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Eukaryotic Cell, June 2008, p. 926-937, Vol. 7, No. 6
1535-9778/08/$08.00+0     doi:10.1128/EC.00037-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Loss of Regulators of Vacuolar ATPase Function and Ceramide Synthesis Results in Multidrug Sensitivity in Schizosaccharomyces pombe{triangledown}

Keren Dawson, W. Mark Toone,{dagger} Nic Jones,* and Caroline R. M. Wilkinson*

Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom

Received 1 February 2008/ Accepted 11 April 2008


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ABSTRACT
 
We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H+)-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.


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INTRODUCTION
 
One of the biggest challenges facing modern medicine is that of multidrug resistance (MDR), a phenomenon whereby cells acquire tolerance to a range of structurally and functionally unrelated drugs, such as those used in chemotherapy or compounds employed to treat fungal infections. The common occurrence of MDR in tumors represents a major problem in the successful chemotherapeutic treatment of cancer. Candida albicans is an opportunistic fungal pathogen that can cause severe infections in humans, particularly in those who are immunocompromised such as AIDS patients and individuals undergoing chemotherapy. Fluconazole is widely used to treat such infections; however, resistance to this drug can occur, resulting in reduced treatment efficacy (33). There is a need, therefore, to identify pathways that control resistance, as their manipulation might restore drug sensitivity to MDR cells.

MDR arises mainly through the increased efflux of drugs from the cell. This transport is mediated through membrane transporters, which fall into a small number of protein superfamilies such as the major facilitator superfamily and the ATP-binding cassette transporters. These transporters have a broad specificity for a variety of structurally unrelated compounds. In some cases, their natural role is to protect the cell against toxins whereas others have more physiological targets but can, upon overexpression or mutation, confer drug resistance (13).

Yeast has long been used as a model organism to study many aspects of cell biology, but recent studies are now utilizing it in anticancer drug discovery (36). Genome-wide screens of budding yeast have been employed to identify the targets and pathways that are acted upon by a particular drug (1, 24, 31).

One pathway that seems to control innate resistance to drugs in budding yeast involves the vacuolar (H+)-ATPase (V-ATPase) (31, 50). This is a large, multisubunit complex found in all eukaryotic cells. It is present in the membranes of several organelles such as the Golgi apparatus, endosomes, and vacuoles or lysosomes and is responsible for the acidification of these compartments by coupling the hydrolysis of ATP to the transport of protons across membranes. The V-ATPase consists of two subcomplexes: the V0 complex, which is embedded in the membrane and forms a channel for protons, and the V1 complex, which is bound to the cytosolic surface of the V0 complex and catalyzes the hydrolysis of ATP. In eukaryotic cells, this enzyme plays a role in many physiological processes, including receptor-mediated endocytosis and protein sorting along the secretory pathway (17, 28). The subunits of the budding yeast V-ATPase are encoded by the VMA genes, mutations in which lead to the so-called Vma– phenotype, whereby cells display sensitivity to high levels of extracellular calcium, high pH, heavy metals, and a variety of drugs (31, 50). A number of studies suggest that the role of the budding yeast V-ATPase in determining resistance to drugs appears to be conserved in mammalian cells (25, 30, 31).

One factor thought to play a role in controlling V-ATPase function is Rav1 (also known as Soi3), an evolutionarily conserved protein that was initially identified in budding yeast, where it was found as part of a Skp1-containing complex called RAVE (35). Also present in the budding yeast RAVE complex is a protein called Rav2. In other eukaryotes, there are homologues of Skp1 and Rav1, but while most fungi have a homologue of Rav2, there is no obvious candidate in fission yeast. To date, the role of Rav1 and RAVE in the regulation of V-ATPase activity has only been studied in budding yeast. The RAVE complex binds to the V1 domain of the V-ATPase, and consistent with a role in regulating this complex, Scrav1{Delta} and Scrav2{Delta} mutants display both a temperature-sensitive Vma– phenotype and defects in vacuolar acidification. RAVE appears to promote the assembly of V-ATPase (35, 40), and Rav1 function is also required for trafficking between the early endosome and the vacuole, presumably through its control of endosome acidification (38).

Here we have identified and characterized two fission yeast genes that, when mutated, give rise to multidrug sensitivity. The first, rav1, encodes the homologue of budding yeast Rav1. The second, lac1, encodes a homologue of two budding yeast proteins, Lag1 and Lac1, which are required for the synthesis of ceramide (11, 34).


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MATERIALS AND METHODS
 
Growth of Schizosaccharomyces pombe. The strains used in this study are listed in Table 1. S. pombe was grown as previously described (26). Gene deletion and epitope tagging were carried out as previously described (4). The sequences of the oligonucleotides used are available upon request. The fission yeast genomic library used to identify rav1 and lag1 was as previously described (27). The lag1, lac1, and vma3 cDNAs were cloned into the pREP1 vector (and the lag1 cDNA was also cloned into pREP41). The sequences of the oligonucleotides used are available upon request. Expression from the nmt41 promoter in pREP41 gives a lower level of expression than expression from the nmt1 promoter in pREP1 (6).


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  		TABLE 1. Yeast strains used in this study

Isolation of rav1-1. Wild-type S. pombe was plated to a density of 3 x 103cells/plate and exposed to a predetermined dose of UV calculated to give one lesion per cell. Sensitivity acquired due to mutation was selected by replica plating resultant colonies to yeast extract (YE) plates with and without 50 µg/ml doxorubicin (D1515; Sigma).

Determination of drug sensitivities. Drug sensitivity was determined by using dilution assays in which 5 µl of a cell suspension containing 2 x 106cells/ml and 10-fold dilutions from this concentration were pipetted onto YE plates containing appropriate ranges for each drug. The drugs used were obtained from the following suppliers: doxorubicin, Sigma (D1515); fluconazole, gift from Pfizer; bleomycin, Fluka (15361); camptothecin, Sigma (C9911); hydroxyurea, Sigma (H8627); cycloheximide, Sigma (C4859). To assess their sensitivity to heat shock, we grew cells to mid-log phase and treated them at 50°C for 15 min before performing a dilution assay.

Microscopy. Imaging of doxorubicin accumulation was performed as already described, except that cells were incubated in doxorubicin for 90 min (41). For indirect immunofluorescence assay, cells were fixed as previously described (48); anti-myc (MCA1929; Serotec) was used at a dilution of 1 to 100. Secondary antibodies were diluted 250-fold (Alexa Fluor 488 anti-mouse, A-11001; Molecular Probes). Filipin staining was done as previously described (45). Quinacrine staining was carried out as previously described (16). Cells were imaged by using the Deltavision system (Applied Precision Instruments); the softworx (API) software was utilized to control image capture.

Protein analysis. Total protein was extracted in radioimmunoprecipitation assay buffer as previously described (21). The antibodies used were antitubulin (T1568; Sigma) at 1 in 1,000, anti-myc (MCA1929; Serotec) at 1 in 1,000, and anti-Skp1 (23).

Immunoprecipitations. For isolating Rav1-interacting proteins with subsequent identification by mass spectrometry, cell extracts were made from rav1-13 myc cells in buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% NP-40, and protease inhibitors (Roche). Identification of Vma2 was done by the Cancer Research UK mass spectrometry service as previously described (8). The peptides matched to Vma2 covered 52.3% of the protein. All other immunoprecipitations were as previously described (21).


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RESULTS
 
Isolation of rav1-1 and lac1{Delta} multidrug-sensitive mutants. In order to define pathways involved in resistance to chemotherapeutic drugs in S. pombe, a screen was performed to isolate mutations resulting in a multidrug-sensitive phenotype by using the anticancer drug doxorubicin. One particular mutant, which we named the rav1-1 mutant for reasons described below, was sensitive to doxorubicin but displayed robust growth on control plates (Fig. 1A). This mutation also resulted in sensitivity to a range of structurally unrelated drugs (Fig. 1A). The rav1-1 mutant was also sensitive to calcium (Fig. 1B), heavy metals (Fig. 2C), and high pH (data not shown). To identify the genomic locus affected in the rav1-1 mutant, it was transformed with a genomic library and two clones were identified which suppressed its doxorubicin and calcium sensitivity. Clone 1 rescued the rav1-1 mutant even at high levels of doxorubicin (50 µg/ml), whereas the second clone only rescued growth at more intermediate levels of the drug (data not shown). Similarly, clone 1 rescued the growth defect on calcium whereas clone 2 only had a partial effect (Fig. 1B). Upon sequencing, clone 1 contained one complete open reading frame (ORF) corresponding to the gene designated SPBC1105.10 that encodes a protein of 1,297 amino acids. The protein encoded by SPBC1105.10 shows 25% sequence identity with the Rav1 protein of Saccharomyces cerevisiae. Sequence analysis of the SPBC1105.10 ORF in the rav1-1 mutant revealed a single C-T transition at nucleotide 79 resulting in an arginine-stop conversion at position 26, and therefore we renamed SPBC1105.10 as rav1 and the R26stop mutant allele of SPBC1105.10 as rav1-1. A rav1 deletion mutant was viable and grew normally but exhibited a drug sensitivity profile similar to that of the rav1-1 mutant strain (Fig. 1C), although the rav1{Delta} mutant strain was slightly more sensitive to drugs than the rav1-1 mutant strain was (Fig. 1C). Western blot analysis revealed that a small amount of truncated Rav1 protein is produced in the rav1-1 mutant, consistent with translation initiating at an internal start site (Fig. 1D), thus providing an explanation for the slight difference in phenotype between rav1{Delta} and rav1-1 mutant cells.


Figure 1
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  		FIG. 1. Characterization of the multidrug-sensitive phenotype of a rav1-1 mutant. (A) Increasing dilutions of wild-type (wt) and rav1-1 mutant cells were plated on YE (control) plates with and without doxorubicin at 20 µg/ml, camptothecin at 50 µg/ml, bleomycin at 0.25 µg/ml, cycloheximide at 7.5 µg/ml, hydroxyurea at 10 mM, and fluconazole at 20 µg/ml. The plates were photographed after incubation at 30°C for 3 days. (B) Isolation of the rav1 gene and the lag1 multicopy suppressor of rav1-1. rav1-1 mutant cells were transformed with the pREP1 vector (vector) or the rav1 or lag1 clone isolated from the S. pombe genomic library. Cells were plated on either minimal medium (control) or minimal medium containing CaCl2. (C) Overexpression of lag1 can partially rescue the drug and calcium sensitivity caused by a rav1{Delta} allele. rav1-1 or rav1{Delta} mutant cells were transformed with the pREP1 vector (vector), the lag1 genomic clone (genomic), or the lag1 cDNA expressed from the nmt1 promoter (cDNA). Cells were plated on either minimal medium (control) or minimal medium containing doxorubicin (20 µg/ml), bleomycin (0.5 µg/ml), or CaCl2 (100 mM). (D) Western blot assay of wild-type and mutant Rav1 proteins tagged with 13 copies of the myc epitope. The upper and lower arrows indicate the full-length and truncated Rav1 proteins, respectively. wt indicates an untagged strain. Loading was assessed with antitubulin antibodies.


Figure 2
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  		FIG. 2. Multidrug- and heavy-metal-sensitive phenotypes of lac1{Delta} and rav1{Delta} mutants. (A) A lac1{Delta} but not a lag1{Delta} mutant is sensitive to a range of drugs. The strains were plated on YE containing bleomycin (0.5 µg/ml), cycloheximide (CHX; 7.5 µg/ml), fluconazole (20 µg/ml), and camptothecin (50 µg/ml). After a week, on the control plate, the lac1{Delta} mutant had grown to the same extent as the wild type (wt) had after 3 days. (B) Wild-type and rav1{Delta}, lac1{Delta}, and lag1{Delta} mutant cells were plated onto YE (control) plates and YE containing 200 mM CaCl2. (C) Wild-type and rav1{Delta} and lac1{Delta} mutant cells were plated onto YE (control) and YE containing either 1 mM ZnSO4 or 0.2 mM CdSO4. (D) rav1{Delta} mutant cells were transformed with the empty vector, the rav1 or lag1 (grav1 or glag1) genomic clone, or a pREP1 vector containing the cDNA of rav1 or lag1 (clag1 or clac1). The cells were plated on minimal medium alone or on minimal medium containing 200 mM CaCl2, doxorubicin (50 µg/ml), or fluconazole (20 µg/ml). (E) lac1{Delta} cells were transformed with the empty vector, pREP1, the pREP41 vector containing the cDNA of lag1, or pREP1lac1. The cells were plated on minimal medium alone or medium containing 100 mM CaCl2. (F) Wild-type cells were transformed with the empty pREP1 vector, pREP1lag1, or pREP1lac1 and plated on minimal medium alone or on medium containing doxorubicin (50 µg/ml), fluconazole (20 µg/ml), bleomycin (0.5 µg/ml), or cycloheximide (15 µg/ml). Plates were incubated for 2 days at 30°C.

Loss of the S. pombe lac1 gene also results in multidrug sensitivity. The second genomic clone (clone 2), which resulted in partial rescue of the rav1-1 mutant, corresponded to the SPAC1A6.09c ORF. This encodes a protein homologous to Lag1 and Lac1 in S. cerevisiae, which are required for the de novo synthesis of ceramide. Only one of the two proteins is required for this process, but loss of both ScLag1 and ScLac1 results in a severe growth defect and the inability to synthesize ceramide (11, 34). Sphingolipids are constituents of the membrane bilayer; their backbone consists of ceramide. Along with sterols, sphingolipids form localized structures in the membrane called lipid rafts which are thought to be involved in the biosynthetic delivery of proteins to the yeast plasma membrane (37). The protein encoded by SPAC1A6.09c displays 38 and 39% identity to ScLag1 and Lac1, respectively, and has previously been assigned the name Lag1 (49; http://www.genedb.org/genedb/pombe/index.jsp). There is also a second S. pombe homologue of ScLag1 and Lac1 encoded by the SPBC4F6.02 ORF. This protein is 40 and 41% identical to ScLac1 and ScLag1, respectively, and 35% identical to SpLag1.We have named the gene that encodes this protein lac1.

In addition to rescuing the multidrug sensitivity of the rav1-1 mutant, we also found that lag1 overexpression resulted in partial rescue of the rav1{Delta} mutant's phenotypes (Fig. 1C). We also overexpressed the lag1 cDNA and found that these higher levels of expression resulted in even better resistance of rav1{Delta} mutant cells to drugs (Fig. 1C). Given that upregulating the levels of lag1 suppressed the drug sensitivity of rav1 mutants, we decided to test whether loss of lag1 would also confer multidrug sensitivity upon S. pombe. However, the lag1{Delta} mutant grew to the same extent as the wild type upon exposure to drugs. Strikingly, though, deletion of its homologue, lac1, gave rise to a multidrug-sensitive phenotype (Fig. 2A). Although the lac1{Delta} mutant grows more slowly than the wild type (Fig. 2A), we do not believe that this explains its multidrug-sensitive phenotype since upon further incubation, lac1{Delta} mutant colonies grew to the same extent as the wild type but remained growth retarded upon drugs (data not shown).

The identification of a Rav1 homologue in S. pombe led us to speculate that Rav1 may be playing a role in this yeast similar to that of its counterpart in S. cerevisiae. Therefore, we assessed the growth of Sprav1{Delta} cells on medium containing high levels of calcium chloride, as mutants defective in vacuolar ATPase function are sensitive to calcium (43, 51). The rav1{Delta} mutant cells were sensitive to calcium, indicating that SpRav1 may also play a role in regulating vacuolar function. Furthermore, loss of Splac1, but not lag1, resulted in sensitivity to calcium, suggesting that Lac1 may influence vacuolar function (Fig. 2B and 1B and C). Vacuolar mutants are also sensitive to heavy metals (10), and accordingly, we found that loss of rav1 resulted in sensitivity to cadmium and zinc ions. In addition, the lac1{Delta} mutant (but not the lag1{Delta} mutant; data not shown) was also sensitive to these metals, again consistent with the hypothesis that vacuolar function might be impaired in this mutant (Fig. 2C). Loss of both rav1 and lac1 is additive, as the double mutants exhibited greater drug sensitivity than either single mutant (Fig. 2A), suggesting that Rav1 and Lac1 act in different pathways to influence innate drug resistance.

In addition to the drug sensitivity assays, two further pieces of data demonstrated the differences between lag1 and lac1. Firstly, overexpression of lac1 from the same heterologous promoter used to drive the expression of lag1 did not result in rescue of the rav1{Delta} mutant's multidrug-sensitive phenotype (Fig. 2D). Secondly, overexpression of lag1 did not rescue the calcium sensitivity (Fig. 2E) or multidrug sensitivity (data not shown) associated with the lac1{Delta} mutant.

The ability of lag1 to rescue the multidrug sensitivity of the rav1{Delta} mutant and the phenotypes associated with loss of lac1 suggested that lag1/lac1 might be involved in controlling the innate drug resistance of wild-type cells. To address this, we overexpressed lag1 and lac1 in the wild type and exposed the cells to medium containing drugs. As shown in Fig. 2F, overexpression of either lag1 or lac1 resulted in a modest increase in the cells' ability to grow upon exposure to drugs.

In summary, we have identified two genes, rav1 and lac1, whose loss results in multidrug sensitivity. Overexpression of lag1, a homologue of lac1, resulted in increasing resistance to drugs in the rav1-1 and rav1{Delta} mutants, whereas overexpression of lag1 and lac1 in the wild type resulted in increased drug resistance, suggesting that altering the dosage of these ceramide synthase components can modulate innate drug resistance.

Mutations in rav1 and lac1 lead to an increased amount of doxorubicin in the cell. A possible explanation for the drug sensitivities of the rav1{Delta} and lac1{Delta} mutants is that they are more permeable to drugs. Alternatively, these mutants may have a reduced capacity to extrude drugs. Both scenarios could lead to increased amounts of drugs in the cell. We tested this by imaging cells treated with doxorubicin. It is a naturally fluorescent compound, a feature that has been used to visualize its uptake into cells (41). The growth of rav1{Delta} and lac1{Delta} mutant cells was impaired on plates containing this drug (Fig. 3A). Moreover, more rav1 and lac1 mutant cells had accumulated doxorubicin compared to the wild type (Fig. 3B). The lag1{Delta} mutant did not appear to accumulate the drug to any significant extent, a finding which is consistent with its lack of a multidrug-sensitive phenotype. We quantified the accumulation of doxorubicin and found that similar percentages of rav1{Delta} and lac1{Delta} mutant cells displayed fluorescence (45 and 49%, respectively). In contrast, during the same period of exposure to doxorubicin, only 2.2% of wild-type cells displayed a fluorescent signal. Interestingly, 7% of lag1{Delta} mutant cells had accumulated the drug, so while this mutant does not display drug sensitivity, it appears that loss of the lag1 gene has resulted in a mild phenotype (Fig. 3C).


Figure 3
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  		FIG. 3. Mutations in rav1 and lac1 lead to increased accumulation of doxorubicin. (A) The strains were plated on YE with 50 µg/ml doxorubicin. (B) Log phase cells were grown in YE containing 50 µg/ml doxorubicin before imaging. The size bar represents 15 µm. Images taken with longer exposure times are shown for wild-type (wt) and lag1{Delta} mutant cells so that the outline of cells that have not taken up the drug can be seen. (C) The numbers of cells displaying a fluorescent signal in panel A were quantified and expressed as a percentage of the total number of cells counted. At least 200 cells were observed for each strain, and the average of the data from three independent experiments is shown with standard deviations. (D) Higher magnification of a lac1{Delta} mutant cell exposed to doxorubicin as in panel B. For this image, the cells had been exposed to doxorubicin and then resuspended in water to promote vacuolar fusion. This enabled clearer observation of the vacuoles so that the cytoplasmic accumulation of doxorubicin and exclusion of the drug from the vacuoles could be visualized.

In rav1{Delta} and lac1{Delta} mutant cells, the drug appears to be distributed throughout the cytoplasm and does not appear to enter the vacuoles (Fig. 3B and enlarged image in panel D). This is in contrast to the drug-sensitive hal4-1 mutant, which sequesters doxorubicin in the vacuoles (41). These are known to be the site where many metabolites are accumulated, a function which has been linked to the activity of the V-ATPase (reviewed in references 17 and 19). This finding suggests that in rav1{Delta} and lac1{Delta} mutant cells, the activity of the V-ATPase is defective. Taken together, our data show that in fission yeast, loss of rav1 or lac1 leads to a multidrug-sensitive phenotype, which is accompanied by, and probably due to, an increased amount of drugs in the cells.

SpRav1 is part of a RAVE-like complex and influences V-ATPase function. In order to determine how loss of SpRav1 results in multidrug sensitivity, we sought to characterize how SpRav1 functions in the cell. First we investigated whether the RAVE complex is conserved between budding and fission yeasts. As shown in Fig. 4A, Skp1 coprecipitates with Rav1-myc. The interaction was abolished when Skp1 was epitope tagged at its amino terminus, suggesting that this tag or the combination of Rav1 and Skp1 tags impedes binding.


Figure 4
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  		FIG. 4. S. pombe Rav1 is part of a RAVE-like complex. (A) SpRav1 binds to Skp1. The Rav1-13myc protein was immunoprecipitated (IP) from whole-cell extracts (WCE) with anti-myc antibodies. A nontagged strain (wt) was used as a control. The experiment was also carried out with a strain where the skp1 gene is fused to the hemagglutinin epitope under the control of the nmt1 promoter. This was performed in the presence or absence of thiamine (Thi) to vary the amount of Skp1 expressed. Extracts were analyzed with anti-Skp1 antibodies. The immunoprecipitated Rav1 protein was detected with anti-myc antibodies. (B) Protein sequence alignment of S. cerevisiae Rav2 with its putative homologues from C. glabrata, C. albicans, and S. pombe. The accession numbers of the latter three sequences, respectively, are as follows: GenBank, CAG59121 [GenBank] .1; Candida DB, CA1654; GenBank, CAA20308 [GenBank] .1. Alignment was performed with ClustalX. Residues identical in all four species are shaded in black; those conserved across two or three species are shaded in dark gray; conservative changes are shaded in light gray. The amino acid positions are indicated on the right. (C) SpRav2 (SPBC3H7.12) interacts with Skp1. The Rav2-13myc protein was immunoprecipitated from whole-cell extracts. A nontagged strain (wt) was used as a negative control, and rav1-13myc was used as a positive control. Skp1 was detected as in panel A. The immunoprecipitated Rav2 protein was detected with anti-myc antibodies. (D) The S. pombe rav2{Delta} mutant is partially sensitive to drugs and calcium. The concentrations used were 50 µg/ml doxorubicin and 100 mM CaCl2.

There is not an obvious candidate for Rav2 in S. pombe, but we did notice an uncharacterized ORF (SPBC3H7.12) with very weak similarity to ScRav2: 13% identity over 224 amino acids. This ORF encodes a protein of 287 amino acids, which is considerably shorter than ScRav2 and its fungal homologues. An alignment of this ORF, which we have renamed Rav2, with the putative Rav2 sequences from C. albicans and C. glabrata along with the S. cerevisiae Rav2 sequence is shown (Fig. 4B). Skp1 coprecipitated with SpRav2, and although we were unable to detect an interaction between epitope-tagged versions of Rav1 and Rav2, we found that the interaction between Rav2 and Skp1 was abrogated in the absence of Rav1 (Fig. 4C), suggesting that Rav1 bridges between these two proteins in a RAVE-like complex. It seems likely that the complex is sensitive to the presence and/or combinations of epitope tags. Furthermore, the topography of RAVE in fission yeast is such that Skp1 appears to bind to Rav2 through Rav1, a finding that is conserved between fission and budding yeasts (35).

A deletion mutant of rav2 displayed a mild sensitivity to a range of drugs including doxorubicin, cycloheximide, and bleomycin, and its growth was slightly retarded by high levels of calcium, consistent with a similar but more muted multidrug-sensitive phenotype than that of the rav1{Delta} mutant (Fig. 4D and data not shown). Taking all of these data together, we propose that, despite its low homology and shorter length, SPBC3H7.12 is the orthologue of ScRav2. Its role in providing resistance to drugs and controlling V-ATPase activity appears to be less critical than that of Rav1. We also found protein sequences that display low similarity to SpRav2 in a number of higher eukaryotes including chickens, zebra fish, and humans (accession numbers XP_424594 [GenBank] , NP_956257 [GenBank] , and NP_078865.1 [GenBank] ). Although the sequence identity is low between these sequences and S. pombe Rav2, it extends across the length of the proteins (data not shown). This finding suggests that the RAVE complex might be conserved in higher eukaryotes.

To gain further insight into the role of the S. pombe RAVE complex, we sought to identify further Rav1-interacting partners by isolating proteins that coprecipitated with Rav1. The resulting material was separated by gel electrophoresis, and we observed one strong band upon silver staining of the gel (Fig. 5A). We observed an inverse staining phenomenon, which can be caused by excess protein. The material was analyzed by mass fingerprinting, which revealed that this band corresponded to the V1 vacuolar ATPase component Vma2. Matched peptides to Vma2 covered 52.3% of the protein (data not shown). This is one of five V1 subunits demonstrated to coprecipitate with ScRav1 (35, 39), findings which suggest that ScRAVE functions via an interaction with intact V1. Our data indicate that the interaction of Rav1 with the V1 subcomplex of the V-ATPase is conserved between the two yeasts. We attempted to immunoprecipitate epitope-tagged Vma2 with epitope-tagged Rav1 but were unable to identify an interaction, possibly due to the combination of tags that we used.


Figure 5
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  		FIG. 5. Rav1 interacts with the V-ATPase and influences its function. (A) Rav1-13myc protein was purified, and coprecipitating proteins were identified by gel electrophoresis and silver staining in comparison to the immunoprecipitate from a nontagged control (wt). The arrow corresponds to the prominent band observed upon silver staining, which was identified as Vma2. (B) Loss of Rav1 results in vacuolar acidification defects. Wild-type and rav1{Delta} mutant cells were exposed to quinacrine and imaged by fluorescence microscopy. Equal exposure times are shown for both strains. The size bar represents 10 µm. (C) Epistasis analysis of the rav1{Delta} and vma1{Delta} mutants. Wild-type and vma1{Delta}, rav1{Delta}, and vma1{Delta}rav1{Delta} mutant cells were plated on 20 µg/ml fluconazole, 100 mM CaCl2, and 50 µg/ml doxorubicin. (D) Rav1 protein is localized throughout the cytoplasm. Indirect immunofluorescence analysis was carried out with either wild-type untagged or rav1-13myc mutant cells with anti-myc. The scale bar represents 10 µm. DAPI, 4',6'-diamidino-2-phenylindole.

Defective V-ATPase activity will result in a reduction in vacuolar acidification; therefore, we assessed the status of the vacuoles in the Sprav1{Delta} mutant by using quinacrine, which is a weakly basic dye that accumulates in acidic compartments (46). In wild-type cells, the dye accumulated in the vacuoles as expected; however, in the rav1{Delta} mutant there was no vacuolar accumulation of quinacrine but instead we observed a weak staining of the cytoplasm and the vacuolar membranes (Fig. 5B). These data suggest that SpRav1 plays a role in controlling the V-ATPase function in fission yeast.

Next we asked whether the multidrug-sensitive phenotype of the rav1{Delta} mutant was due to defective V-ATPase activity or to an additional role for Rav1. Mutants lacking V1 subunits in budding yeast are known to have a disrupted V1 structure and abolished V-ATPase function (14, 18, 29, 44). A deletion mutant of the S. pombe homologue of Vma1 (which contains subunit A of the V1 subcomplex) was multidrug sensitive (Fig. 5C and data not shown). A vma1{Delta} rav1{Delta} double mutant was no more sensitive than the vma1{Delta} single-mutant strain upon exposure to fluconazole or calcium, which suggests that, with regard to these reagents, Rav1 and Vma1 lie on the same pathway and that the multidrug sensitivity of the rav1{Delta} mutant is caused by reduced V-ATPase activity. The vma1{Delta} mutant was more sensitive to drugs than was the rav1{Delta} mutant, suggesting that some residual V-ATPase function exists in the absence of Rav1 function. Interestingly, we observed a slight additive effect of the two mutations upon exposure to doxorubicin.

The interaction of Rav1 with the V-ATPase in budding yeast has been proposed to be transient, and consistent with this, ScRav1 has been found to be localized throughout the cytoplasm (35) and also found in punctate structures consistent with early endosomal membranes (38). We found that SpRav1 is also localized to the cytoplasm, where it appeared to be distributed largely in discrete punctate structures, reminiscent of the latter budding yeast study (Fig. 5D).

Taking all of the above data together, we conclude that SpRav1 is part of a RAVE-like complex in fission yeast. SpRav1 interacts with and appears to regulate the V-ATPase; lack of this function results in defective V-ATPase activity, which renders the cells sensitive to a variety of drugs.

Loss of S. pombe lac1 results in heat shock sensitivity and disruption of plasma membrane sterol distribution. We also sought to characterize the lac1{Delta} mutant. In budding yeast, loss of LAG1 or LAC1 results in viable cells whereas the double mutant is inviable or very sick, suggesting a redundancy of function between these two genes. This lethality is believed to result from an inability to synthesize ceramide (5, 11, 34).

Our drug sensitivity assays showed that loss of lac1 but not lag1 resulted in sensitivity to drugs (Fig. 2). Several other assays also illustrated that loss of SpLac1 is more detrimental to the cell. Firstly, we found that the lac1{Delta} mutant displayed a general growth defect (Fig. 6A). Secondly, we found that lac1{Delta} mutant cells are sensitive to heat stress. In addition to their structural role in membranes, sphingolipids have been proposed to act in signal transduction pathways during various stress responses, including heat shock (9). Shifting yeast cells to a high temperature results in increased levels of ceramide that occur via de novo synthesis (47). We found that the lac1{Delta} mutant, but not the lag1{Delta} mutant, was sensitive to heat shock (Fig. 6B). Thirdly, we found that lac1{Delta} mutant cells have an aberrant plasma membrane, as judged by examining sterol distribution. The localization of sterols in S. pombe can be assessed by using the fluorescent probe filipin. In growing cells, sterols are detected at the plasma membrane and enriched specifically at the growing tips of the cell and also at the site of cytokinesis in cells undergoing division (45). Strikingly, in lac1{Delta} mutant cells, but not rav1{Delta} and lag1{Delta} mutant cells, the pattern of filipin fluorescence was distributed evenly around the entire cell (Fig. 6C), suggesting that the structure of the plasma membrane is abnormal in this mutant.


Figure 6
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  		FIG. 6. Characterization of the lac1{Delta} mutant. (A) lac1{Delta} mutant cells display a slow-growth phenotype. Wild-type (wt) and rav1{Delta}, lac1{Delta}, and lag1{Delta} mutant cells were diluted in YE to 2 x 106/ml, and their growth was assayed by measuring absorbance at 600 nm. Symbols: filled circles, wild type; empty circles, lag1{Delta} mutant; filled squares, lac1{Delta} mutant; filled squares, rav1{Delta} mutant. (B) The lac1{Delta} mutant is sensitive to heat shock. Cells were shifted to 50°C for 15 min, plated on YE, and then incubated at 30°C. Control samples were grown at 30°C before plating. All strains were grown on the same plates; a mutant not relevant to this study has been removed (between the lag1{Delta} and lac1{Delta} mutants). After a week, on the control plate, lac1{Delta} mutant cells had grown to the same extent as the wild type had after 3 days, but not those exposed to heat shock. (C) lac1{Delta} mutant cells display an abnormal distribution of sterols in their plasma membrane. Wild-type and lac1{Delta}, lag1{Delta}, and rav1{Delta} mutant cells were treated with filipin and examined by fluorescence microscopy. (D) The rav1{Delta} mutant was transformed with the lag1 genomic clone (lag1), the rav1 genomic clone (rav1), or the empty vector. Transformants were exposed to quinacrine and imaged by fluorescence microscopy. Equal exposure times are shown for all strains. The size bar represents 10 µm. (E) The vma3{Delta} mutant was transformed with the empty vector, the pREP1 vector containing the lag1 (lag1) or vma3 (vma3) cDNA, and the pREP41 vector containing the lag1 cDNA (41lag1). Cells were plated on minimal medium and minimal medium containing 100 mM CaCl2 (100 mM) or fluconazole (50 µg/ml). (F) The vma3{Delta} mutant was transformed with the pREP1 vector (Vector), the pREP1lag1 cDNA (lag1), or the pREP1vma3 cDNA (vma3). Transformants were exposed to quinacrine and imaged by fluorescence microscopy. Equal exposure times are shown for all strains. The size bar represents 10 µm.

Our data suggested that loss of lac1 also resulted in defective V-ATPase activity, as the lac1{Delta} mutant displayed Vma– phenotypes such as sensitivity to calcium and heavy metals (Fig. 2B and C). We examined the lac1{Delta} mutant for vacuolar acidification defects by using quinacrine as described above. The lac1{Delta} mutant cells did not accumulate quinacrine in their vacuoles, consistent with a defect in vacuolar acidification, but the procedure resulted in the cells displaying gross morphological defects (data not shown). Despite the phenotypic differences between the lac1{Delta} and lag1{Delta} mutants, there appears to be a certain overlap in the functions of the two mutated genes, as we found that the lag1{Delta} lac1{Delta} double-deletion mutant is inviable (data not shown). We conclude that while Splac1 and lag1 display some degree of redundancy, as suggested by their sequence identity and the inviability of the lac1{Delta} lag1{Delta} double mutant, loss of lac1 results in more apparent defects than loss of lag1.

We found that the multidrug sensitivity of lac1{Delta} mutant cells is not merely due to misregulation of the V-ATPase, as a lac1{Delta} vma1{Delta} double mutant displayed more severe phenotypes than either single mutant (data not shown).

Based on sequence homology to the budding yeast counterparts, we propose that in the Splac1{Delta} mutant, ceramide synthesis is highly compromised, resulting in misregulation of sphingolipid metabolism. In addition, lac1/lag1 function seems to play an important role in regulating V-ATPase activity, as the lac1 mutant displays Vma– phenotypes and defects in vacuolar acidification while overexpression of lag1 rescues the rav1{Delta} Vma– and multidrug-sensitive phenotypes. Indeed, overexpression of lag1 was able to restore acidification to the vacuoles as quinacrine accumulated in the vacuoles of a rav1 mutant as it also did in cells where the expression of rav1 had been restored (Fig. 6D). In contrast, in cells transformed with the empty vector, quinacrine remained in the cytoplasm and around the vacuolar membranes.

We hypothesized that overexpression of lag1 rescues the multidrug sensitivity of rav1{Delta} through the restoration of vacuolar acidification (which is defective due to inefficient V-ATPase assembly). Overexpression of lag1 would not, therefore, be expected to rescue the drug sensitivity of a V-ATPase subunit deletion mutant, as in this case, assembly and thus activity of the V-ATPase would be completely abolished (18, 44). To test this, we overexpressed lag1 in a mutant with a deletion of vma3, which encodes a subunit of the V0 subcomplex. Overexpression of lag1 from two different strengths of the heterologous nmt promoter did not rescue sensitivity to fluconazole (Fig. 6E) or to bleomycin or cycloheximide (data not shown). Interestingly, there was a weak rescue of sensitivity to calcium (Fig. 6E). To determine if this rescue might be related to changes in vacuolar acidification, we stained cells with quinacrine.

As shown in Fig. 6F, overexpression of lag1 did not restore acidification to the vacuoles (compare cells where lag1 has been overexpressed to cells where the vma3 gene has been overexpressed). We conclude that overexpression of lag1 does not rescue the multidrug-sensitive phenotype of the vma3{Delta} mutant, a strain where V-ATPase assembly would not be expected to occur. Although we see slight effects, on calcium, for example, there is no concomitant rescue of vacuolar acidification and thus we propose that these effects result from the overexpression of lag1 affecting other functions in the cell, perhaps the activity or trafficking of plasma membrane transporters that selectively mediate the extrusion of certain substances.


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DISCUSSION
 
In this study, we have identified two fission yeast genes, rav1 and lac1, that play an important role in determining the innate resistance of fission yeast to a variety of toxic compounds. This is the first study to demonstrate that loss of either of these evolutionarily conserved proteins, in any organism, results in such a multidrug-sensitive phenotype. These genes were not identified in genome-wide screens in budding yeast for mutants that gave rise to multidrug sensitivity, illustrating the usefulness of carrying out such studies with S. pombe as well as S. cerevisiae. In the case of Scrav1{Delta}, a mild sensitivity to fluconazole but not to other drugs was previously described (31), but neither the LAG1 nor the LAC1 gene has been identified in such screens (1, 24). In addition, we found that overexpression of the lag1 gene in rav1 mutants resulted in increasing resistance to drugs. To the best of our knowledge, this is the first demonstration that increasing the gene dosage of a ceramide synthase component through overexpression is able to modulate drug resistance. Indeed, the ability of the rav1 mutants to grow on drugs was greatly enhanced when the lag1 cDNA was highly overexpressed under the control of the heterologous nmt1 promoter compared to the genomic clone (Fig. 1C).

Conservation of the RAVE complex. We have shown that the RAVE complex, as identified in budding yeast, is likely to be conserved at very least among fungi. Moreover, the function of RAVE also seems to have been conserved, as loss of Sprav1 results in defective V-ATPase activity. We propose, therefore, that Rav1 also serves to regulate the assembly of the V-ATPase in fission yeast. Our genetic analysis suggested that the multidrug-sensitive phenotype of the rav1{Delta} mutant is due to reduced V-ATPase activity. Indeed, although it has been implicated in other organisms, this is the first study to demonstrate that loss of fission yeast V-ATPase activity results in multidrug sensitivity. Intriguingly, however, in the case of doxorubicin, there appeared to be an increased sensitivity in the rav1{Delta} vma1{Delta} double mutant (Fig. 5C). The reason for this is unclear, as vma1 encodes the catalytic component of the V-ATPase and its loss would be expected to abolish V-ATPase activity. It is possible that Rav1/RAVE has a function other than regulation of the V-ATPase and that loss of this specifically results in increased sensitivity to doxorubicin; alternatively, free V-ATPase subunits or partial complexes could result in a gain of function which now renders the cell more sensitive to doxorubicin.

Overexpression of lag1 rescues the Vma– phenotypes of a rav1 mutant. We found that overexpression of lag1 rescued the Vma– phenotypes and the vacuolar acidification defect of a rav1 mutant. Given the likely role of lag1 in promoting ceramide synthesis, this suggests a link between sphingolipid metabolism and V-ATPase function. It is possible that altering membrane composition might promote the assembly of the V-ATPase. Alternatively, it might upregulate the activity of any residual V-ATPase already assembled. Sphingolipids with a C26 acyl group are required for the generation of V1 subcomplexes with ATPase activity (7). One possibility that could explain both our findings and those of Chung et al. is that some aspect of RAVE function or assembly requires a specific sphingolipid composition. It will be interesting to determine whether overexpression of lag1 promotes V-ATPase assembly in the absence of Rav1.

The multidrug-sensitive phenotype of rav1{Delta} and lac1{Delta} mutants. Why are rav1 and lac1 mutants sensitive to a range of drugs? We propose that it is unlikely that each of these mutants could simultaneously modulate the multiple processes affected by the range of drugs to which they are sensitive. It seems more probable that loss of rav1 or lac1 affects the efflux or influx of drugs, as indicated by the increased accumulation of doxorubicin in these mutants (Fig. 3B). It is possible that loss of rav1 or lac1 affects either the levels or activities of key plasma membrane proteins that serve to take up or extrude drugs from the cell. In the rav1{Delta} mutant, intracellular trafficking might be affected by the loss of pH regulation in the biosynthetic-secretory pathway as a consequence of misregulated V-ATPase activity. This could result in plasma membrane proteins being rerouted for degradation as a result of incorrect targeting.

While Lac1/Lag1 function may affect the activity of the V-ATPase, lac1{Delta} and Vma subunit double mutants showed severely retarded growth in the absence of drugs, suggesting that other functions are affected in these cells besides V-ATPase activity (data not shown). An alteration in the lipid composition of the membrane could change the rate of passive uptake of drugs into the cell. Indeed, mutations in various ERG genes encoding components of the ergosterol biosynthesis pathway render budding yeast sensitive to a number of drugs (31). On the other hand, a number of studies with budding yeast have linked defective sphingolipid and ergosterol synthesis to the inefficient delivery of transporters to the plasma membrane, suggesting that trafficking is dependent upon the cellular lipid composition (2, 3, 22, 32, 42). We propose that trafficking defects may be occurring in the lac1{Delta} mutant due to a disruption of membrane composition, in addition to any defects caused by a reduction in V-ATPase activity. Indeed, in budding yeast, Lag1/Lac1 is required for normal delivery of glycosylphosphatidylinositol-anchored proteins to the plasma membrane (5). Interestingly, there is a link between MDR and the expression of genes involved in lipid metabolism in budding yeast. The transcriptional activators Pdr1 and -3 regulate the expression of multiple genes encoding components of the sphingolipid biosynthesis pathway; this includes LAC1 but not LAG1. Pdr1 and -3 also control the expression of transporters that mediate the efflux of a variety of drugs; therefore, these transcriptional regulators appear to coordinate the control of both drug transporters and the membrane environment in which they function (12, 20).

Homologues of both Rav1 and Lac1/Lag1 can be identified in mammalian cells, as well as in a number of pathogenic fungi, including C. albicans. Thus, these factors could be potential targets, the modulation of which could be used to combat MDR that arises in tumors and fungal infections.


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ACKNOWLEDGMENTS
 
We thank Takashi Toda for anti-Skp1 antibodies, Sara Mole and Kaoru Takegawa for strains, Pfizer for fluconazole, Steve Bagley for help with microscopy, Patty Kane for helpful advice, and Pall Jonsson and Crispin Miller for help with bioinformatic analysis.


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FOOTNOTES
 
* Corresponding author. Mailing address for N. Jones: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom. Phone: 44 161 446 3129. Fax: 44 161 446 3109. E-mail: njones{at}picr.man.ac.uk. Mailing address for C. R. M. Wilkinson: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom. Phone: 44 161 446 3129. Fax: 44 161 446 3109. E-mail: cwilkinson{at}picr.man.ac.uk Back

{triangledown} Published ahead of print on 25 April 2008. Back

{dagger} Present address: Samuel Lunenfeld Research Institute, Toronto, Ontario M5G1X5, Canada. Back


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Eukaryotic Cell, June 2008, p. 926-937, Vol. 7, No. 6
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