal
acidification (10), and bone resorption (11).
The V-ATPases are multisubunit complexes composed of at least 13 subunits organized into two structural domains (1-8). The peripheral
V1 domain contains eight subunits (subunits A-H) with molecular mass of 70-14 kDa and is responsible for ATP hydrolysis. The
integral V0 domain is composed of five subunits (subunits a, c, c', c", and d) with molecular mass of 100-17 kDa and is responsible for translocation of protons across the membrane. The
V-ATPases thus resemble the F-ATPases (or ATP synthases), which
function in ATP synthesis (12-17). Unlike the F1 and
F0 domains, however, the separate V1 and
V0 domains are not capable of ATP hydrolysis or passive
proton translocation under normal physiological conditions (18).
Because of the variety of functions served by V-ATPases in the cell, it
is likely that the activity of V-ATPases is tightly controlled. Several
mechanisms have been proposed for controlling V-ATPase activity
in vivo, including reversible disulfide bond formation
between conserved cysteine residues at the catalytic site (19, 20),
changes in the tightness of coupling between proton transport and ATP
hydrolysis (21, 22), and changes in the density of V-ATPases through
selective targeting (10, 23). Among the regulatory mechanisms for which
there is the most compelling evidence is reversible dissociation of the
V-ATPase complex. Kane (24) has shown that in yeast, the V-ATPase
undergoes dissociation into its component V1 and
V0 domains in response to removal of glucose from the
medium. This dissociation occurs rapidly, does not require new protein
synthesis, and is rapidly reversed upon re-addition of glucose to the
medium (24). Dissociation of the V-ATPase has also been shown to occur
in insects during molting (25) and has been suggested to occur in
mammalian cells based upon the existence of pools of free
V1 and V0 domains (26, 27). Dissociation may
thus represent a general mechanism for regulating V-ATPase activity.
Several recent studies have suggested an important link between
V-ATPases and the cytoskeleton. A temperature-sensitive mutation in
VMA4 was reported to cause a defect in actin distribution
and bud morphology (28). In renal cells, the V-ATPase has been shown to
interact with the PDZ protein NHE-RF, which in turn is able to bind
actin-associated proteins such as ezrin (29). Finally, direct binding
of V-ATPase to actin filaments has been demonstrated in osteoclasts
(30). These findings have led us to investigate the possible role of
interaction between the V-ATPase and cytoskeletal elements in
glucose-dependent dissociation of the V-ATPase in yeast.
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EXPERIMENTAL PROCEDURES |
Materials and Yeast Strains--
Leupeptin, aprotinin, and
pepstatin were obtained from Roche Molecular Biochemicals. Zymolyase
100T was from Seikagaku America, Inc. Yeast extract, dextrose, peptone,
and yeast nitrogen base were from Difco. Molecular biological reagents
were from New England Biolabs Inc., Promega, and Life Technologies,
Inc. Monoclonal antibodies 8B1-F3 against subunit A and 10D7 against
subunit a, rhodamine-conjugated phalloidin, and the
actin-depolymerizing agent latrunculin were obtained from Molecular
Probes, Inc. Rabbit antiserum against yeast
-tubulin was a kind gift
of Dr. Frank Solomon (Department of Biology, Massachusetts Institute of
Technology). Cy2-labeled goat anti-rabbit antibody was obtained from
Jackson ImmunoResearch Laboratories, Inc. Nocodazole and most other
chemicals were from Sigma. Saccharomyces cerevisiae strains
used in this study and their genotypes are listed in Table
I.
Drug Treatment of Cells in Culture--
Yeast strains were grown
in YEPD medium (yeast extract/peptone/dextrose) to mid-log phase and
converted to spheroplasts. Spheroplasts were resuspended in medium with
1.2 M sorbitol. Nocodazole was added from a 5 mg/ml stock
solution in Me2SO to a final concentration to 12.5 µg/ml
(40 µM). Cells were incubated in the absence or presence
of nocodazole for 2.5 h with shaking at 30 °C. Latrunculin was
added from a 10 mM stock solution in Me2SO to a
final concentration of 0.2 mM. Cells were incubated in the
absence or presence of latrunculin for 15 min with shaking at 30 °C.
Control cells were treated with an equal amount of
Me2SO.
In Vivo Dissociation and Reassembly of the V-ATPase in Response
to Glucose Depletion and Re-addition--
Dissociation and reassembly
of the yeast V-ATPase were induced by glucose depletion or re-addition
to the medium (24) and was detected by Western blotting as previously
described (33). Briefly, yeast cells were grown overnight to mid-log
phase, and then cells were converted to spheroplasts by treatment with
zymolyase (24). For measurement of the effects of inhibitors on
dissociation, spheroplasts were incubated in the absence or presence of
inhibitors as described above and then resuspended in YEPD or YEP
(yeast extract/peptone) medium containing 1.2 M sorbitol
and the same concentration of inhibitors, followed by incubation for 40 min at 30 °C. For measurement of the effects of inhibitors on
reassembly, spheroplasts were first incubated in YEP medium containing
sorbitol for 40 min at 30 °C to induce dissociation, then incubated
in the absence or presence of inhibitors as described above, and finally resuspended in YEPD or YEP medium containing sorbitol and the
indicated inhibitors for 40 min at 30 °C. Spheroplasts were then
solubilized with 1% C12E9 (polyoxyethylene
9-lauryl ether) in the presence of 1 mM
dithiobis(succinimidyl propionate) (24), and complexes containing
subunit A (located in the V1 domain) were
immunoprecipitated using monoclonal antibody 8B1-F3 (34) and protein
A-Sepharose. The samples were separated by SDS-PAGE on 10% acrylamide
gels and transferred to nitrocellulose, and Western blotting was
performed using both anti-subunit A antibody 8B1-F3 and monoclonal
antibody 10D7 directed against the 100-kDa subunit a of the
V0 domain (35). Western blots were developed using a
secondary antibody conjugated to horseradish peroxidase and visualized
by an enhanced chemiluminescence detection system (ECL, Kirkegaard & Perry Laboratories, Inc.). For temperature-sensitive cdc mutants, yeast strains were grown overnight to
mid-log phase at 25 °C and converted to spheroplasts as described
above. Spheroplasts were then incubated at 25 or 37 °C for 4 h.
Dissociation of the V-ATPase in response to glucose depletion was
measured at both 25 and 37 °C as described above.
Fluorescence Microscopy--
Actin filaments were visualized in
fixed cells using rhodamine-conjugated phalloidin as previously
described (36). Briefly, cells were fixed for 1 h in 3.7%
formaldehyde, and the fixed cells were washed and incubated in
phosphate-buffered saline containing 6 µM
rhodamine-conjugated phalloidin for 1 h in the dark. Cells were
then washed extensively and resuspended in mounting solution (90%
glycerol and 0.1 mg/ml p-phenylenediamine). Microtubules were visualized by immunofluorescence as previously described (37).
Cells were fixed by incubation in 3.7% formaldehyde for 1 h,
washed, and permeabilized by incubation with zymolyase and Glusulase.
The permeabilized cells were attached to polylysine-coated slides, and
the microtubules were visualized by incubation with a rabbit polyclonal
antibody against
-tubulin followed by a Cy2-conjugated goat
anti-rabbit secondary antibody. Immunofluorescence was observed using a
Axiovert 10 fluorescence microscope (Zeiss) equipped with a rhodamine
filter for actin staining and a fluorescein isothiocyanate filter for
microtubule staining. In both cases, a 40× objective was used, and the
images were recorded using a CCD camera.
Other Procedures--
Vacuolar membrane vesicles were isolated
as previously described (38). ATPase activity was measured using a
continuous spectrophotometric assay, and proton transport was measured
using ATP-dependent quenching of
9-amino-6-chloro-2-methoxy-acridine fluorescence quenching, both
as previously described (39). Activities were measured in both the
absence and presence of the specific V-ATPase inhibitor concanamycin A
(1 µM).
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RESULTS |
Recent studies have suggested that there may exist interactions
between the V-ATPase and cytoskeletal elements (28-30). Because dissociation of the V-ATPase has been proposed to represent an important mechanism of regulating V-ATPase activity in vivo
(24, 25), we wished to determine whether interactions with the
cytoskeleton might play a role in dissociation of the V-ATPase complex.
Dissociation of the V-ATPase in yeast was induced by incubation of
spheroplasts in glucose-free medium (24) and was detected by
solubilization of the V-ATPase with detergent and immunoprecipitation
with an antibody directed against subunit A of the V1
domain (34). Western blotting was then performed using antibodies
against both subunit A and the 100-kDa subunit a of the V0
domain (35). Dissociation appeared as a reduction in the amount of the
100-kDa subunit a immunoprecipitated with the antibody directed against
subunit A, as previously described (33).
As shown in Fig. 1, preincubation of
spheroplasts with the actin filament-disrupting agent latrunculin (40)
had no effect on dissociation of the V-ATPase in response to glucose
depletion. By contrast, preincubation with the microtubule-disrupting
agent nocodazole (41) dramatically reduced the amount of
glucose-dependent dissociation observed. To determine the
effects of these agents on in vivo reassembly of the
V-ATPase, spheroplasts were first incubated in glucose-free medium to
induce dissociation; incubation was continued in the absence or
presence of the inhibitors; and finally, glucose was added back to the
medium to induce reassociation of V1 and V0
domains. The assembly status of the V-ATPase was assessed as described
above. As can be seen from the data in Fig. 2, neither latrunculin nor nocodazole
blocked reassembly of the V-ATPase upon re-addition of glucose. These
results suggest that in vivo dissociation, but not
reassembly, of the V-ATPase is dependent upon the presence of intact
microtubules, but that neither process requires intact actin
filaments.

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Fig. 1.
Effect of nocodazole and latrunculin on
glucose-dependent dissociation of the yeast V-ATPase.
Yeast strain YPH499 was grown to mid-log phase; cells were converted to
spheroplasts; and the spheroplasts were incubated in the absence or
presence of 40 µM nocodazole for 2.5 h or of 0.2 mM latrunculin for 15 min at 30 °C. Control spheroplasts
were incubated with 1% Me2SO for 2.5 h at 30 °C.
Spheroplasts were then incubated for 40 min at 30 °C in YEP medium
with or without glucose and in the continued presence or absence of the
corresponding inhibitor. The spheroplasts were solubilized with 1%
C12E9 in the presence of 1 mM
dithiobis(succinimidyl propionate), and the V-ATPase was
immunoprecipitated using monoclonal antibody 8B1-F3 against subunit A
of the V1 domain. The proteins were then separated by
SDS-PAGE on 10% acrylamide gels, and Western blotting was performed
using both 8B1-F3 and monoclonal antibody 10D7 against the 100-kDa
subunit a of the V0 domain. Blots were developed as
described under "Experimental Procedures."
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Fig. 2.
Effect of nocodazole and latrunculin on
glucose-dependent reassembly of the yeast V-ATPase.
Wild-type cells (YPH499) were grown to mid-log phase and converted to
spheroplasts as described under "Experimental Procedures."
Spheroplasts were incubated for 40 min at 30 °C in YEP medium
without glucose to induce dissociation of the V-ATPase. Spheroplasts
were then incubated in the absence or presence of 40 µM
nocodazole for 2.5 h or of 0.2 mM latrunculin for 15 min at 30 °C, followed by incubation for 40 min at 30 °C in YEP
medium with or without glucose and in the continued presence or absence
of the corresponding inhibitor. Spheroplasts were solubilized with
detergent; the V-ATPase was immunoprecipitated with the anti-subunit A
antibody; and Western blotting was performed using antibodies
against both subunits A and a as described in the legend to Fig.
1.
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To test the effects of latrunculin and nocodazole on the cytoskeleton
in yeast, cells were treated with either latrunculin or nocodazole, and
the actin and microtubular networks were visualized using staining with
rhodamine-conjugated phalloidin or a polyclonal antibody against
-tubulin, respectively. As shown in Fig.
3, although latrunculin completely
disrupted the actin cytoskeleton in yeast, nocodazole had almost no
effect on actin. By contrast, treatment with nocodazole completely
disrupted the microtubular network, whereas latrunculin had relatively
little effect on microtubules (Fig. 4).
These results suggest that nocodazole is affecting dissociation of the V-ATPase through disruption of microtubules, rather than through
a more general disruption of the cytoskeleton.

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Fig. 3.
Effect of nocodazole and latrunculin
treatment on actin staining in yeast. Wild-type yeast cells
(YPH499) were grown to mid-log phase and then incubated with 2%
Me2SO for 2.5 h (control), 0.2 mM latrunculin for 15 min, or 40 µM
nocodazole for 2.5 h. After incubation, the cells were fixed with
formaldehyde, and actin filaments were labeled with
rhodamine-conjugated phalloidin as described under "Experimental
Procedures."
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Fig. 4.
Effect of nocodazole and latrunculin
treatment on microtubule staining in yeast. Wild-type yeast cells
(YPH499) were grown to mid-log phase and then incubated with 2%
Me2SO for 2.5 h (control), 0.2 mM latrunculin for 15 min, or 40 µM
nocodazole for 2.5 h. After incubation, the cells were fixed with
formaldehyde and permeabilized by treatment with zymolyase, and the
microtubules were visualized using a rabbit polyclonal antibody against
-tubulin followed by a Cy2-conjugated goat anti-rabbit secondary
antibody as described under "Experimental Procedures."
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To further test whether the inhibitory effect of nocodazole is due to
disruption of microtubules, glucose-dependent dissociation of the V-ATPase was measured in two yeast strains bearing mutations in
tubulin that result in resistance to nocodazole. DBY7051 and DBY8154
were selected for resistance to the structurally related compound
benomyl (31), but the mutation in the TUB2 gene present in
these strains also results in resistance of growth to nocodazole (data
not shown). As shown in Fig. 5,
glucose-dependent dissociation of the V-ATPase was
unaffected by nocodazole in both the DBY7051 and DBY8154 strains. These
results suggest that nocodazole exerts its effect on dissociation of
the V-ATPase through disruption of microtubules, rather than through
some nonspecific target.

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Fig. 5.
Effect of nocodazole on
glucose-dependent dissociation of the V-ATPase in
nocodazole-resistant yeast strains. Yeast strains DBY7051 and
DBY8154 bearing mutations in the TUB2 gene that confer
resistance to nocodazole were grown to mid-log phase and converted to
spheroplasts as described under "Experimental Procedures."
Spheroplasts were incubated in the absence or presence of nocodazole,
followed by incubation in the absence or presence of glucose as
described in the legend to Fig. 1. Spheroplasts were then solubilized
with detergent; the V-ATPase was immunoprecipitated with the
anti-subunit A antibody; and Western blotting was performed using
antibodies against both subunits A and a as described in the legend to
Fig. 1.
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Nocodazole inhibits mitosis and arrests yeast cells in the
G2 phase of the cell cycle through disruption of
microtubules involved in formation of the spindle body (42). It was
therefore possible that nocodazole inhibited dissociation of the
V-ATPase indirectly by blocking progression of cells through the cell
cycle. To test this possibility, glucose-dependent
dissociation was examined in yeast strains bearing
temperature-sensitive mutations in CDC genes that result in
arrest of cells in different stages of the cell cycle. Mutations in the
CDC2, CDC8, CDC15, and
CDC28 genes result in arrest of yeast cells at the
nonpermissive temperature in the G2, S, M, and
G1 phases of the cell cycle, respectively (32). Yeast cells
were grown to mid-log phase, incubated at either the permissive
(25 °C) or nonpermissive (37 °C) temperature for 4 h,
converted to spheroplasts, and then incubated in either the absence or
presence of glucose at the same temperature as the original incubation
for an additional 40 min. The V-ATPase was solubilized and
immunoprecipitated, and assembly was assessed as described above. As
shown in Fig. 6,
glucose-dependent dissociation of the V-ATPase was nearly
identical at the permissive and nonpermissive temperatures in all four
yeast strains, although somewhat reduced immunoprecipitation of subunit
A was observed in the strain bearing a mutation in CDC8
incubated in the absence of glucose at both temperatures. These results
suggest that nocodazole does not inhibit dissociation of the V-ATPase
in yeast by blocking progression of cells through the cell cycle. The
results also indicate that glucose-dependent dissociation
of the V-ATPase can occur at any stage of the cell cycle.

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Fig. 6.
Glucose-dependent dissociation of
the V-ATPase in yeast strains bearing temperature-sensitive mutations
in CDC genes resulting in cell cycle arrest.
Yeast strains bearing temperature-sensitive mutations in
CDC2, CDC8, CDC15, and
CDC28 were employed in this experiment. At the nonpermissive
temperature (37 °C), these strains are arrested in the
G2, S, M, and G1 phases of the cell cycle,
respectively. Cells were grown to mid-log phase at the permissive
temperature (25 °C) and converted to spheroplasts as described under
"Experimental Procedures." Spheroplasts were then incubated at
either 25 or 37 °C for 4 h, followed by continued incubation at
the corresponding temperature for 40 min in the presence or absence of
glucose. Spheroplasts were solubilized; the V-ATPase was
immunoprecipitated; and Western blotting was performed using antibodies
against both subunits A and a as described in the legend to Fig.
1.
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Disruption of microtubules has been shown to lead to fragmentation of
the central vacuole in yeast (42), whereas glucose depletion leads to
coalescence of vacuolar membrane vesicles into a single large vacuole
(38). This suggests that the assembly status of the V-ATPase may be
sensitive to vacuolar morphology and that nocodazole inhibits
dissociation by preventing formation of a single vacuole on glucose
depletion. To test this idea, assembly of the V-ATPase was measured in
response to exposure of cells to two other stress conditions shown to
lead to formation of a single large vacuole, namely ethanol shock and
nitrogen starvation (43, 44). As shown in Fig.
7, neither of these conditions led to
dissociation of the V-ATPase complex. Incubation of spheroplasts in
medium lacking nitrogen for up to 3 h also induces no V-ATPase dissociation (data not shown). These results indicate that although formation of a single large vacuole in yeast may be a necessary condition for dissociation of the V-ATPase to occur, it is not a
sufficient condition.

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Fig. 7.
Effect of ethanol shock and nitrogen
depletion on assembly of the V-ATPase. Yeast strain YPH499 was
grown to mid-log phase; cells were converted to spheroplasts; and the
spheroplasts were incubated in YEPD medium for 30 min at 30 °C in
the presence or absence of 6% ethanol (a) or were incubated
in YEPD medium (+Nitrogen) or 2% glucose
( Nitrogen) for 30 min at 30 °C
(b). Spheroplasts were then solubilized with detergent; the
V-ATPase was immunoprecipitated with the anti-subunit A antibody; and
Western blotting was performed using antibodies against both subunits A
and a as described in the legend to Fig. 1.
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Because mutations that lead to inhibition of V-ATPase activity have
been shown to inhibit dissociation of the V-ATPase in response to
glucose depletion (45, 46), it was possible that nocodazole might
inhibit dissociation through some direct effect on the activity of the
V-ATPase. To test this, concanamycin-sensitive ATPase activity and
ATP-dependent proton transport were examined in vacuoles
isolated from spheroplasts incubated in the absence or presence of
nocodazole. As shown in Table II, no
differences in either ATP hydrolysis or proton transport were observed
following treatment with nocodazole, suggesting that nocodazole does
not block dissociation by irreversibly inhibiting V-ATPase
activity.
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Table II
Effect of nocodazole on ATPase activity and proton transport by the
yeast V-ATPase in isolated vacuoles
Wild-type yeast cells (YPH499) were grown to mid-log phase and
converted to spheroplasts as described under "Experimental
Procedures." Spheroplasts were then incubated in YPD medium in the
absence or presence of 40 µM nocodazole for 2.5 h,
followed by lysis and isolation of vacuoles as described under
"Experimental Procedures." ATPase activity and
ATP-dependent proton transport were measured for isolated
vacuoles in the absence and presence of 1 µM concanamycin
A using a coupled spectrophotometric assay and uptake of acridine
orange, respectively. Values are expressed relative to activities
measured for control vacuoles in the absence of both nocodazole
treatment and concanamycin A. The specific activity of the ATPase in
control vacuoles in the absence of concanamycin A was 1.2 µmol of
ATP/min/mg of protein.
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To test whether the dependence of in vivo dissociation
on microtubules resulted from some direct interaction of the V-ATPase with tubulin, spheroplasts were incubated in the absence or presence of
glucose and the absence or presence of nocodazole; the V-ATPase was
immunoprecipitated using the antibody against subunit A; and Western
blotting was performed using both antibodies against subunit A and
-tubulin. As can be seen from the data in Fig.
8 (lanes 2-5), no association
of
-tubulin with the V-ATPase was detectable under any of the
conditions tested. Because both the intact V-ATPase and the
V1 domain would be immunoprecipitated using the antibody against subunit A, this result also indicates that
-tubulin does not
directly bind to the V1 domain. In addition, no
-tubulin was detectable in vacuoles isolated from spheroplasts incubated in the
absence or presence of nocodazole (Fig. 8, lanes 6 and 7). These results suggest either that the V-ATPase does not
directly associate with microtubules or, if it does, that this
association does not persist following cell disruption or vacuole
isolation.

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Fig. 8.
The yeast V-ATPase does not directly
associate with -tubulin. Lane
1, wild-type yeast cells (YPH499) were grown to mid-log phase and
then lysed; an aliquot of the whole cell lysate (40 µg of protein)
was separated by SDS-PAGE on a 10% acrylamide gel; and Western
blotting was performed using either the anti-subunit A antibody or a
rabbit polyclonal antibody against -tubulin. The blot was developed
as described under "Experimental Procedures." Lanes
2-5, wild-type yeast cells (YPH499) were grown to mid-log phase
and converted to spheroplasts, and the spheroplasts were incubated for
2.5 h at 30 °C in the absence or presence of 40 µM nocodazole, followed by incubation for 40 min at
30 °C in YEP medium either with or without glucose and in the
continued presence or absence of nocodazole. The spheroplasts were then
solubilized with 1% C12E9 in the presence of 1 mM dithiobis(succinimidyl propionate), and the V-ATPase was
immunoprecipitated using monoclonal antibody 8B1-F3 against subunit A
of the V1 domain. The proteins were separated by SDS-PAGE,
and Western blotting was performed as described above. Lane
2, +glucose/ nocodazole; lane 3,
glucose/ nocodazole; lane 4, +glucose/+nocodazole;
lane 5, glucose/+nocodazole. Lanes 6 and
7, vacuoles were isolated as described under "Experimental
Procedures" from spheroplasts incubated in the absence or presence of
nocodazole as described above. An aliquot of vacuoles (5 µg of
protein) was then separated by SDS-PAGE, and Western blotting was
performed as described above. Lane 5, nocodazole;
lane 6, +nocodazole.
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DISCUSSION |
Although dissociation of the V-ATPase complex has been shown to
represent an important mechanism of regulating activity in both yeast
(24) and insects (25), the mechanism by which dissociation is
controlled remains unknown. In yeast, dissociation of the V-ATPase in
response to glucose depletion has been shown to require catalytic activity of the enzyme (45, 46). On the other hand, many of the
signaling pathways that are activated upon removal of glucose from the
medium are not involved in dissociation of the V-ATPase. These include
the Ras-cAMP pathway, the Snf1p kinase-regulated pathway, the protein
kinase C-dependent pathway, and the protein phosphatase
2A-dependent stress response pathway (45). In addition, the
assembly status of the V-ATPase does not appear to be controlled by the
level of glucose 6-phosphate in the cell, the earliest intracellular
metabolite of glucose, as is the case for at least some of the other
processes controlled by glucose in yeast (45). Rather, V-ATPase
assembly is dependent upon the presence and further metabolism of
rapidly fermentable carbon sources, including glucose, fructose, and
mannose (45).
Dissociation of the V-ATPase in response to glucose depletion has
been suggested to represent a mechanism of conserving cellular stores
of ATP (24). In fact, there appears to exist a considerable surplus of
V-ATPase activity in the cell that is not essential to maintain a
wild-type growth phenotype (i.e. growth at pH 7.5) since
mutants possessing as little as 20% of wild-type V-ATPase activity
still show normal growth at pH 7.5 (47, 48). Thus, cells may retain
sufficient capacity to acidify their intracellular compartments even
upon glucose depletion. A transient decrease in cellular ATP levels has
been measured in yeast upon glucose depletion and has been suggested to
serve as a possible trigger in the dissociation process (45). However,
V-ATPase assembly cannot be directly tracking ATP levels in the cells
since ATP concentrations are restored within 20 min of glucose removal, whereas the V-ATPase remains dissociated (45).
We demonstrate in this study that dissociation of the V-ATPase in
response to glucose depletion is dependent upon intact microtubules, but not on actin filaments. This result was somewhat unexpected given
previous reports of V-ATPase association with actin (30) and
actin-associated proteins such as the ezrin-binding protein NHE-RF
(29). The latter protein has been shown to bind to the B1
isoform of the V-ATPase in renal B-type intercalated cells, and this
interaction has been suggested to be involved in the selective
targeting of V-ATPases to the apical or basolateral membrane in these
cells. Moreover, direct interaction between the V-ATPase and actin has
been demonstrated in osteoclasts (30), cells that have the capacity to
target V-ATPases to the plasma membrane (11). Thus, actin/V-ATPase
interactions may be important only in cells that target V-ATPases to
the cell surface. Since V-ATPases are restricted to intracellular
compartments in yeast, interactions between V-ATPases and actin
filaments may not exist or may be less important than in mammalian cells.
It is interesting that although dissociation of the V-ATPase in yeast
requires intact microtubules, reassembly of the V-ATPase upon
re-addition of glucose does not require microtubules. This suggests
that dissociation and reassembly of the V-ATPase may represent
independently controlled processes. It is possible that microtubules
may be involved in movement of dissociated V1 domains away
from the vacuole or in movement of as yet unidentified signaling molecules to the vacuole. In fact, many signaling processes are dependent upon an intact microtubular network (49). Polarized delivery
of intact V- ATPases in renal intercalated cells has also been shown
to depend upon an intact microtubular network (50), but this result
most likely represents the dependence of vesicular movement on
microtubules. In contrast to the reported association of the V-ATPase
with actin in osteoclasts (30), no direct association of tubulin with
the V-ATPase could be detected in yeast (Fig. 8).
Because disruption of microtubules leads to fragmentation of the
central vacuole (42), whereas glucose depletion leads to formation of a
single large vacuole (38), it is possible that nocodazole inhibits
dissociation by preventing vacuolar coalescence. However, dissociation
cannot be occurring as a direct result of vacuolar coalescence since
other stress conditions that lead to formation of a single large
vacuole (i.e. ethanol shock and nitrogen depletion (43, 44))
do not cause dissociation of the V-ATPase complex. Thus, formation of a
single large vacuole in yeast may be a necessary, but not sufficient,
condition for dissociation of the V-ATPase to occur.
We have also found that dissociation of the V-ATPase is not dependent
upon a particular stage of the cell cycle. Thus, nocodazole treatment
does not affect V-ATPase dissociation through disruption of the cell
cycle. This suggests that the signaling pathways involved in activating
dissociation are not specific to a particular stage of the cell cycle.
Further work will be required to identify these signaling pathways and
to determine whether an intact microtubular network is required for
dissociation of the V-ATPase in other systems.
 |
ACKNOWLEDGEMENTS |
We thank Dr. David Botstein (Department of
Genetics, Stanford University) for providing yeast strains DBY7051 and
DBY8154, Dr. Leland Hartwell (Fred Hutchinson Cancer Center) for
providing the cdc mutant yeast strains, and Dr. Frank
Solomon for providing a rabbit anti-yeast
-tubulin antiserum and
protocols for performing immunofluorescence staining of yeast microtubules.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM34478 (to M. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail:
michael.forgac@tufts.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M100637200
 |
ABBREVIATIONS |
The abbreviations used are:
V-ATPases, vacuolar
[H+]-ATPases;
PAGE, polyacrylamide gel
electrophoresis.
 |
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