INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

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, V_o, 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 (vma4-1^ts) 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 vma4-1^ts 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 vma4-1^ts 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.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

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.

MAT, ade6, leu2-3, leu2-112, ura3-52, pep4-3, gal2

MAT, ade6, leu2-3, leu2-112, ura3-52, gal2

Plasmid 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 vma4-1^ts allele integrated at the VMA4 locus (JWY1) was generated by a two-step gene replacement (32). The vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts allele (JWY1/2µ-vma4-1^ts) was generated by first cloning the BamHI fragment containing the vma4-1^ts allele from pRS315 into YEp24 which had been digested with BamHI to form plasmid 2µ-vma4-1^ts, 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-vma4-1^ts 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.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

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 (vma4-1^ts) proved to be temperature conditional, exhibiting the Vma phenotype only at elevated temperature (37 °C). As shown in Fig. 1A, vma4-1^ts cells grew normally at the permissive temperature (25 °C or 30 °C). When cells were grown at 37 °C, however, the vma4-1^ts cells failed to grow on YEPD, pH 7.5, + 60 mM CaCl₂ medium, but remained capable of growth in pH 5.0 medium. The vma4-1^ts 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 vma4-1^ts mutation borne on a CEN-plasmid. Wild-type cells transformed with the vma4-1^ts mutant on a CEN-plasmid showed normal growth at pH 7.5 at 37 °C, suggesting that the mutation is recessive.

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Genetic characteristics of the vma4-1ts mutant. A, the vma4-1ts mutant exhibits a Vma phenotype at elevated temperature. Cells were streaked as indicated on YEPD, pH 7.5, + 60 mM CaCl2 or YEPD, pH 5.0, plates and incubated at 25 °C or 37 °C for 3 days. Strains shown are wild-type (SF838-1D); vma4 (SF838-1Dvma4); vma4//pVMA4 (SF838-1Dvma4 carrying the wild-type VMA4 gene on the pRS315 (CEN) plasmid); vma4/pvma4ts (SF838-1Dvma4 carrying the vma4-1ts allele on the pRS315 plasmid). B, multiple sequence alignment of the region surrounding the mutation in the yeast vma4-1ts mutant. The vma4-1ts has a single amino acid change at Asp145  Gly; the aspartate mutated is conserved in eight genes for the 27-kDa subunit that have been cloned from different species.

Biochemical Analysis of the vma4-1^ts 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 vma4-1^ts mutant might be related to protein stability. Western blot analysis of whole cell lysates revealed that vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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).

View larger version (62K): [in this window] [in a new window]   Fig. 2.   The 27-kDa subunit is destabilized in the vma4-1ts mutant. A, steady-state levels of the 27- and 69-kDa subunits in wild-type (1) and vma4-1ts mutant (2) cells grown overnight in YEPD, pH 5, medium at the indicated temperature. B and C, time course of decay of the mutant 27-kDa subunit at 37 °C. The vma4-1ts mutant (SF838-1Dvma4/pvma4-1ts; B, and wild-type cells (SF838-1D; C, were grown overnight at 25 °C, then diluted to the same density in YEPD, pH 5.0, medium, and growth continued at 25 or 37 °C for the indicated times. For A-C, samples corresponding to a constant number of cells were removed from each culture and whole cell lysates were prepared after the indicated period of growth. The lysates were separated by SDS-polyacrylamide gel electrophoresis and V-ATPase subunits were identified by Western blotting. The 69-, 60-, and 42-kDa subunits were detected with monoclonal antibodies 8B1, 13D11, and 7A2, respectively, and the 27-kDa subunit was detected with subunit-specific polyclonal antisera as described under "Experimental Procedures."

View larger version (18K): [in this window] [in a new window]   Fig. 3.   The newly synthesized 27-kDa subunit protein is rapidly degraded in the vma4-1ts mutant, but can be protected from degradation by assembly at 25 °C. A, wild-type cells (SF838-1D) were converted to spheroplasts and pulse-labeled for 5 min at 25 () or 37 °C () with Tran[35S]-label, then chased with an excess of unlabeled methionine and cysteine at the same temperature for the indicated times. The spheroplasts were rapidly lysed and the 27-kDa subunit was immunoprecipitated as described under "Experimental Procedures." Immunoprecipitates were analyzed by SDS-PAGE and the 27-kDa band was quantitated on a PhosphorImager. The amount of Vma4 protein is expressed as a percentage of the Vma4 protein present immediately after the 5-min pulse at 25 °C. Error bars represent the range of duplicate samples. B, the vma4-1ts mutant (JWY1) was subjected to the same pulse and chase conditions and then quantitated as in A. Parallel samples were again labeled and chased at 25 () or 37 °C (). C, a strain overproducing the vma4-1ts allele (JWY1/2µ-vma4-1ts) was subjected to the same pulse and chase conditions and then quantitated as in A. Parallel samples were again labeled and chased at 25 () or 37 °C (). D, assembly of the 27-kDa subunit into V-ATPase complexes protects the subunit from degradation. The overproducing strain used in C (JWY1/2µ-vma4-1ts) was converted to spheroplasts, labeled for 60 min with Tran35S-label at either 25 or 37 °C, and then chased in the presence of excess unlabeled methionine and cysteine under the following conditions: 60-min pulse at 25 °C, no chase (lane 1); 60-min pulse at 25 °C, 60-min chase at 25 °C (lane 2); 60-min pulse at 25 °C, 60-min chase at 37 °C (lane 3); 60-min pulse at 37 °C, no chase (lane 4); 60-min pulse at 37 °C, 60-min chase at 37 °C (lane 5). Each sample was then solubilized under nondenaturing conditions, cross-linked with DSP, and immunoprecipitated with the 8B1 antibody, which recognizes the 69-kDa subunit, followed by Protein A-Sepharose (35). Immunoprecipitates were then separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.

vma4

View larger version (47K): [in this window] [in a new window]   Fig. 4.   The peripheral subunits are present at reduced levels in vacuoles isolated from the vma4-1ts mutant. Vacuolar vesicles were prepared from wild-type cells (SF838-5A) carrying the YEp24 plasmid and cells overproducing the vma4-1ts allele (JWY1/2µ-vma4-1ts). A, quantitative comparison of peripheral subunits in wild-type and mutant vacuoles. The indicated masses of vacuolar protein (0.063-1.0 µg for wild-type, 1.0 µg for the mutant) were separated by SDS-polyacrylamide gel electrophoresis and subjected to Western blotting as described under "Experimental Procedures." B, thermal stability of the 27-kDa subunit in isolated wild-type and mutant vacuoles. 5 µg of vacuolar protein was loaded to detect 100- and 42-kDa subunits, and 1.5 µg to detect 69-, 60-, and 27-kDa subunits. Proteins were separated by SDS-PAGE, and analyzed by Western blotting with monoclonal antibodies against 100-, 69-, 60-, 42-kDa subunits and polyclonal antibodies against 27-kDa subunit. Vacuolar vesicles from each cell type were incubated for varying times at 25 and 37 °C before solubilization, as follows: lane 1, no incubation at 25 or 37 °C; lane 2, 30-min incubation at 25 °C; lane 3, 60-min incubation at 25 °C; lane 4, 30-min incubation at 37 °C; lane 5, 60-min incubation at 37 °C.

Loss of Acidification at Elevated Temperature in vma4-1^ts 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 vma4-1^ts 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). vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Quinacrine uptake by wild-type and mutant cells grown under varied conditions. vma4-1ts mutant cells (SF838-1Dvma4/pvma4-1ts) were grown to log phase in YEPD, pH 5, at 25 °C, then shifted to YEPD, pH 5, at 25 °C for an additional 1 h (A) or 3 h (C), YEPD, pH 7.3, at 37 °C for 1 h (B) or 3 h (D), YEPD, pH 5, at 37 °C for 3 h (E), or YEPD, pH 5, containing 100 mM CaCl2 at 37 °C for 3 h (F). After the indicated time in each growth condition, cells were harvested, incubated with 200 µM quinacrine at pH 7.5 for 5 min, washed, and visualized immediately. Wild-type cells (G and I) and SF838-1Dvma4 mutants (H and J) were visualized after growth in YEPD, pH 7.3, at 37 °C for 3 h (G and H) or in YEPD, pH 5.0, at 25 °C for 3 h (I and J), and stained with quinacrine as described for the vma4-1ts mutant. For each condition, immunofluorescence micrographs obtained under fluorescein isothiocyanate optics are shown on the left and Nomarski images of the same cells are shown on the right. Each panel represents two different fields of cells arranged using Adobe Photoshop.

The vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Quantitation of the cellular morphologies under different growth conditions. Wild-type cells (SF838-1D; open bar), vma4 cells (SF838-1Dvma4, black bar), and vma4-1ts cells (SF838-1Dvma4/pvma4-1ts, gray bar) were grown to log phase at 25 °C and then shifted to YEPD, pH 5, at 25 °C or YEPD, pH 7.3, at 37 °C for the indicated time before examining their morphology under Nomarski optics. Percentages of cells with the following morphologies are shown: UB, unbudded; SB, small budded (daughter cell less than 50% of the diameter of the mother cell); LB, large budded (daughter cell greater than 50% of the diameter of the mother cell); and AM, showing abnormal morphology, specifically multiple buds and/or elongated buds. At least 150 cells from each strain under each condition were scored to obtain the indicated percentages.

vma4

vma4

View larger version (30K): [in this window] [in a new window]   Fig. 7.   A, vma4-1ts mutant cells retain viability during incubation in YEPD, pH 7.5, at 37 °C. Wild-type (SF838-1D), vma4 (SF838-1Dvma4), and vma4-1ts (SF838-1Dvma4/pvma4-1ts) cells were grown in liquid cultures under permissive conditions, YEPD, pH 5.0, at 25 °C, then diluted into either YEPD, pH 5.0, and grown at 25 °C or YEPD, pH 7.5, and grown at 37 °C. At the indicated times, 106 cells were removed from each culture, the cells were 5-fold serially diluted 8 times (top to bottom for each condition) and then a constant fraction of the diluted mixture was spotted on YEPD, pH 5.0, plates and grown at 30 °C. B, growth curves of wild-type (, ), vma4 (, ), and vma4-1ts (, ) cells in YEPD medium buffered to pH 5.0 at 25 °C (filled symbols) or YEPD medium buffered to pH 7.5 at 37 °C (open symbols). All three cell types were grown at pH 5.0 under permissive conditions, then diluted into the indicated medium at time 0. Samples were removed at the indicated times and cell density quantitated from the absorbance at 600 nm (OD600 nm).

View larger version (59K): [in this window] [in a new window]   Fig. 8.   A-F, chitin deposition is delocalized in vma mutant cells grown at pH 7.3. Wild-type (A and B), vma4 (C and D) strain, and vma4-1ts (E and F) cells were grown in YEPD, pH 5, at 25 °C (A, C, and E) or in YEPD, pH 7.3, at 37 °C (B, D, and F) for 16 h. Whole cells were labeled with calcofluor (A-F) as described under "Experimental Procedures," and observed by fluorescence microscopy under a UV filter set. G-H, nuclear localization in vma4-1ts mutants. vma4-1ts mutants were grown in YEPD, pH 5, at 25 °C (G) or YEPD, pH 7.3, at 37 °C (H) for 16 h, and DNA was labeled with DAPI as described under "Experimental Procedures." All pictures were photographed and printed using identical magnifications and exposure times. Images were arranged using Adobe Photoshop. The strains used were the same as in Fig. 7.

vma4

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Actin delocalization in vma4 and vma4-1ts mutant cells. vma4-1ts mutant cells (A-F), wild-type (G and H), and vma4 mutant cells (I and J) were grown in YEPD, pH 5, at 25 °C (E, G, and I) or in YEPD, pH 7.3, at 37 °C (A, C, H, and J). Cells were grown under the indicated conditions for 2 h (A and B) or 16 h (C-J). Whole cells were labeled with rhodamine/phalloidin in order to stain F-actin, as described under "Experimental Procedures" in frames A, C, E and G-J, and H. The morphology of the same vma4-1ts mutant cells shown in A, C, and E, are shown under Nomarski optics in B, D, and F. All pictures were photographed by using identical magnifications, and exposure times for the immunofluorescence micrographs were also the same. Images were arranged using Adobe Photoshop. Genotypes of strains are the same as in Fig. 7.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts cells. The morphological phenotypes of the vma4 cells are not identical to those of the vma4-1^ts mutant, either. Although both vma4 and vma4-1^ts mutants show actin and chitin delocalization at pH 7.3, the vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts mutants are able to display more dramatic morphological aberrations because they maintain viability. A third possibility is that the vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts 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 vma4-1^ts mutant and other vma mutants. In future experiments we will attempt to address the specific connections between the vacuolar H⁺-ATPase and actin functions.