Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
* Author for correspondence (e-mail: nelson{at}post.tau.ac.il )
Accepted 11 February 2002
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Summary |
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Key words: V-ATPase, subunit a, yeast, Saccharomyces cerevisiae, biogenesis, endocytosis, proton pumping
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Introduction |
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In common with the evolutionarily related F-ATPase, whose primary function
in eukaryotic cells is to form ATP, V-ATPase is composed of two functional
domains. The peripheral V1 domain, which is responsible for ATP
hydrolysis, contains at least eight subunits, denoted as subunits A through H
(Stevens and Forgac, 1997;
Nelson and Harvey, 1999
). The
membrane Vo domain consists of at least five subunits (a, c,
c', c'' and d) and functions in proton translocation
(Powell et al., 2000
;
Stevens and Forgac, 1997
). In
most of the eukaryotic organisms studied, a null mutation in the V-ATPase gene
results in a lethal phenotype. In contrast, yeast cells lacking V-ATPase
activity due to the same null mutation can survive, but only in a buffered
medium at pH 5.5 (Nelson and Nelson,
1990
). These mutations exhibit a conditionally lethal phenotype in
which cells cannot grow at a pH higher than 7.0 or various other medium
conditions (Nelson and Nelson,
1990
). This feature makes yeast one of the favorite organisms for
V-ATPase studies. Furthermore, the yeast genome has one gene encoding each
subunit of the V-ATPase, except for VPH1 and STV1, which
encode two isoforms of the 100 kDa a subunit (Vph1p and Stv1p). Disruption of
both subunits is necessary to produce the null V-ATPase mutant phenotype
(Manolson et al., 1992
,
1994
), which creates a unique
opportunity to study the role of isoforms in the V-ATPase subunits. In other
organisms, many of the V-ATPase subunit isoforms were demonstrated to be
tissue-specific (Futai et al.,
2000
), yet some were found in specific locations in the same cells
(Toyomura et al., 2000
). In
yeast, Vph1p was assigned to the vacuole, and a Golgi and/or endosome
localization was proposed for Stv1p
(Manolson et al., 1994
). These
observations suggest that subunit a may be responsible for the V-ATPase
localization in specific subcellular compartments and/or regulation of
enzymatic activity, which implies that there should be specific and distinct
phenotypes for both mutants. The vph1
mutant is unable to
accumulate Quinacrine in the vacuole, and therefore appears to be defective in
vacuolar acidification, but no phenotype has been described for absence of
Stv1p, and the stv1
mutant was reported to be identical to the
wild type (Manolson et al.,
1994
).
Properties of V-ATPase complexes containing both disrupted Vph1 and Stv1
genes (double-deletion mutant) were recently studied with overexpression of
each gene cloned into a yeast shuttle vector
(Kawasaki-Nishi et al., 2001).
It was demonstrated that a complex containing Stv1p showed lower assembly with
the catalytic subunits and a lower ratio of proton transport to ATP hydrolysis
than the V-ATPase complex containing Vph1p.
In this paper, we reinvestigate the subcellular locations of the Vph1p and
Stv1p and also define the characteristics of the separate vph1
and stv1
phenotypes in comparison to the wild type and
double-deletion mutant. We used a new pH-sensitive LysoSensor Green DND-189
dye to determine the extent of acidification within the intracellular
compartments in both wild type and null mutants for each of the two isoforms
of subunit a. The V-ATPase was suggested to be directly involved in
endocytosis and membrane fusion (Wendland
et al., 1998
; Ungermann et
al., 1999
; D'Hondt et al.,
2000
; Peters et al.,
2001
). Using the endocytic marker FM 4-64 we show that the
endocytic process is inhibited whenever there is a decrease in the cellular
V-ATPase activity.
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Materials and methods |
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Preparation of yeast strains
All the yeast strains used in this work were prepared as previously
described (vma3, Nelson
and Nelson, 1990
; vma8
and vtc1
,
Cohen et al., 1999
). Constructs
for disruption of the VPH1 and STV1 genes were prepared as
follows. Each gene was amplified from yeast genomic DNA by polymerase chain
reaction (PCR) with specific primers, cloned into pGEM-T easy or pBluescript
SK, respectively. For VPH1, a fragment of 610 base pairs (bp) was
deleted by digestion with HincII and EcoRV, and a blunt-end
LEU2 marker was inserted in its place. For STV1 (which was
cloned into the KpnI-BamHI sites of pBluescript) a 2360 bp
fragment was deleted by digestion with EcoR1 and XhoI,
filled in and a blunt-end TRYP1 or URA3 marker inserted. The
constructs were excised using appropriate enzymes at either end and used for
yeast W303 or vph1
(haploid strains) transformation.
Homologous recombination and null V-ATPase phenotypes were checked as
previously described (Cohen et al.,
1999
).
Antibody preparation and western analysis
Polyclonal antibody against Stv1p was obtained by injecting rabbits with a
chimeric protein containing the maltose-binding protein and hydrophilic
sequence of amino acid residues 231-436 of Stv1p. The DNA fragment encoding
these amino acids was amplified by PCR with introduced EcoRI and
HindIII restriction sites. The amplified DNA fragment was cloned
in-frame to the maltose-binding protein in the plasmid PMAL-C (New England
Biolab). Following sequence verification, 500 ml of bacterial culture was
grown to A600=0.5, induced with
isopropyl-ß-D-thiogalactoside (IPTG) for 3 h and harvested by
centrifugation at 4000g. The cells were broken using a French
press and the protein purified on a column containing maltoseagarose.
The fractions containing the chimeric protein were dissociated by SDS, loaded
on a preparative gel and electrophoresed. The gel was briefly stained by
Coomassie Blue, the identified protein band cut out and the fusion protein was
electroeluted. About 0.25 mg fusion protein was injected into rabbits as
previously described (Nelson,
1983). Polyclonal antibody against Sec14p was prepared as a
chimeric protein containing the maltose-binding protein and the whole
open-reading frame sequence of the Sec14p was prepared using a procedure
similar to that described for the Stv1p antibody. Monoclonal antibodies that
recognize Vph1p (10D7-A7-B2), CPY (10A5-B5) and Pep12p (2C3G4) were from
Molecular Probes, Inc. Polyclonal antibody against the purified Pma1p was
raised in rabbits and used at a dilution of 1:10,000. Antibody against Sed5p
was a generous gift from Dr Randy Schekman (University of Berkeley, USA).
Polyclonal antibodies against Vma8p and Vma5p were raised in guinea pigs and
used at a dilution of 1:1000. The antibody detection system (ECL) was from
Amersham. Western blots were performed as previously described
(Cohen et al., 1999
).
Membrane preparations and protein subcellular fractionation
Whole-cell extracts from the strains were prepared as described by Lyons
and Nelson (1984) with several
modifications. Briefly, yeast cells (20 ml) were grown to late logarithmic
phase, centrifuged and washed once with water. Cells were resuspended in 0.45
ml water. Sodium hydroxide and 2-mercaptoethanol were added to a final
concentration of 0.2 mol l-1 and 0.5 %, respectively. The
suspension was placed on ice for 20 min, then 0.125 ml of dissociation buffer
(2 % SDS, 80 mmol l-1 Tris-HCl, pH 6.8, 1 % 2-mercaptoethanol, 10 %
glycerol and 0.01 % Bromophenol Blue) was added and adjusted immediately with
HCl to pH 7.5-8. The proteins were extracted for 1 h at room temperature and
insoluble material was removed by centrifugation in an Eppendorf centrifuge
(18,000 g) at room temperature for 10 min.
For membrane preparation, yeast cells were grown in 500 ml YPD medium (pH
5.5) to A600=1.0. The suspension was centrifuged at 3000
g for 5 min and the pellet was washed once with water and again with
1 mol l-1 sorbitol. The cell wall was digested by 2.5 U zymolyase
in 10ml solution containing 10 mmol l-1 Hepes, pH 7.5, and 1 mol
l-1 sorbitol. After 30 min incubation at 30 °C, the suspension
was centrifuged in 15 ml Corex tubes at 3000 g for 5 min. Glass beads
(1 ml) were added to the pellet, together with 1 ml of a solution containing
30 mmol l-1 Mops, pH 7.0, 10 µl protease inhibitor cocktail
(Sigma), 1 mmol l-1 phenylmethylsulfonyl fluoride (PMSF), 1 mmol
l-1 EDTA and 1 mmol l-1 EGTA. The suspension was
vortexed five times for 30 s each with incubation on ice for 30 s inbetween.
The solution was removed from the glass beads and placed in a fresh Corex tube
for centrifugation at 1000 g for 5 min, forming a pellet containing
the cell debris and nuclei. The supernatant was centrifuged at 115,000
g for 30 min and the pellet was suspended in 0.3-0.5 ml of solution
containing 10 mmol l-1 Tris-Cl, pH 7.5, 1 mmol l-1 EDTA,
2 mmol l-1 dithiothreitol (DTT), 25 % glycerol, and stored as the
membrane fraction at -80 °C. Sucrose gradients were also used to estimate
the relative density of various membrane fractions. The gradients (20 %-60 %
sucrose) were made as described by Lupashin et al.
(1997), and were centrifuged
in a Beckman SW-40 rotor for 14h at 150,000 g.
Differential centrifugation analysis and sucrose gradient fractionation of
Golgi membranes were performed as previously described
(Graham et al., 1994;
Graham and Krasnov, 1995
).
Briefly, spheroplasts (300 A600 units) obtained by
zymolyase treatment (0.125 mg ml-1 in 50 mmol l-1
Tris-HCl, pH 7.5, 1.4 mol l-1 sorbitol, 40 mmol l-1
2-mercaptoethanol) were lysed by sevenfold dilution in hypoosmotic buffer (0.1
mol l-1 sorbitol, 10 mmol l-1 TEA, pH 7.5, 1 mmol
l-1 EDTA) followed by Dounce homogenisation (25 strokes). The
lysate was centrifuged at 1000 g for 6 min to generate P1 (pellet)
and S1 (supernatant) fractions, and the latter was centrifuged at 13,000
g to generate P13 and S13 fractions. The S13 fraction was layered
onto a two-step sucrose cushion consisting of 1 ml of 66 % sucrose and 1 ml of
20 % sucrose, then centrifuged in a Beckman SW-40 rotor at 120,000 g
for 2h at 4 °C. The membranes present at the 20 %-66 % sucrose interface
(P120) were collected in a volume of approx. 1.2 ml and adjusted to approx. 48
% sucrose using a 66 % sucrose solution. The membrane sample (1.5 ml) was
layered on top of a 60 % sucrose cushion (0.5 ml) and a sucrose step gradient
consisting of 47.5 % (1.5 ml), 45 % (1.0 ml), 42 % (2.0 ml), 40 % (2.0 ml), 38
% (1.0 ml), 36 % (1.0 ml) and 32 % (1.5 ml) sucrose was layered on top of the
sample. All sucrose solutions were prepared in 10 mmol l-1
Na-Hepes, pH 7.5. The gradients were centrifuged in an SW-40 rotor at 31,000
g for 17 h at 4 °C. Fifteen fractions (approx. 0.7 ml) were
collected, starting from the bottom of the gradient. The proteins from each
fraction were precipitated by addition of 0.13 ml of 5x SDS-sample
buffer and 1.0 ml of ice-cold ethanol. After incubation on ice for 30 min, the
samples were centrifuged at 18,000 g for 30 min at 4 °C. After
aspiration of the supernatants, the pellets were resuspended in 0.05 ml of
SDS-sample buffer and stored at -20 °C.
Preparation of yeast vacuoles
For preparation of vacuoles, cells were grown in YPD medium adjusted to pH
5.5 with HCl and harvested at a cell density of about 0.8
A600 units. Vacuolar membranes were prepared according to
the method of Uchida et al.
(1985), except that the 8 %
Ficoll gradient purification step was omitted, the homogenization buffer
contained no magnesium and the vacuoles were washed only once with the EDTA
buffer (10 mmol l-1 Tris-HCl, pH 7.5, 1 mmol l-1 EDTA, 2
mmol l-1 DTT). ATP-dependent proton uptake activity was assayed by
following the absorbency changes of Acridine Orange at 490-540 nm as
previously described (Supek et al.,
1994
). The reaction mixture (1 ml) contained 20 mmol
l-1 Mops-Tris, pH7, 15 mmol l-1 KCl, 135 mmol
l-1 NaCl and 15 µmol l-1 Acridine Orange. Isolated
yeast vacuoles (10-30 µg) were added to the reaction mixture followed by 10
µl of 0.1 mol l-1 MgATP. The reaction was terminated by the
addition of 1 µl of 1 mmol l-1 carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP).
Vacuole staining with fluorescent probes
For FM4-64 vacuolar staining, cells were grown in YPD to
A600=0.8-1.6. Cells (20-40 A600 units
ml-1 in YPD medium) were incubated on ice for 30 min with 30
µmol l-1 FM4-64 dye (Molecular Probes Inc.), washed once with
YPD and incubated for 60 min for steady-state experiments as described
(Vida and Emr, 1995).
For Quinacrine staining, yeast cells were grown in YPD to A600=0.8. The cells were cooled on ice for 5 min and 1 ml of cell suspension was sedimented by centrifugation and resuspended in 100 µl YPD containing 100 mmol l-1 Hepes, pH 7.6, and 200 µmol l-1 freshly prepared Quinacrine. The suspension was incubated for 5-10 min at 30 °C and cooled on ice for 5 min. The cells were sedimented by centrifugation and resuspended in 1 ml 100 mmol l-1 Hepes, pH 7.6, 2 % glucose. The cells were washed twice with the same cold buffer and resuspended in 0.1 ml of the same solution. 4 µl of the cell suspension was mixed on the microscope slide with 4 µl of 1 % low-melting agarose kept at about 45 °C and covered with a glass cover. The cells were examined using a Zeiss LSM510 confocal laser microscope. Fluorescence profiles were generated using a Zeiss LSM Image browser.
For LysoSensor Green DND-189 staining (Molecular Probes Inc), all strains were grown at 30 °C to A600=0.7-1. Cells were harvested and washed with uptake buffer (YPD containing 100 mmol l-1 Hepes, pH 7.6). The cell pellets were resuspended at a concentration of 15 A600 units ml-1 in uptake buffer and dye was added to a final concentration of 4 µmol l-1 from a stock solution of 1 mmol l-1 in DMSO. Cells were then incubated for 5 min at 30 °C and washed once with the same buffer. Cell pellets were resuspended in fresh YPD, pH 7.6, at 15 A600 units ml-1, placed on standard slides with low-melting agarose and photographed immediately after staining. The samples were viewed using a confocal laser scanning microscope (Zeiss) equipped with an Argon 458 nm laser and a C-Apochromat 63x water immersion objective. A LP505 filter was used for LysoSensor Green DND- 189 fluorescence. The images were recorded, merged and processed using the Zeiss LSM Image browser.
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Results |
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Immunolocalization studies of Vph1p and Stv1p in yeast suggested that Vph1p
is localized in the central vacuole, whereas Stv1p is localized in some other
intracellular compartments, possibly the Golgi or endosomes
(Manolson et al., 1994).
Several studies showed that V-ATPase is present along the Golgi complex
(Moriyama and Nelson, 1989
;
Ying et al., 2000
;
Grunow et al., 1999
).
Subcellular fractionation of the Golgi membranes using the technique of Graham
and Krasnov (1995
) was used to
determine the location of the Stv1p subunit more precisely. This technique
separates the vacuoles more efficiently and allows better detection of the
V-ATPase complex in the different Golgi and endosomal fractions. The cell
membranes were applied to the bottom of a sucrose gradient and centrifuged to
equilibrium. Fractions were collected from the bottom and probed with
antibodies against Stv1p, Vph1p, Pep12p, Sec14p and Sed5p. Sed5p is the marker
for early Golgi compartments, Sec14p for the late Golgi vesicles and Pep12p
for endosomes. In the fractionation profile shown in
Fig. 2, Vph1p and Stv1p were
broadly distributed throughout the gradient. Furthermore, the Vph1p profile
matches that of the Pep12p profile, suggesting an endosomal distribution on
the gradient. ALP (vacuolar membrane protein) marks the vacuolar membrane
contamination. Stv1p is located in two peaks along the gradient. The first
peak (fractions 3-5) coincides slightly better than Vph1p with the Sec 14p
(trans-Golgi) distribution, and the second peak (fractions 12,13)
matches the broad profiles of Pep12p and Sed5p
(Fig. 2). We also found that
the V1 subunit, Vma5p, peaked in the first four fractions. The peak
of Vma5p overlapped mostly with that of Sec14p, raising the possibility that
the assembled V-ATPase complex present in a late Golgi compartment is more
stable. A similar distribution was also observed for Vma8p, another
V1 subunit (not shown). Assuming that the amount of assembled
V-ATPase is an indicator of its ATPase activity, these results support
previous reports that acidification develops along the Golgi complex and is
maximal in the trans-Golgi compartment
(Llopis et al., 1998
;
Schapiro and Grinstein, 2000
).
Fractionation in yeast does not yield clean separation of compartments, so it
is difficult to draw exact conclusions about localization. Assuming that the
two subunit a isoforms are in separate complexes, and that this western
profile represents assembled and unassembled proteins, we can cautiously say
that from the relative amounts, at least in fraction 3, Stv1p is the V-ATPase
isoform in the Golgi complex.
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Stv1p localization in vph1 strain
It was proposed that Stv1p does not normally reside on the vacuolar
membrane in wild-type cells, and only overexpressed Stv1p-HA in
vph1/stv1
and vph1
strains could be
found in the vacuolar membrane (Manolson
et al., 1994
; Kawasaki-Nishi
et al., 2001
). On the other hand, detectable levels of Vma1p and
Vma2p were found to be associated with vacuoles from the vph1
strain (Manolson et al.,
1992
). Our antibody revealed that purified vacuoles from wild-type
cells also contain Stv1p. Fig.
3A shows a western analysis of purified vacuoles from wild-type,
stv1
and vph1
probed with antibodies against
Stv1p, Vph1p, Vma5p and Vma8p. Stv1p is detected in vacuolar membranes from
the wild-type and vph1
cells. These data also demonstrate that
the level of Stv1p in the vph1
mutant strain increased in
comparison to the wild-type strain, as was found for Vph1 in
stv1
mutant (Fig.
1B). On the other hand, the amount of V-ATPase complex in the
vph1
was reduced significantly (based on amounts of the
V1 subunits of V-ATPase, Vma5p and Vma8p present in those vacuolar
membranes) as compared to the wild type.
|
Although vacuolar membranes isolated from the vph1 strain
contained an evidently assembled V-ATPase complex (see
Fig. 3A), it was still possible
that other V-ATPase-containing membranes also contaminated the vacuoles of
vph1
strain and wild-type. We could detect Golgi and endosomal
proteins in isolated vacuoles with antibodies against Sec14p and Pep12p,
respectively (data not shown). Fig.
3B depicts two fractions of the gradient (8 and 13) showing,
respectively, endosomal and intact vacuolar fractions, which were obtained
from a sucrose gradient fractionation of vph1
strain,
decorated with antibody against CPY (a soluble vacuolar marker), Pep12p
antibody (an endosomal marker), and Vma8p and Stv1p antibodies. Fraction 8
represents the peak of the endosomal fractions and fraction 13 the peak of the
vacuolar fractions. CPY is a vacuolar-soluble protein, so we also probed both
fractions with antibody against ALP, a resident of the vacuolar membrane, that
was present throughout the gradient (not shown). These data are in agreement
with the assumption that an assembled V-ATPase complex is present in the
vacuole in vph1
strain with no detectable endosomal
contamination.
V-ATPase proton uptake activity in vph1 and stv1
strains
Previous work has demonstrated that vacuoles of a vph1
mutant with neutral lumenal pH do not accumulate Quinacrine and have no
detectable bafilomycin A1-sensitive V-ATPase activity
(Manolson et al., 1994
).
Because we showed that small amounts of assembled V-ATPase complex are present
in vacuoles from our vph1
strain, it was of interest to assess
their proton pumping activity. Fig.
4 shows the ATP-dependent proton uptake activity of vacuoles
isolated from wild-type, stv1
and vph1
strains. The ATP-dependent proton uptake activity from the
vph1
mutant (26 nmol min-1 mg-1) was
30-fold less than the specific activity of wild-type (817 nmol
min-1 mg-1), but nevertheless measurable. This explains
the observation that Quinacrine fluorescence, which is dependent on higher
proton concentrations, was not observed in the vph1
strain.
Strains that overexpress Stv1p showed a marked reduction in the assembled
V-ATPase complex, even though there were appreciable amounts of Stv1p in the
vacuolar membrane (Kawasaki-Nishi et al.,
2001
). It appears that deletion of the VPH1 gene results
in naturally overexpressed Stv1p in vacuolar membranes (Figs
1B,
3A). These results also suggest
that the compensation of a vph1
mutant by Stv1p is only
partial.
|
As a control for V-ATPase activity we isolated vacuoles from a
stv1 strain. Surprisingly, the initial rate of V-ATPase
activity in these vacuoles was diminished by 40-50 % (see
Fig. 4). This highly
reproducible result led us to do a more careful analysis of the amounts of
V1 subunits upon the vacuolar membranes in this strain, which are
more indicative of the enzyme's activity. A reduction in Vma5p and Vma8p is
apparent on western blot analysis (Fig.
3A), whereas the Vph1p level is slightly higher than in wild type.
This pattern suggests that in wild type the Stv1p in its post-Golgi location
plays a role in facilitating the assembly process of the holoenzyme in the
vacuolar membrane.
LysoSensor Green DND-189 staining of V-ATPase null mutants
LysoSensor Green DND-189 is an acidotropic probe that accumulates in the
membranes of acidic organelles as a result of protonation. It was used to
investigate the acidic compartments in cultured cerebellar granule cells and
plant tissue (Cousin and Nicholls,
1997; Guttenberger,
2000
). We used this dye to stain the acidic compartments of yeast
(see Materials and methods). The dye labeled the vacuolar membranes of
wild-type cells in less than 5 min, and the signal remained stable for more
than 1h. The staining is more efficient than with Quinacrine, as shown in
Fig. 5A, which shows parallel
labeling of wild-type cells with LysoSensor Green DND-189 and Quinacrine (a
vital dye for the vacuolar lumen). LysoSensor Green detects acidic vacuoles in
almost 100 % of the stained cells. The vacuolar staining was confirmed by
double labeling with FM4-64 lipophilic styryl dye, which selectively stains
endocytic compartments and yeast vacuolar membranes.
Fig. 5B shows double-staining
images at different intervals. After 25 min the green staining of the vacuolar
membrane is clearly visible, and the red staining by FM 4-64 is concentrated
mainly in cytoplasmic vesicles, which may correspond to endosomes
(Vida and Emr, 1995
). Some of
these vesicles are definitely not stained by LysoSensor Green; conversely some
vesicles are stained by LysoSensor Green but not by FM4-64. After 45 min the
staining of the vacuolar membrane by the FM4-64 is almost complete. Double
staining of the vacuolar ring (Fig.
5B) also demonstrates that LysoSensor Green DND-189 is an
efficient dye for the staining of acidic compartments in yeast.
|
LysoSensor Green was used to examine vacuolar membrane staining of wild
type, vph1 and stv1
mutants and the double
mutant vph1
/stv1
cells. The vacuolar membrane was
stained normally in wild-type cells, but no staining was observed in the
double mutant vph1
/stv1
, other V-ATPase null mutants
(Fig. 6), or in wild-type cells
treated with the specific V-ATPase inhibitor concanamycin A (not shown).
LysoSensor Green staining and V-ATPase activity are correlated in the
vtc1
mutant, which was shown to have diminished V-ATPase
activity in vitro (Cohen et al.,
1999
), while its staining with the vital dye was much less intense
than that of the wild-type cells (not shown). Our results obtained with the
stv1
mutant, which grows well in YPD medium, pH 7.5, showed
that the intensity of the vacuolar staining was lower than in the wild type
(Fig. 6). This observation is
in agreement with the data in Fig.
4, which demonstrate reduced V-ATPase activity in the
stv1
strain. Interesting results were observed in
vph1
cells. Half of the cells were weakly stained and the
others, when the signal was intensified, showed a clear difference between
vph1
and the double mutant in vacuolar staining
(Fig. 6). This observation
suggests that, in vph1
cells, Stv1p might reach the vacuolar
membrane, as also indicated by western analysis
(Fig. 3). Moreover, when grown
on a medium buffered at pH 7.5, the staining of vacuoles in the
vph1
cells is intensified
(Fig. 6).
|
Protein sorting in vph1 and stv1
mutants
We recently investigated the effect of vacuolar ATPase null mutations on
the targeting of the plasma membrane H+-ATPase (Pma1p) through the
secretory pathway (Perzov et al.,
2000). We showed that the amounts of Pma1p in the plasma membranes
of V-ATPase-depleted mutants were markedly reduced, and a large amount of the
protein was accumulated in the ER-Golgi in a non-active form. If Stv1p and
Vph1p are part of separate V-ATPase complexes in distinct compartments, we
might expect differential effects on Pma1p distribution in both mutants. We
therefore performed sucrose gradient fractionation on total membranes prepared
from the wild-type, vph1
, stv1
and
vph1
/stv1
strains. As shown in
Fig. 7A, the distribution of
Pma1p in the sucrose gradient fractions of the membrane preparation was not
altered in stv1
and vph1
strains, but was
altered in the vph1
/stv1
null mutant, as is usually
observed in other null V-ATPase mutants. These results indicate that only
complete inactivation of the V-ATPase in all cellular compartments leads to
redistribution of Pma1p. In contrast, we observed defects in processing of
vacuolar hydrolyses in vph1
cells, while in the
stv1
strain the vacuolar proteins mature as in the wild type
(Fig. 7B).
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Effect of V-ATPase null mutations on FM4-64 internalization
FM4-64 is a fluorescent lipophilic dye, which serves as a marker for
endocytosis and has been used for this purpose in yeast
(Vida and Emr, 1995). The dye
is initially incorporated into the plasma membrane and, through endocytosis,
reaches and accumulates in the vacuolar membrane. It has been suggested that
the acidification of endocytic organelles by V-ATPase is essential for
endosomal trafficking in mammalian cells
(Clague, 1998
). We used FM4-64
dye to investigate the role of acidification in endosomal trafficking in
yeast. The wild-type and null V-ATPase strains were stained with FM4-64 and
the localization of the dye recorded after 20, 40, 60 and 90 min.
Fig. 8A shows that, in the
wild-type strain, the dye reaches the vacuole after 20 min and stains it
strongly after 40 min. Vacuolar staining in the vma3
mutant is
inhibited, and after 60 min the cells do not even show the vacuolar staining
which the wild-type cells do after 20 min. After 60 min of internalization,
the dye stained vacuolar membranes only in the wild type, whereas in the
vma3
mutant, vph1
/stv1
cells or other
null V-ATPase mutants, much of the internalized dye only reaches vesicular
intermediates in the cytoplasm and levels do not change much during this time.
Only after 90 min does some of the dye reach the vacuolar membrane.
Fig. 8B summarizes the staining
data obtained with vph1
and stv1
cells. The
transport of the dye to the vacuole in vph1
cells resembles
that in null mutants, but slight staining of the vacuolar membranes is
observed in some cells (Fig.
8B). In stv1
cells, FM4-64 reached the vacuolar
membrane with kinetics similar to the wild type, but a mix of fragmented
vacuoles was observed very frequently (a similar vacuolar pattern was obtained
by staining the stv1
strain with LysoSensor Green dye, see
Fig. 6). Fragmented vacuoles
are a component of the phenotype in the stv1
strain.
|
Fragmented vacuoles and no defect in endocytic delivery of the dye to the
vacuole were also detected in vps1 cells
(Raymond et al., 1992
;
Nothwehr et al., 1995
;
Vida and Emr, 1995
). Vps1p is
a dynamin-like protein required for formation of endosome-bound vesicles from
the Golgi, and in vps1
mutants endosomal-bound transport is
diverted to the cell surface (Nothwehr et
al., 1995
).
These data indicate that the null V-ATPase mutants, as well as the
vph1 mutant, markedly slow down the endocytic process in the
yeast cells. To test this further, we stained the wild-type strain in the
presence of 3 µmol l-1 concanamycin A, a specific inhibitor of
V-ATPase. Fig. 9 shows that the
drug inhibited the internalization of FM4-64, making the wild type similar to
the stained V-ATPase null mutant: endosomal-like structures near the vacuolar
membrane are stained, but the stain does not reach the vacuole membrane.
|
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Discussion |
---|
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---|
V-ATPase is present along the secretory pathway and plays a central role in
its proper function, especially in the Golgi complex
(Moriyama and Nelson, 1989;
Grunow et al., 1999
;
Ying et al., 2000
;
Schoonderwoert et al., 2000
).
To demonstrate the subcellular location of Stv1p we used a Golgi fractionation
method (Graham and Krasnov,
1995
), which separates between cis- and
trans-Golgi. Using this gradient we found that Stv1p and Vph1p in the
wild type showed different profiles: Vph1p exhibited mainly an endosomal
pattern, and was probably on its way to the vacuole, whereas Stv1p peaked
nearer to trans-Golgi. V1 subunits Vma5p and Vma8p showed
a main peak in trans-Golgi, which might indicate that this location
has the most stable V-ATPase complex. The pH along the secretory pathway falls
from the ER through Golgi and reaches maximum acidity in lysosomes
(Grabe and Oster, 2001
;
Llopis et al., 1998
). In the
Golgi complex the trans-Golgi is the most acidic compartment, which
is in good agreement with the high levels of V-ATPase amounts described
above.
By disrupting one of the a subunits we can demonstrate that the other
partially compensates for the lack of its counterpart. Therefore, both mutants
of one isoform lack some features of the null V-ATPase mutant growth profile.
However, it may be that the ability of Stv1p to compensate for the lack of
Vph1p in the vph1 mutant is at much lower efficiency (Figs
3,
4) because it is less stable in
the vacuolar membrane (Kawasaki-Nishi et
al., 2001
). This is seen in the higher rate of growth at pH 7.5,
as well as in the processing of the vacuolar proteins ALP and CPY (see
Fig. 7B) when compared to
vph1
. We do not see the same results for the reciprocal mutant
(stv1
), which might indicate that Vph1p functions normally in
place of Stv1p (Fig. 7B). It
was therefore interesting to see that the specific activity of proton pumping
by V-ATPase in vacuoles isolated from the stv1
mutant was
diminished in comparison to the wild-type strain. Even though Vph1p itself is
overexpressed in the vacuolar membrane in the stv1
mutant
(Figs 3A,
4), there is less holoenzyme
present in it, hence the decrease in activity. Stv1p is therefore not
redundant in the wild type. The complex containing it has a role in its proper
functioning, as does the complex containing the Vph1p.
The same results were obtained by in vivo staining of these yeast
strains with a fluorescent pH indicator, LysoSensor Green. Examination of
stained yeast cells revealed residual staining in vacuolar membranes, in the
Vph1p null mutant (but not in the double mutant), which correlates with the
small but measurable H+ translocating activity of V-ATPase in the
vacuoles of the vph1 mutant
(Fig. 6).
The relationship between V-ATPase function and the endocytosis processes is
intriguing. On the one hand, endocytosis is responsible for the viability of
yeast V-ATPase null mutants, while on the other, some of the endocytosis
mutants were described to have a very similar phenotype to the V-ATPase null
mutant (Munn and Riezman,
1994; D'Hondt et al.,
2000
; Yoshida and Anraku,
2000
). The acidification of endosomal compartments due to V-ATPase
activity and its relation to endocytic processes, e.g. ligandreceptor
dissociation during receptor-mediated endocytosis, is well-established in
mammalian cells (Mellman et al.,
1986
). Other studies showed that endosomal carrier vesicle
formation, as well as transfer between late endosomes and lysosomes, are
pH-sensitive processes inhibited by the specific V-ATPase inhibitor
bafilomycin A (Clague et al.,
1994
; van Weert et al.,
1995
; van Deurs et al.,
1996
). In yeast, the same relationship has yet to be demonstrated.
The FM4-64 dye staining of yeast cells was used to visualize endocytosis
in vivo (Vida and Emr,
1995
), and to stain the V-ATPase null mutant in comparison to the
wild-type strain. We found inhibition of FM 4-64 internalization in the
V-ATPase null mutants. After treatment for 60 min, during which time all the
dye concentrated in the vacuolar membrane in the wild type, much of the
internalized dye in the mutants only reached endosomes surrounding the
vacuolar membrane. We could mimic the mutant in the wild-type cells by
addition of concanamycin A, which specifically inhibits V-ATPase activity
(Fig. 9).
Since the staining kinetics of this dye are used as a measure for
endocytosis, we conclude that V-ATPase mutants inhibit endocytosis in yeast as
well. We see that the staining of the plasma membrane (not shown) and of the
cytoplasmic vesicles, presumably endosomes, is similar for both the wild type
and null V-ATPase mutants. However, the final step of internalization of the
dye in the vacuolar membrane is markedly dependent on proper V-ATPase
functioning. FM4-64 was used to examine whether the stv1 or
vph1
might have a phenotype in endocytosis.
Fig. 8 shows that the Vph1p
null mutant displays the double-mutant phenotype, while the Stv1p null mutant
has similar staining kinetics to wild type but exhibits prevalent vacuolar
localization.
As expected for such a fundamental enzyme, its null mutation, if viable,
should affect a vital pathway or pathways of the cell. We conclude that the
V-ATPase function is to determine the pH conditions, not only in the lumen of
their respective organelles, but also in processes involved in intervesicular
activities connected with membrane fusion. A similar conclusion was reached by
Ungermann et al. (1999), who
showed that V-ATPase activity is needed for in vitro homotypic fusion
processes in yeast. This may relate to the recently published data on the
participation of the Vma3p of V-ATPase's Vo sector in membrane
fusion processes (Peters et al.,
2001
). Numerous protein complexes are involved in membrane fusion,
a process that is not yet completely resolved
(Wickner and Haas, 2000
;
Pelham, 2001
). The fact that
V-ATPase is suggested as taking part in various stages of this process, and
that there are multiple spontaneous revertants of V-ATPase null mutants
(Cohen et al., 1999
), hint that
the suppressor mutations come from the vast pool of the fusion machinery.
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Acknowledgments |
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