From the Department of Biochemistry & Molecular Biology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210
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ABSTRACT |
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The 27-kDa E subunit, encoded by the
VMA4 gene, is a peripheral membrane subunit of the yeast
vacuolar H+-ATPase. We have randomly mutagenized the
VMA4 gene in order to examine the structure and function of
the 27-kDa subunit. Cells lacking a functional VMA4 gene
are unable to grow at pH > 7 or in elevated concentrations of
CaCl2. Plasmid-borne, mutagenized vma4 genes
were screened for failure to complement these phenotypes. Mutants
producing Vma4 proteins detectable by immunoblot were selected; one
(vma4-1ts) is temperature conditional, exhibiting
the Vma phenotype only at elevated temperature
(37 °C). Sequencing revealed that a single point mutation, D145G,
was responsible for the phenotypes of the
vma4-1ts allele. The unassembled
27-kDa subunit made in the vma4-1ts
cells is rapidly degraded, particularly at 37 °C, but can be protected from degradation by prior assembly into the V-ATPase complex.
In purified vacuolar vesicles from the mutant cells, the peripheral
subunits are localized to the vacuolar membrane at decreased levels and
a comparably decreased level of ATPase activity (14% of the activity
in wild-type vesicles) is observed. When vma4-1ts
mutant cells are shifted to pH 7.5 medium at 37 °C, the cells become
enlarged and exhibit multiple large buds, elongated buds, and other
abnormal morphologies, together with delocalization of actin and
chitin, within 4 h. These phenotypes suggest connections between
the vacuolar ATPase, bud morphology, and cytokinesis that had not been
recognized previously.
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INTRODUCTION |
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Acidification of the intracellular compartments comprising the
vacuolar network plays an important role in membrane trafficking, protein sorting, protein degradation, ion homeostasis, and nutrient storage (reviewed in Refs. 1-3). Vacuolar-type ATPases
(V-ATPases)1 are present in
the vacuole/lysosome, Golgi apparatus, coated vesicles, chromaffin
granules, and synaptic membrane vesicles (1, 3). The yeast vacuolar
proton-translocating ATPase (H+-ATPase) is a multisubunit
complex that acidifies the yeast vacuole (4, 5), and biochemical and
genetic characterization indicates that it consists of at least 13 polypeptides (3). The enzyme complex is divided into a peripheral
cytoplasmic domain, V1, which contains the sites for ATP
hydrolysis, and an integral membrane domain, Vo, which
forms the proton channel. With the exception of the vph1
and stv1
mutants, which lack two different isoforms of
the 100-kDa subunit (6, 7), disruption of any of the ATPase subunit
genes in yeast results in an identical set of Vma
(vacuolar membrane
H+-ATPase) phenotypes, including a complete
loss of vacuolar acidification and bafilomycin A1-sensitive
ATPase activity in isolated vacuoles, an inability to grow in medium
buffered higher than pH 7 (with optimal growth at pH 5), sensitivity to
high extracellular Ca2+ concentration, and a number of
other physiological alterations (8-10).
The overall physiological consequences of loss of vacuolar acidification have recently been examined in a number of organisms other than yeast, either by construction of a chromosomal disruption of a V-ATPase subunit gene (11, 12), or by growth of cells in the presence of concanamycin A, a specific inhibitor of V-ATPases (13, 14). Disruption of the gene encoding the B subunit of the Drosophila V-ATPase resulted in a larval-lethal phenotype, with evidence that many or most different cell types required the gene product for growth (12), and disruption of the catalytic A subunit in Neurospora crassa also proved to be lethal (11). Growth of N. crassa in the presence of inhibiting concentrations of concanamycin A resulted in a conditional phenotype; growth of wild-type cells was inhibited at pH 7, but cells were able to grow in medium buffered to pH 5.8. This growth phenotype could be suppressed by mutations in the plasma membrane proton pump, but under all conditions, the cells showed gross morphological alterations in the presence of concanamycin A (14). Dictyostelium also exhibited gross morphological alterations in its endomembrane system when cells were grown in the presence of concanamycin A (13). These experiments suggest that V-ATPases play a critical but poorly defined role in overall cell physiology and morphology, in addition to their specific roles in endocytosis and protein sorting.
Even though the yeast Vma growth phenotype has been used
in screening for mutants showing defects in vacuolar acidification (8,
15, 16), the underlying mechanism for the growth arrest of yeast cells
at elevated pH or in high concentrations of CaCl2 is still
not known. The cell division cycle (CDC) of the yeast Saccharomyces cerevisiae involves several sequential
morphogenetic events before cytokinesis, including formation of a ring
of chitin (the "bud scar") at the site of the previous budding
events, and concentration of patches of actin filaments at the bud site
(17). During bud formation, the location of the pre-bud site is
specified by the mating type of the cell and the site of the previous
cytokinesis, and the cell polarity is maintained during bud growth. A
very large number of genes have been identified that affect progression of these morphological changes with the cell cycle. Cells with defects
in either CDC42 or CDC24 genes, which are
essential for the establishment for cell polarity, lose the asymmetric
location of actin filaments and the cell is arrested as large, round,
unbudded cell (18, 19). During the pre-bud site assembly, the actin rearrangement is triggered by the activations of CDC28 and
CLNs (20). Mutant cells defective in CDC3, CDC10,
CDC11, or CDC12 are unable to complete cytokinesis but
undergo multiple cycles of budding, DNA synthesis, and nuclear
division. The buds formed in these mutants are abnormally elongated
(21). There are also indications that alterations in cytoplasmic pH may
be linked to the cell cycle. Some plasma membrane H+-ATPase
(pma1) mutants fail to complete bud enlargement and
cytokinesis, and the cells accumulate nucleated small buds (22). A
significant change in cytoplasmic pH has been demonstrated during the
early part of the cell cycle (23), so the ATPase-mediated regulation of
internal pH may play a central role in regulating cell division.
The VMA4 gene encodes the 27-kDa E subunit of the peripheral
(V1) sector of the yeast vacuolar ATPase (24). Null mutants (vma4) revealed that the 27-kDa subunit is essential for
the assembly and activity of the vacuolar ATPase (15). The null vma4 mutants are viable, but exhibit a typical
Vma
phenotype (24). Vacuolar membranes prepared from the
vma4
mutants lacked the 69-, 60-, 42-kDa and other
peripheral subunits, whereas a significant portion of the 100-kDa
subunit (and/or the 75-kDa breakdown product) was still present on the
vacuolar membrane (15), suggesting that the 27-kDa subunit is required
for stable assembly of the V1 sector onto the vacuolar
membrane. Tomashek et al. (25) demonstrated that stability
of the Vma4 protein depended on the presence of the VMA10
gene product and that the VMA4 and VMA10 gene
products could be cross-linked, suggesting that these two
V1 subunits may interact as part of a "stalk" in the
V-ATPase.
In this study, we have randomly mutagenized the VMA4
gene in order to study the structure and function of the 27-kDa
subunit. One of the mutants (vma4-1ts) is
temperature sensitive, and only shows the Vma phenotype
at 37 °C. The 27-kDa subunit was made in this mutant, but rapidly
degraded at the non-permissive temperature. Assembly of the mutant
27-kDa subunit into V-ATPase complexes protected it from degradation
and the assembled complexes showed ATPase activity. When the
vma4-1ts cells were shifted to medium buffered to
pH > 7 at 37 °C, the cells show abnormal bud morphologies and
the delocalization of actin and chitin, suggesting the growth arrest of
vma4-1ts at elevated pH may result from the defects
in secretion and polarized growth. These experimental results reveal a
possible connection between the vacuolar ATPase, bud formation, and
polarized growth during the cell division cycle of budding yeast that
could be mediated by intracellular pH or Ca2+ changes.
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EXPERIMENTAL PROCEDURES |
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Materials, Strains, and Media-- Zymolyase 100T and Tran35S-label were purchased from ICN. Dithiobis(succinimidylpropionate) was obtained from Pierce. Rhodamine-phalloidin was purchased from Molecular Probes, and calcofluor white and DAPI (4',6-diamidino-2-phenylindole) were purchased from Sigma. Enzymes for molecular biology were obtained from Boehringer Mannheim and New England Biolabs; Sequenase was purchased from U. S. Biochemical Corp. All other reagents were purchased from Sigma.
The yeast strain SF838-1D (genotype: MATPlasmid and Strain Construction--
A 1.8-kilobase
BamHI fragment, containing the 699-base pair open reading
frame of the VMA4 gene, was cloned into the pRS315 shuttle
vector (28) at the BamHI site to generate plasmid pVMA4. A
BglII site was introduced into the VMA4 gene at
nucleotide 688 of the open reading frame. The deletion plasmid (pJW2)
was constructed by replacing the 650-base pair
HindIII-BglII fragment within the VMA4
open reading frame with a 1.1-kilobase URA3 gene fragments from YEp24 vector digested at the ClaI and SmaI
sites. The resulting plasmid was linearized with BamHI, and
the vma4::URA3 allele was integrated
into the VMA4 locus of both wild-type strains, replacing the
VMA4 gene by one-step gene disruption (29), to give deletion
strains SF838-5A
vma4
and SF838-1D
vma4
. Deletion of VMA4 was confirmed by
polymerase chain reaction of chromosomal DNA prepared from yeast cells
(30), using primers: 5'-TAGGTATACAAGCTGCTG and
5'-TTTCGGCCGACTTGTCCCTCGTTGCT. DNA sequencing was performed using a
Sequenase kit (U. S. Biochemical Corp.) and the dideoxy chain
termination method using the same primers (31). The plasmid pvma4-1 (consisting of the pVMA4 plasmid with the D145G
mutation in the VMA4 gene) was isolated from a yeast strain
expressing the temperature conditional Vma
phenotype
after screening as described below. A strain containing the
vma4-1ts allele integrated at the
VMA4 locus (JWY1) was generated by a two-step gene
replacement (32). The vma4-1ts allele was
cut from the pRS315 vector with XhoI and SacII
and then ligated to integrating vector pRS305 (28) digested with SalI and SacII to form plasmid pJW6. pJW6 was
digested with SphI to linearize the plasmid. SF838-5A
vma4
cells were transformed with the digested plasmid,
and integrants were selected by growth on supplemented minimal medium
lacking leucine. The integrants were then grown non-selectively (on
YEPD, pH 5.0 at 25 °C), and cells that became auxotrophic for uracil
were identified by growth on plates containing 5-fluoroorotic acid
(27). Integration of the vma4-1ts allele
at the VMA4 locus to generate strain JWY1 (with loss of the
vma4
::URA3 allele) were confirmed by the
presence of a fragment of the appropriate size by polymerase chain
reaction from genomic DNA, followed by sequencing of this polymerase
chain reaction fragment. The strain overproducing the
vma4-1ts allele
(JWY1/2µ-vma4-1ts) was generated by first cloning
the BamHI fragment containing the vma4-1ts
allele from pRS315 into YEp24 which had been digested with
BamHI to form plasmid 2µ-vma4-1ts, and
then transforming the JWY1 strain with this plasmid and selecting
uracil prototrophs.
Mutagenesis of VMA4 Gene and Screening for Vma
Cells--
The VMA4 gene on the pRS315 plasmid was
propagated in XL 1-Red cells, an Escherichia coli strain
which is deficient in three of the primary DNA repair pathways
(mutS, mutD, mutT) (Stratagene). The mutagenized plasmids
were transformed to the vma4
yeast strain by an overnight
lithium acetate procedure (33). Transformants were selected on
supplemented minimal medium lacking leucine. Vma
cells
were screened by checking the growth on YEPD, pH 7.5, 60 mM
CaCl2 and YEPD, pH 5.0, plates at 25, 30, and 37 °C.
Transformants that grew on pH 5.0 but failed to grow on pH 7.5 Ca2+ plates were selected for further characterization as
described (34).
Immunoprecipitation-- To study the assembly of vacuolar ATPase, immunoprecipitations were carried out under nondenaturing conditions in the presence of 0.6 mM dithiobis(succinimidylpropionate) added at the time of solubilization as described (35), except that cells were converted to spheroplasts at room temperature and shaken in supplemented minimal medium lacking methionine containing 1.2 M sorbitol for 20 min at 25 or 37 °C before labeling. ATPase complexes were immunoprecipitated with the 8B1 monoclonal antibody (36). To study the turnover of Vma4 protein, cells were converted to spheroplasts and labeled as for the nondenaturing immunoprecipitations and the chase was initiated by addition of unlabeled methionine and cysteine to 0.33 mg/ml each. At each time point, spheroplasts were pelleted by centrifugation and lysed in 100 µl of pre-warmed immunoprecipitation buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 1% SDS, 20 mM EDTA) by incubation at 75 °C for 20 min, the lysis mixture was diluted to 0.5 ml with water, then the mixture was placed on ice and pretreated with Protein A-Sepharose Cl-4B. After centrifugation to pellet the Protein A-Sepharose, the supernatant was incubated overnight with 3 µl of rabbit anti-27-kDa polyclonal antiserum (a generous gift from Tom Stevens and Margaret Ho). Protein A-Sepharose was then added to precipitate the immune complexes, and immunoprecipitated proteins were washed, solubilized, and analyzed by SDS-PAGE as described (35). Duplicate samples for each time point were quantitated on a Molecular Dynamics PhosphorImager (Model 425E).
Isolation of Vacuolar Vesicles and ATPase Activity
Assay--
Vacuolar vesicles were prepared as described (37), except
that the temperature-sensitive mutant cells were grown overnight at
25 °C and all of samples were converted to spheroplasts at room
temperature. JWY1/2µ-vma4-1ts cells and
SF838-5A cells transformed with YEp24 were grown in supplemented
minimal medium lacking uracil to an optical density at 600 nm
(OD600) of 0.8-1.0 before harvesting. The measurement of
vacuolar ATPase activity was performed on a Beckman DU640
spectrophotometer using a coupled enzyme assay at 25 °C (38). To
examine the thermal stability of the V-ATPase, both types of vesicles
were incubated at 25 and 37 °C for 30 and 60 min, then the activity
was assayed at 25 °C and samples were taken for Western blotting. In
addition, the stability of the activity in the mutant vesicles was
monitored by performing the coupled enzyme assay at 37 °C. No
decrease in activity was observed over 30 min. Protein concentrations
were determined by the Lowry assay (39). Concanamycin A-sensitive ATPase activity was determined by comparing the ATPase activity with
and without a 20-min preincubation with 100 nM concanamycin A (40).
Western Blotting-- To determine the levels of the 69-, 60-, 42- and 27-kDa subunits in whole cells, the cells were grown overnight at 25 °C, then diluted to equivalent density in YEPD, pH 5.0, medium and grown at both 25 and 37 °C. Equal numbers of cells (~107) were removed at various times, and whole cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting as described previously (41). Vacuolar proteins were also detected as described previously (41). The 10D7, 7D5, 13D11, and 7A2 monoclonal antibodies were used to detect the 100-, 69-, 60-, and 42-kDa subunits (41), and polyclonal antisera raised against the 27-kDa subunit were used to detect Vma4p (15).
Fluorescence Microscopy--
For staining with quinacrine, cells
were grown under various conditions at 25 or 37 °C to logarithmic
phase, then 0.5 × 107 cells were harvested, washed
with phosphate-buffered saline, pH 7.5, containing 2% glucose,
incubated with 200 µM quinacrine, pH 7.5, for 5 min, and
visualized immediately as described (37). To visualize actin in whole
cells (42), cells were fixed in 3.7% formaldehyde for 1 h after a
defined period of growth at 25 or 37 °C. Cells were sonicated,
pelleted, washed, and resuspended in phosphate-buffered saline. Then
fixed cells were incubated with 0.6 µM
rhodamine-phalloidin for 2 h in the dark. Finally, cells were
washed extensively with phosphate-buffered saline and resuspended in
mounting medium (90% glycerol, 0.1 mg/ml -phenylenediamine). Immunofluorescence micrographs were obtained by using an Axioskop (Zeiss) microscope under fluorescein isothiocyanate optics for observation of quinacrine staining and rhodamine optics for observation of actin with a × 100 objective. To detect chitin localization, cells were grown overnight at 25 °C to the mid-logarithmic phase, then diluted to the same density in YEPD, pH 5.0, or YEPD, pH 7.3, medium and grown at 25 or 37 °C. Constant numbers of cells (0.3 × 107) were taken at various times and briefly sonicated
in microcentrifuge tubes to disperse clumps, then pelleted and washed
with water. Chitin labeling of bud scars was observed after incubation
of cells in a 300 µg/ml solution of calcofluor for 5 min in the dark (42). For visualization of nuclei, 1 µg/ml DAPI was included in the
mounting medium (42). Chitin and DAPI staining were observed using a UV
filter set. Photographs were taken using Tmax 400 film (Kodak). Images
were printed on Rapitone paper and arranged using Adobe Photoshop 3.0. Morphology of the cells was observed under Nomarski optics. In
scoring populations of wild-type and mutant cells under various
conditions, at least 150 cells for each condition were analyzed and
large budded cells were defined as cells where the diameter of the
smallest spheroid was greater than half of the diameter of the
largest one.
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RESULTS |
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Identification of a Temperature Conditional Mutation in
VMA4--
In order to better characterize the structural and
functional roles of the VMA4 gene product, we randomly
mutagenized the gene in vitro and then screened for
mutations that affected vacuolar H+-ATPase function
in vivo. Deletion of the VMA4 gene leads to the loss of growth on medium buffered to pH greater than 7 or medium containing high concentrations of CaCl2, with the most
severe growth defects occurring in medium that has both elevated
calcium concentrations and elevated pH (8, 9). The Vma
phenotype can be complemented by transforming the vma4
strain with the plasmid carrying VMA4 gene, as shown in Fig.
1A. We randomly mutagenized
the VMA4 gene by transforming the mutator E. coli strain XL 1-Red with a plasmid-borne copy of the gene. The random mutation rate in the mutator strain was reported to be about one base
change per 2000 nucleotides (43). The mutagenized vma4 plasmids were used to transform a vma4
yeast strain.
Transformants were selected by leucine prototrophy and mutant plasmids
failing to complement Vma
phenotypes were identified.
About 2,300 transformants were screened and 6 colonies exhibiting a
Vma
phenotype were selected. From this collection, only 5 mutants producing stable Vma4 protein based on Western blot analysis
were identified. When the mutants were screened further for temperature dependence of the pH-sensitive growth phenotype, one of these mutants
(vma4-1ts) proved to be temperature conditional,
exhibiting the Vma
phenotype only at elevated temperature
(37 °C). As shown in Fig. 1A, vma4-1ts cells grew
normally at the permissive temperature (25 °C or 30 °C). When
cells were grown at 37 °C, however, the vma4-1ts
cells failed to grow on YEPD, pH 7.5, + 60 mM
CaCl2 medium, but remained capable of growth in pH 5.0 medium. The vma4-1ts mutation was then integrated
into the genome by pop-in, pop-out gene replacement (32). The resulting
mutant (JWY1) showed the same temperature and pH conditional growth
phenotype as the vma4-1ts mutation borne on a
CEN-plasmid. Wild-type cells transformed with the
vma4-1ts mutant on a CEN-plasmid showed normal
growth at pH 7.5 at 37 °C, suggesting that the mutation is
recessive.
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Biochemical Analysis of the vma4-1ts Mutant-- Production of 27-kDa subunit protein in cells grown at 30 °C was one of the criteria in our mutant screen, but we investigated whether the temperature dependence of the vma4-1ts mutant might be related to protein stability. Western blot analysis of whole cell lysates revealed that vma4-1ts cells growing at the permissive temperature (25 °C) have a somewhat lower steady-state level of the Vma4 protein (27-kDa) than wild-type cells, but mutant cells grown overnight at 37 °C have no detectable 27-kDa subunit (Fig. 2A). Both cell lines have comparable levels of the 69-kDa V1 subunit at both temperatures (Fig. 2A). The time course for the loss of the 27-kDa subunit with a temperature shift is shown in Fig. 2B. After a shift to 37 °C, there was a dramatic loss of the 27-kDa subunit in vma4-1ts cells over several hours, with about 50% of the protein seen at 25 °C remaining after 8 h at 37 °C, and none detectable after 24 h. Other peripheral subunits of the V-ATPase, the Vma1 (69-kDa), Vma2 (60-kDa), and Vma5 (42-kDa) proteins remained at the same levels in whole cell lysates from vma4-1ts cells at both permissive and nonpermissive temperatures (Fig. 2B). Wild-type cells show no decrease in the level of the 27-kDa subunit during incubation at 37 °C (Fig. 2C).
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Loss of Acidification at Elevated Temperature in vma4-1ts
Mutants--
Uptake of the fluorescent lysosomotropic amine quinacrine
into the yeast vacuole provides a qualitative measure of vacuolar acidification in vivo (37). In order to determine the
approximate rate at which vacuolar acidification is lost in the
vma4-1ts mutant after a shift to 37 °C, we
stained cells grown under various conditions with quinacrine and
visualized the staining by fluorescence microscopy (Fig.
5). As shown in Fig. 5, G and
I, wild-type cells show bright staining with quinacrine
(left) that colocalizes with vacuoles visualized under
Nomarski optics (right) whether they are incubated at
37 °C in pH 7.3 medium or at 25 °C in pH 5 medium. vma4 mutants show no quinacrine uptake under either
condition (Fig. 5, H and J, left),
despite the presence of vacuoles of normal morphology
(right). vma4-1ts mutants accumulate
quinacrine into the vacuole and show staining comparable to that of
wild-type cells when grown at 25 °C (Fig. 5, A and
C). After 1 h of growth at 37 °C, however, the
vma4-1ts cells showed slightly diminished quinacrine
staining (Fig. 5B), and after 3 h at 37 °C,
quinacrine staining of the vacuole was completely gone (Fig. 5,
D-F). After 3 h at 37 °C, some of the vma4-1ts cells appeared to show some cytoplasmic
staining (Fig. 5, D and F), but this staining was
excluded from the vacuole and was also seen in a small population of
vma4
cells. These results suggest that the V-ATPase had
lost its ability to maintain a proton flux across the vacuolar membrane
in the vma4-1ts mutant after 3 h at 37 °C,
regardless of whether the mutant was grown at pH 7.3 (Fig.
5D), pH 5.0 (Fig. 5E), or in 100 mM
CaCl2 (Fig. 5F). We cannot eliminate the
possibility that mutant cells growing at pH 5.0 maintain some
acidification of the vacuole by a means other than proton pumping by
the V-ATPase (for example, endocytosis (46)) because the actual
quinacrine staining must be carried out at elevated pH to allow
deprotonation of the amine.
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The vma4-1ts Cells Also Show Defects in Bud Morphology and
Cytokinesis--
Although the Vma phenotype has been
used frequently for the identification of mutants lacking vacuolar
H+-ATPase activity, the basis of the pH and
Ca2+-sensitive growth accompanying loss of V-ATPase
activity is not well understood. The vma4-1ts mutant
is the first temperature conditional vma mutant to be characterized, and it could be an ideal tool for examining the onset of
Vma
phenotypes and determining the biological basis of
these phenotypes. To examine the onset of the Vma
phenotypes, haploid vma4-1ts cells were first grown
overnight to logarithmic phase at 25 °C in medium buffered to pH 5, then cells were transferred to pH 7.3 medium and shifted to 37 °C.
Control experiments were done by growing the cells in pH 5 medium at 25 and 37 °C and in pH 7.3 medium at 25 °C. Microscopic analysis of
cells shifted to pH 7.3 medium and incubated at 37 °C revealed that
up to 25% of the vma4-1ts cells showed abnormally
elongated or multiple buds after 4 h (Fig.
6). Under similar conditions, only 4% of
vma4
cells had abnormal buds, and almost none of the
wild-type cells showed the aberrant morphologies. There was a
pronounced increase in the number of unbudded cells after
vma4
cells were shifted to pH 7.3 medium for 24 h
(Fig. 6), although even at pH 5.0, the vma4
mutant
appears to contain a higher proportion of unbudded cells than the other
strains.
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DISCUSSION |
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The yeast vacuolar H+-ATPase closely resembles the vacuolar-type ATPases from other fungi, plant, and animal cells (3, 4). Although all of the vacuolar H+-ATPases that have been characterized appear to have at least eight subunits, the functions of only three of these subunits, the catalytic ATP-hydrolyzing subunit of approximately 70-kDa, the regulatory ATP-binding subunit of approximately 60-kDa, and the 17-kDa proteolipid involved in formation of the proton pore, have been clearly defined (3, 5). These three subunits exhibit homology to subunits of the F1F0-ATPase that reflects an evolutionary relationship between the two enzymes (47).
The 26-30-kDa E subunit of the vacuolar ATPase has been cloned from a
number of species (Ref. 45, and references therein). The secondary
structure of all of the sequenced E subunits is predicted to be
predominantly -helical, with conserved NH2 and COOH
termini, even though the overall sequence of Vma4 protein is not highly
conserved (showing roughly 25% overall identity) (44, 48). Although
the primary sequence of the Vma4 subunit does not show significant
similarity to any subunit of the F-type ATPase, it has been suggested
that this subunit may have an analogous role in the V-type ATPase to
that of the
-subunit in the F-type ATPase, which also has highly
conserved
-helical structure near the COOH and NH2
termini (44). The
-subunit is part of the stalk region of
F1-ATPases and interacts directly with
- and
-subunits through the
-helical regions (49), presumably to help
communicate conformational changes in these subunits to the proton
pore. Tomashek et al. (25) have provided recent structural evidence that the yeast Vma4p may also behave as a stalk subunit in
combination with the Vma10p, Vma7p, and Vma8p subunits of the V-ATPase.
Asp145 of S. cerevisiae VMA4 is highly conserved
among sequences cloned from other species, and is near the beginning of
the COOH-terminal -helices predicted by computer analysis (44). The
D145G mutation responsible for the phenotypes of the
vma4-1ts mutant appears to act primarily by
destabilizing the unassembled protein. Although the mutant 27-kDa
subunit is less stable than wild-type at both the permissive and
nonpermissive temperatures, a larger proportion of the protein appears
to be immediately degraded at 37 °C and the fraction of the protein
that is not immediately degraded is not competent for assembly. At
25 °C, the stable fraction of the protein appears to be fully
competent for assembly and, in fact, to be protected by assembly from
degradation during a subsequent incubation at 37 °C (Fig. 3).
Complementation of the Vma
phenotype implies that
V-ATPases assembled in the vma4-1ts mutant at
25 °C must exhibit some V-ATPase activity, and mutant vacuoles
isolated from cells grown at 25 °C contain 14% of the concanamycin
A-sensitive activity of wild-type vacuoles and a comparably reduced
level of the peripheral subunits (Fig. 4). Previous results have
indicated that complementation of Vma
phenotype requires
approximately 25% of the wild-type V-ATPase activity (34), but the
vacuole isolation takes several hours and it is possible that there is
more loss of activity from the mutant enzyme during this time than
there is from the wild-type enzyme. Incubation of the mutant vacuolar
membranes at 37 °C indicated that both the ATPase activity and the
V1 subunits present in these membranes were stable for at
least 1 h, but we cannot eliminate the possibility that there is
some loss of ATP-driven proton pumping during incubation at elevated
temperature. Consistent with this possibility, in Fig. 5 there seems to
be some loss of quinacrine staining in the mutant strain after 1 h
at 37 °C. When the mutant V-ATPase is synthesized at 37 °C,
assembly of newly synthesized 27-kDa subunit is almost completely
eliminated and V-ATPase complexes lacking the 27-kDa subunit but
containing most or all of the other subunits appear, at least in
relatively short-term experiments (Fig. 3D). In contrast,
immunoprecipitations from the vma4
strain with antibodies
against the 60- and 69-kDa subunits detected only a complex containing
these two subunits (50) after 60 min labeling, although we have more
recently detected an interaction with the 100-kDa subunit as
well.3 We have not fully
explored the possibility that the unstable 27-kDa subunit synthesized
at 37 °C in vma4-1ts cells supports assembly of
different partial complexes than those present in the
vma4
strain; however, the fact that the mutation is
recessive suggests that any partial complexes formed are not functional.
The vma4-1ts strain exhibits distinct phenotypic
differences from the vma4 strain. vma4
cells lose viability much more rapidly during an incubation at pH 7.5 than the vma4-1ts cells. The morphological
phenotypes of the vma4
cells are not identical to those
of the vma4-1ts mutant, either. Although both
vma4
and vma4-1ts mutants show actin
and chitin delocalization at pH 7.3, the vma4-1ts
mutant showed much more dramatic morphological phenotypes, including elongated and multiple buds, while vma4
cells accumulated
primarily unbudded cells (Figs. 6, 8, and 9). There are several
possible explanations for these differences. First, there may be a
physiological difference between "long-term" loss of vacuolar
H+-ATPase function in the vma4
mutant and the
more sudden loss of ATPase function caused by shifting the
vma4-1ts mutant to elevated temperature. As
described below, the effects of the vma mutants on
morphology probably result from changes in cellular ion balances. It
may be that other ion channels or pumps are recruited to partially
compensate for loss of the V-ATPase during long-term growth of
vma4
mutant, but there is no time for such an adaptation
when the vma4-1ts mutant is shifted to elevated
temperature. Consistent with this explanation, Bowman et al.
(14) have reported that mutations in the plasma membrane proton pump of
Neurospora can partially compensate for the defects of a
strain grown in concanamycin A, and Temesvari et al. (13)
have also observed adaptation to concanamycin A in
Dictyostelium. It could be that some of these adaptations, which help sustain the vma4
mutant yeast strain at pH
5.5, may accelerate its demise at pH 7.3. Alternatively, the long-term growth defects of the vma4
strain, which cause retarded
growth even under optimal conditions, pH 5.5, may weaken the
cells to such an extent that they rapidly decline at pH 7.3, while the vma4-1ts mutants are able to display more dramatic
morphological aberrations because they maintain viability. A third
possibility is that the vma4-1ts mutant is not a
true "loss of function" mutation, but this possibility seems
inconsistent with the observation that the mutation is recessive.
The pH-dependent growth of vma mutants has been extensively exploited in screening for vma mutants, but the physiological reason that cells lacking vacuolar H+-ATPase activity lose viability at elevated extracellular pH is not understood. Characterization of the vma mutants has focused primarily on changes inside the vacuolar lumen or in the later stages of the vacuolar network, including loss of organelle acidification, defects in protein sorting or processing, and depletion of Ca2+ and metabolite stores (8, 10, 51, 52), but none of these changes has been directly connected to a pH-dependent growth defect. Based on the phenotypes of the vma4-1ts mutant at 37 °C, it appears that the vacuolar H+-ATPase is also essential for several functions occurring outside the vacuolar network, including maintenance of a polarized actin distribution, normal bud morphology, and cytokinesis when cells are at elevated pH (pH > 7).
The observation that the vma4-1ts mutant affected
these processes under the nonpermissive conditions was somewhat
surprising but not unprecedented. Previous studies have suggested a
role for the vacuolar H+-ATPase in actin distribution and
cell morphology. vma mutants exhibit synthetic lethality
with several end mutants, including the end3
and end4
mutants, and this has been attributed to a requirement for endocytosis in mutants lacking a functional V-ATPase (46). An additional explanation, however, is that both the
end mutants and the vma mutants impair certain
functions of the actin cytoskeleton, affecting its roles in cell growth
as well as in endocytosis, such that the combination of mutants is
lethal. Specifically, the END4 gene is allelic to
SLA2, a gene identified from synthetic lethal interactions
with actin mutants. sla2
mutants resemble the
vma4-1ts mutants in that cells become enlarged and
show actin delocalization at 37 °C (53). end3
mutants
also show delocalization of actin and chitin and budding defects
indicative of disrupted actin function (54). Growth of
Neurospora in the presence of concanamycin A results in a
number of dramatic morphological phenotypes that could be
associated with cytoskeletal disruption (14). In addition, certain mutations in the PMA1 gene, which encodes the yeast
plasma membrane H+-ATPase, cause defects in cellular
morphology and cytokinesis, suggesting these processes might be
generally connected to pH homeostasis. In the pma1-105
mutant, cells arrest with multiple, small nucleated buds (22), and two
mutants containing PMA1 promoter mutations that reduce
activity of the enzyme appear to cause an elongated bud morphology
(55).
It is unlikely that a V-ATPase subunit plays a direct role in
morphological or cell cycle processes, but loss of V-ATPase function
has the potential to change a number of other cellular parameters that
have been linked directly to the cell cycle. The yeast vacuolar
H+-ATPase is involved in Ca2+ and pH
homeostasis and in sequestration of a number of other metal ions. The
vacuole is the major Ca2+ store in yeast cells, and uptake
of Ca2+ into the vacuole is mediated by a
Ca2+/H+ antiporter driven by the proton
gradient that is established by the vacuolar H+-ATPase
(56). vma mutants have been shown to have elevated levels of
cytoplasmic Ca2+, apparently as a result of inability to
take up Ca2+ into the vacuole. A vma4 mutant
was shown to have a cytosolic free ionized calcium level
([Ca2+]c) of 1.8 µM when cells were
preincubated with glucose and exposed to 10 mM
extracellular CaCl2 (57); in the absence of added
extracellular CaCl2, other vma mutants exhibit a
[Ca2+]c of 900 nM under conditions
when the wild-type [Ca2+]c was 150 nM
(8). The role played by the vacuolar H+-ATPase in
regulating cytosolic pH in yeast has not been clearly defined, but
vacuolar H+-ATPases are involved in pH homeostasis in other
systems (58). Wild-type yeast cells maintain a relatively constant
cytosolic pH with extracellular pH values varying from 3.0 to 7.5 (59), and are also able to grow over a wide range of pH values. It has not
been established whether vma mutants lose control of
cytosolic pH at elevated pH (>7) where growth of the mutants is
affected. The vacuole is also a store for a number of other divalent
cations and other metabolites, most of which require the vacuolar pH
gradient for uptake (60), so loss of vacuolar H+-ATPase
activity would be predicted to elevate cytosolic levels of these ions
and metabolites as well. Because the vacuolar pH gradient is a common
currency for movement of a number of ions from cytosol to vacuole, it
will be difficult to separate the roles of the vacuolar
H+-ATPase in pH regulation and regulation of other ion
movements.
Cytosolic pH and Ca2+ concentrations can potentially affect
yeast cell morphology and cell cycle progression in a number of ways
(61, 62), many of which could account for the phenotypes of the
vma4-1ts mutant. Depletion of intracellular and
extracellular Ca2+ has been shown to generate first a pause
in the cell cycle at G1, and subsequently a full arrest at
G2/M, resulting in an accumulation of small-budded 2N cells
(61). This result suggests that Ca2+ regulation may be
important at multiple places in the cell cycle, and therefore, the
influence of various regulators of cytosolic Ca2+,
including the vacuolar H+-ATPase, may vary at different
points in the cell cycle. The morphological defects of the
vma4-1ts mutant, including multiple and elongated
buds that contain nuclei, suggest a defect in G2/M phase.
The essential targets for Ca2+ are not clear, but a number
of potential targets have been identified. CDC24, which is
essential for establishment of cell polarity and organization of actin
toward the bud site (19), is predicted to be a Ca2+-binding
protein based on its sequence (63). Interestingly, a vma5
mutation was recently demonstrated to show synthetic lethality with the
cdc24-4 allele (64). Actin itself exhibits Ca2+
binding properties that may influence its function, and a number of
actin-binding proteins also appear to bind Ca2+ (65). The
link between intracellular pH in regulation of cellular differentiation
and proliferation is also well established in a number of cell types
(66, 67). Although intracellular pH changes are difficult to measure in
yeast cells and changes during the cell cycle have not been studied
extensively, it has been demonstrated that starved yeast cells
re-entering the cell cycle experience a rise in cytosolic pH of as much
as 0.55 pH units (cytosolic pH 6.75 rising to pH 7.3) at approximately
the time of DNA synthesis (23). One particularly intriguing target of pH regulation, given the phenotypes of the vma4 and
vma4-1ts mutants, is actin itself. In
Dictyostelium, intracellular pH may affect the dynamics of
the cytoskeleton by modulating the interaction of EF-1
, which acts
as an F-actin-binding protein, with F-actin (68, 69). In hamster lung
fibroblast cells, the activation of the Na+-H+
exchanger, NHE1, which plays a central role in intracellular pH
homeostasis, is necessary for RhoA-mediated assembly of stress fibers
(70).
Many of the defects seen in the vma4-1ts mutant cells, including actin and chitin delocalization, enlargement of cells, and abnormal bud morphology are very similar to those seen in yeast actin and actin-binding protein mutants (Ref. 71, and references therein). One relatively simple hypothesis, consistent with the phenotypes of the vma4-1ts mutants, is that when extracellular pH values rise above 7, the vacuolar H+-ATPase plays an essential role in maintaining proper actin localization and that loss of organization in the actin cytoskeleton results in the other phenotypes of the vma4-1ts mutant and other vma mutants. In future experiments we will attempt to address the specific connections between the vacuolar H+-ATPase and actin functions.
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ACKNOWLEDGEMENTS |
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We thank Dave Amberg, Dave Gilbert, and Dave Turner for the use of their microscopes, Dave Amberg for helpful suggestions on this work, and Tom Stevens and Margaret Ho for polyclonal antisera against Vma4 protein.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01-GM50322, American Heart Association, New York State Affiliate Grant 930325, and matching funds from Presidential Young Investigator Award MCB-9296244 (to P. M. K.).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.
American Heart Association Established Investigator. To whom
correspondence should be addressed. Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}hscsyr.edu.
1 The abbreviations used are: V-ATPase, vacuolar-type ATPase; DAPI, 4',6-diamidino-2-phenylindole; H+-ATPase, proton-translocating ATPase; Vma, vacuolar membrane H+-ATPase; YEPD, yeast extract, peptone, 2% dextrose medium; V1, peripheral sector of the vacuolar H+-ATPase; Vo, membrane sector of the vacuolar H+-ATPase; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; CDC, cell division cycle.
2 M. Tarsio and P. Kane, unpublished data.
3 P. Kane, M. Tarsio, and J. Liu, manuscript in preparation.
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REFERENCES |
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