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Vol. 18, Issue 4, 1480-1489, April 2007

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A Phenomics Approach in Yeast Links Proton and Calcium Pump Function in the Golgi

Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Submitted November 29, 2006; Revised January 26, 2007; Accepted February 1, 2007
Monitoring Editor: Vivek Malhotra

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The Golgi-localized Ca²⁺- and Mn²⁺-transporting ATPase Pmr1 is important for secretory pathway functions. Yeast mutants lacking Pmr1 show growth sensitivity to multiple drugs (amiodarone, wortmannin, sulfometuron methyl, and tunicamycin) and ions (Mn²⁺ and Ca²⁺). To find components that function within the same or parallel cellular pathways as Pmr1, we identified genes that shared multiple pmr1 phenotypes. These genes were enriched in functional categories of cellular transport and interaction with cellular environment, and predominantly localize to the endomembrane system. The vacuolar-type H⁺-transporting ATPase (V-ATPase), rather than other Ca²⁺ transporters, was found to most closely phenocopy pmr1, including a shared sensitivity to Zn²⁺ and calcofluor white. However, we show that pmr1 mutants maintain normal vacuolar and prevacuolar pH and that the two transporters do not directly influence each other's activity. Together with a synthetic fitness defect of pmr1vma double mutants, this suggests that Pmr1 and V-ATPase work in parallel toward a common function. Overlaying data sets of growth sensitivities with functional screens (carboxypeptidase secretion and Alcian Blue binding) revealed a common set of genes relating to Golgi function. We conclude that overlapping phenotypes with Pmr1 reveal Golgi-localized functions of the V-ATPase and emphasize the importance of calcium and proton transport in secretory/prevacuolar traffic.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A family of ATP-powered pumps drive the uphill transport of Ca²⁺ across membranes to maintain resting cytosolic Ca²⁺ at submicromolar levels as a prerequisite for ubiquitous and diverse signaling events essential to biology, such as sperm motility, fertilization, T-cell activation, synaptic vesicle fusion, and muscle contraction, to name a few. One such Ca²⁺-ATPase localizing to the Golgi is evolutionarily conserved from yeast to mammals, and it plays a critical role in secretory pathway functions, including protein processing, sorting, and glycosylation. A unique property of the secretory pathway Ca²⁺-ATPase (SPCA) is that it also transports Mn²⁺ with high affinity, serving the dual function of Mn²⁺ detoxification via exocytosis and providing Mn²⁺ for Golgi-localized enzymes such as Kex2 protease and mannosyltransferases (Ton et al., 2002). Disruption of one allele of ATP2C1, encoding human SPCA1, leads to Hailey Hailey disease (HHD), an ulcerative skin disorder characterized by disruption of desmosomal contacts in keratinocytes (Foggia and Hovnanian, 2004). Whereas the molecular etiology of the disease is not clear, it is thought that in HHD patients abnormally high cytosolic ion concentrations, abnormally low luminal ion concentrations, or both, could lead to defective expression, modification, and trafficking of desmosomal proteins. Homozygous null mutations in ATP2C1 seem to be inviable in mammals, although they are tolerated in lower eukaryotes, including fungi and Caenorhabditis elegans (Rudolph et al., 1989; Cho et al., 2005), where compensatory mechanisms presumably suffice to allow viability. A particularly tractable model for understanding such mechanisms is the Saccharomyces cerevisiae orthologue Pmr1 (for review, see Ton and Rao, 2004). The identification of diverse pmr1 mutant phenotypes in yeast has been invaluable in guiding studies on metazoan SPCA orthologues (e.g., see Cho et al., 2005; Ramos-Castaneda et al., 2005), although we cannot yet understand the complex network of gene interactions that lead to many of these phenotypes, including HHD in human. The use of ion-supplemented media (Durr et al., 1998) and ion-selective Pmr1 mutants (Mandal et al., 2000), provides the opportunity to distinguish between Ca²⁺- and Mn²⁺-specific phenotypes.

Conventional genome-wide approaches have identified global patterns of gene expression (transcriptome), physical interactions between gene products (proteome), gene interactions (e.g., synthetic lethal screens), and enzyme function (metabolome). By analogy, the phenome describes genome-wide phenotypic profiles, usually of growth. Herein, we analyze the yeast phenome to find pathways and genes relating to the cellular function of Pmr1. We found that any single phenotypic screen of the haploid yeast deletion collection elicited a broad response, with genes functioning in diverse pathways, making it difficult to discern the underlying gene network leading to the common phenotype with pmr1. In contrast, the subset of genes sharing multiple, overlapping phenotypes with pmr1 was highly enriched in distinct functional categories and subcellular localization. Unexpectedly, this approach did not identify other Ca²⁺ transporters, pointing to a unique, nonredundant role for Golgi-localized ion homeostasis. Instead, mutants of the vacuolar-type H⁺-transporting ATPase (V-ATPase) were found to most closely phenocopy pmr1. Although it is established that the V-ATPase is found in multiple compartments, including the Golgi (Manolson et al., 1994; Kawasaki-Nishi et al., 2001), it has not yet been possible to distinguish between organelle-specific functions, and most vma phenotypes in yeast are attributed to a vacuolar function by default. Our findings lead us to propose that shared phenotypes of vma mutants with pmr1, including multidrug and ion sensitivity, are evidence for Golgi-localized functions of the V-ATPase. We suggest that this simple approach of overlapping multiple, distinct phenotypes can be applied to any gene to reveal new insights into cellular function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Yeast Strains, Growth Screens, and Analysis
A library of 4828 S. cerevisiae strains with deletion of each nonessential gene in a haploid background (BY4742; MAT) was purchased from Research Genetics (ResGen; Invitrogen, Carlsbad, CA). Each strain has a complete replacement of one open reading frame with the kanMX cassette. To make double knockouts, replacement of the PMR1::kanMX cassette with the NAT marker was made in the BY4741 background (MATa) as described previously (Tong et al., 2001), and the resulting strain mated with vma2, vma5 or snf6 from the BY4742 collection. Diploids were selected on YPD plates supplemented with both 200 µg/ml kanamycin and 100 µg/ml clonNAT, sporulated, and dissected. Haploid double mutants resistant to both drugs were identified.

To screen for pmr1 phenotypes, 52 96-well plates representing the collection of mutant strains were thawed, and 5 µl of each culture was used to inoculate 200-µl seed cultures grown 18–36 h in synthetic complete (SC) medium at 30°C. Four microliters of each seed culture was then used to inoculate 96-well microtiter plates each containing 200 µl of SC medium with and without addition of either 1.5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 10 mM MnCl₂, or 10 µM amiodarone (AMD). Concentrations were chosen based on results from serial dilution experiments showing the greatest difference between wild type (WT) and pmr1 growth. On each microtiter plate, two wells were reserved for a WT culture and for growth medium alone, representing positive and negative controls, respectively. Growth was monitored after incubation for 19 h at 30°C by measuring A_{600 nm} on a BMG FLUOstar Optima multimode plate reader with BMG FLUOstar Optima version 1.20 software (BMG Labtechnologies, Durham, NC). Immediately before recording, cultures were rapidly resuspended using an electromagnetic microtiter plate shaker (Union Scientific, Randallstown, MD), and all recordings were made at 30°C. A_{600 nm} values were background subtracted and normalized to average WT growth (mean calculated from 52 separate cultures spanning all microtiter plates per screen). Strains showing <20% of WT growth (<1% of the collection) under control conditions (SC) were omitted from further analysis. Of the strains that were included, low A_{600 nm} values of the seed cultures did not correlate with growth defects observed under experimental conditions. For each screen, conditional growth values were normalized to A_{600 nm} values obtained under control conditions to estimate sensitivity or tolerance. Data were organized, plotted, and analyzed using Excel X (Microsoft, Redmond, WA), S-Plus 6.2 (Insightful, Seattle, WA), and SPSS 12.0 (SPSS, Chicago, IL) software.

Using the Munich Information center for Protein Sequences (MIPS) database (http://mips.gsf.de/projects/funcat), we acquired the annotated Functional Categorization (Fun Cat) and Cellular Location (Cell Loc) of genes identified in the phenocopy screens. Resulting data from all screens were integrated and visualized as network diagrams by using Osprey network visualization software http://biodata.mshri.on.ca/osprey/servlet/Index. Illustrations were created using Adobe Illustrator CS software (Adobe Systems, Mountain View, CA).

⁴⁵Ca Uptake
Total cellular accumulation of calcium was measured by growing yeast to logarithmic phase in SC media, followed by harvesting and resuspending in fresh SC media (3 x 10⁷ cells/ml) supplemented with tracer quantities of ⁴⁵CaCl₂ (26.1 µCi/ml). After 2 h of incubation at 30°C, in the absence or presence of the indicated amounts of drugs, cells were harvested rapidly by filtration on to nitrocellulose membrane filters (HAWP, 0.45 µm; Millipore, Billerica, MA), washed with ice-cold wash buffer (10 mM HEPES and 150 mM KCl), placed in scintillation vials, and processed for liquid scintillation counting by using CytoScint scintillation cocktail (MP Biomedicals, Aurora, OH).

Vacuole Staining and Microscopy
Vacuolar accumulation of quinacrine was examined as described previously (Roberts et al., 1991). Briefly, 3 x 10⁷ log-phase yeast cells were harvested and resuspended in 500 µl of YPD buffered with 50 mM Na₂HPO₄, pH 7.6, containing 200 µM quinacrine. After incubation at room temperature for 5 min, cells were sedimented at 10,000 x g for 5 s, washed once with 500 µl of 2% glucose buffered with 50 mM Na₂HPO₄, pH 7.6, and resuspended in 100 µl of the same solution. Samples were applied to a microscope slide and viewed immediately in the fluorescence microscope by using a fluorescein filter. To stain with N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM4-64), yeast cells were grown to logarithmic phase in YPD, and 16 µM FM4-64 (Invitrogen) was added from a stock solution in dimethyl sulfoxide (DMSO) for 30 min. Samples were applied to a microscope slide and viewed immediately in the fluorescence microscope by using a Texas Red filter.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Pmr1 Mutants Show Multiple Drug-sensitive Phenotypes That Arise from Calcium Homeostasis Defects
The best-known growth phenotypes of pmr1 mutants are directly related to cellular Ca²⁺ or Mn²⁺ homeostasis, as expected for the loss of a Ca²⁺, Mn²⁺-transporting ATPase. Thus, pmr1 strains are hypersensitive to removal of divalent cations from the medium by the chelator BAPTA. We have shown that BAPTA sensitivity in pmr1 mutants corresponds to calcium starvation in the early part of the secretory pathway and that it can be complemented by heterologous expression of phylogenetically diverse Ca²⁺-ATPases of the endoplasmic reticulum (ER), plasma membrane, or Golgi subtypes (Ton et al., 2002). Likewise, hypersensitivity of pmr1 strains to high MnCl₂ corresponds to accumulation of toxic levels of cellular Mn²⁺ and can be corrected specifically by Mn²⁺-transporting pumps unique to the Golgi subtype, including human SPCA1 and SPCA2. The recent observation that pmr1 mutants are sensitive to the antifungal agent amiodarone (Gupta et al., 2003) prompted us to search the database for other reports of drug sensitivities. We found that pmr1 was identified in genome-wide screens of the yeast deletion library for hypersensitivity to wortmannin, tunicamycin, and sulfometuron (Zewail et al., 2003; Parsons et al., 2004). These drugs target diverse and unrelated cellular pathways: wortmannin is an inhibitor of PI-4 kinase in yeast, tunicamycin blocks protein glycosylation, sulfometuron targets branched chain amino acid synthesis, and amiodarone, an antiarrhythmic agent, has been proposed to cause calcium-mediated cell death in yeast.

To evaluate whether the multiple drug-sensitive phenotypes associated with pmr1 null mutants arose from loss of Ca²⁺ or Mn²⁺ transport, or both, we made use of a previously described Pmr1 mutant that has an ion-selective defect. Mutant Q783A was shown to retain ⁴⁵Ca transport and Ca²⁺-ATPase activity at nearly wild-type levels but was severely attenuated in all Mn²⁺-dependent functions examined previously (Mandal et al., 2000). We introduced Q783A mutant and wild-type PMR1 genes into yeast strain K616 (pmr1pmc1cnb1; Cunningham and Fink, 1994), which shows robust ion-sensitive growth phenotypes, and we assessed drug sensitivity of the strains (Figure 1). As expected, wild-type Pmr1 improved growth, relative to the vector control, under all conditions tested. Mutant Q783A could not correct Mn²⁺ hypersensitivity of the host strain, but it improved growth in BAPTA similar to wild type (Figure 1, A and B), demonstrating the Mn²⁺-selective defect of this mutant. We show that expression of mutant Q783A complements drug hypersensitivity to wild-type (amiodarone; Figure 1C), or nearly wild-type levels (sulfometuron methyl, wortmannin, and tunicamycin; Figure 1, D–F), suggesting that Ca²⁺ transport plays a major role in mediating the requirement for Pmr1 in the cellular response to multiple drugs. We note that partial complementation by mutant Q783A in tunicamycin (Supplemental Figure 1) is consistent with a role for both Ca²⁺ and Mn²⁺ ions; thus, tunicamycin leads to accumulation of unfolded proteins, a form of cell stress known to require Ca²⁺ ions (Bonilla et al., 2002), however, tunicamycin toxicity may be exacerbated by depletion of Mn²⁺ ions from the Golgi, because endoglycosidases are Mn²⁺-requiring enzymes. Similarly, there may be overlapping requirements for Ca²⁺ and Mn²⁺ in other drug responses. A potential target, the ATM kinase, mutated in human ataxia telangiectasia, is a Mn²⁺-requiring enzyme that is sensitive to wortmannin (Chan et al., 2000). These observations underscore the importance of Pmr1-mediated ion homeostasis in multiple cellular pathways that impact drug sensitivity.

View larger version (29K): [in this window] [in a new window]   Figure 1. Ion specificity of pmr1 growth phenotypes. K616 (pmr1 pmc1 cnb1) was transformed with a CEN plasmid carrying URA3 alone (K616), wild-type ScPmr1 (PMR1), or ScPmr1 with Q783A mutation (Q783A). Cells (OD0.025) were cultured overnight in 1 ml of SC medium supplemented with varying concentrations of MnCl2 (A), BAPTA (B), amiodarone (C), sulfometuron methyl (D), tunicamycin (E), or wortmannin (F). Growth, expressed as percentage of control (without drug addition), was measured at OD600 and subtracted from background. Every data point is an average of four replicates. Plots are representative of at least two different experiments.

View larger version (15K): [in this window] [in a new window]   Figure 2. Drug stress induces 45Ca uptake. 45Ca accumulation was measured in whole cells after 2-h incubation with 45CaCl2 and the appropriate drug as follows: 40 µM amiodarone, 100 µg/ml sulfometuron methyl, 1 µg/ml tunicamycin, and 0.25 µg/ml wortmannin), as described under Materials and Methods. The total counts are expressed as percent normalized to the no drug control of BY4742 (WT). Data are averages of quadruplicates and representative of two independent experiments.

Genome-wide Screens for Multiple pmr1 Growth Phenotypes Are Enriched in Specific Cellular Pathways

View this table: [in this window] [in a new window]   Table 1. Genes sharing two or more of six pmr1 growth phenotypes, including hypersensitivity to amiodarone, wortmannin, tunicamycin, sulfometuron methyl, MnCl2, and BAPTA

View larger version (46K): [in this window] [in a new window]   Figure 3. Cell function and location of gene deletions that phenocopy pmr1. Graphical representation of genes that share three or more (3, outer ring; 34 genes) or two or more (2, middle ring; 118 genes) of six pmr1 growth sensitivities relative to the entire yeast proteome (inside ring; 6300 genes) classified by function (A) or cellular localization (B). Categories significantly enriched are indicated with numbers that represent the percentage of total genes in that data set.

Of the gene deletions displaying at least two pmr1 phenotypes (Table 1), only three shared all six drug and ion-related growth sensitivities with pmr1: of these three phenotypes, HUR1 is a dubious open reading frame (ORF) encoded on the opposite strand to that of PMR1, so that hur1 essentially recapitulates pmr1. Expression of HUR1 has not been detected at transcript or protein level, and it has no recognizable orthologues. Because reintroduction of PMR1 complements all six phenotypes (Figure 1), HUR1 represents a false positive of the screening strategy; however, it serves as an internal control and validates our findings. Deletion of VMA5, encoding a subunit of the vacuolar H⁺-ATPase complex, also shares all six growth phenotypes with pmr1. YKL118w is a dubious ORF that overlaps another subunit of the V-ATPase on the opposite strand, that of VMA12/VPH2. In all, 12 of the 18 known VMA subunits and assembly factors shared two or more knockout phenotypes with pmr1: VMA5, VMA2, TFP3/VMA11, VMA13, VMA7, VMA4, VMA12, VMA8, VMA10, VMA22, VMA6, and CUP5/VMA3, making this the single-most significant functional module of the collective phenotypes. We note that the shared multidrug sensitivity of pmr1 and vma mutants was recently extended to DNA damaging agents, including cisplatin and hydroxyurea (Pan et al., 2006; Liao et al., 2007).

Remarkably, chromatin remodeling complexes were also abundantly represented (Table 1). This included three components of the SWI/SNF nucleosome-remodeling complex (HAF4/SNF5, SNF6, and SNF2) and five subunits of the SAGA/ADA histone acetyltransferase coactivator complex (GCN5/ADA4, SPT20/ADA5, ADA1, SPT3, and SPT7/GIT2). Two other knockouts would also result in disruption of SWI/SNF components: YJL175w overlaps with SWI13 on the opposite strand, and YLR322w is a dubious ORF that would disrupt SFH1, a SNF5 homolog. Also included in this functional category are SET3, a histone deacetylase, and NHP10, which is related to mammalian high-mobility group proteins and a likely component of the INO80 ATP-dependent chromatin remodeling machinery. Together with transcription factors (GAL11, SIN4, GCN4, OPI1, and RPN4), these are likely to represent cellular stress response pathways. There is evidence that the SAGA complex is required for the expression of roughly 10% of the genome that is involved in stress response (Huisinga and Pugh, 2004; van Voorst et al., 2006). Our findings indicate that although ion stress (Ca²⁺, Mn²⁺, or H⁺) and diverse cytotoxic drugs (amiodarone, wortmannin, sulfometuron, or tunicamycin) may target different pathways, they converge to elicit similar downstream cell survival responses.

Multiple genes in the ergosterol biosynthesis pathway, including ERG2, ERG3, ERG4, ERG6, and SAC1, encoding a lipid inositol phosphoinositide phosphatase, were found to share two or more knockout phenotypes with PMR1. These genes would be expected to impact membrane integrity and permeability not only by altering lipid composition but also by affecting the activity of ion and drug transporters as well as protein and vesicular trafficking pathways. Indeed, the latter were represented by genes directing traffic to or from compartments of the Golgi, endosomes, and vacuole (VPS45, VPS51, VPS52, VPS54, SNF8/VPS22, SNF7/VPS32, and COG5).

Notably missing from the list of gene mutations that phenocopy pmr1 were known Ca²⁺ transporters (PMC1, VCX1) and Ca²⁺ channels (CCH1, MID1, YVC1), pointing to a distinct, nonredundant cellular role of Pmr1. Although large, compensatory increases in expression of Pmc1, the vacuolar ATP-driven Ca²⁺ pump, have been reported (Marchi et al., 1999; Locke et al., 2000), pmr1 mutants retain distinct phenotypes. This may be because of the unusual ion selectivity of Pmr1, which transports both Mn²⁺ and Ca²⁺ ions, and more importantly, its unique localization in the Golgi.

pmr1 Mutants Maintain Normal Vacuolar pH Despite Aberrant Vacuolar Morphology
Because the vacuolar ATPase has to traffic through the early secretory pathway and Golgi, where Pmr1 activity is important, we considered the possibility that the V-ATPase was dysfunctional in pmr1 mutants, resulting in overlapping vma and pmr1 phenotypes. The diagnostic test for loss of acidic vacuoles is the inability to grow at alkaline pH or to handle calcium stress at alkaline pH, as seen for vma5 (Figure 4, A and B). However, pmr1 was similar to wild type in the growth response to alkaline pH and high calcium (Figure 4, A and B), suggesting that vacuolar pH was normal in this mutant. In vma mutants, a consequence of defective acidification within a post-Golgi endosomal compartment is the failure to load the multicopper oxidase Fet3 with copper, resulting in iron starvation and growth sensitivity to the iron-specific chelating agent bathophenanthroline disulfonate (BPS) (Davis-Kaplan et al., 2004). We show that the pmr1 mutant is not hypersensitive to BPS, indicating that loss of the Golgi Ca²⁺, Mn²⁺ pump does not significantly affect the pH-dependent transport and availability of copper to Fet3 (Figure 4C). Furthermore, vacuoles of wild-type and pmr1 cells readily took up the fluorescent weak base quinacrine, which becomes protonated and trapped within acidic compartments (Figure 5, A and B). In contrast, the vma5 mutant failed to accumulate quinacrine, characteristic of defective vacuolar acidification (Figure 5C). However, quinacrine labeling did reveal aberrant vacuolar morphology in the pmr1 mutant, as has been reported previously (Kellermayer et al., 2003). This was more readily visualized using the vacuolar membrane dye FM4-64 (Figure 6), which showed the appearance of multiple vacuolar lobes or fragmented vacuolar compartments in pmr1, suggesting a membrane fusion defect. Interestingly, addition of amiodarone caused a rapid fusion of these compartments, resulting in a single large vacuole within a few minutes (Figure 6C). We hypothesized that the amiodarone-induced calcium burst (Courchesne and Ozturk, 2003; Gupta et al., 2003) promoted membrane fusion of pmr1 vacuoles. Consistent with this idea, addition of 5 mM CaCl₂ also promoted vacuolar fusion in the pmr1 mutant (Figure 6D). We conclude that despite the aberrant vacuolar morphology, the pmr1 mutant has normal vacuolar pH and pH-related vacuolar functions.

View larger version (21K): [in this window] [in a new window]   Figure 4. The pmr1 mutant does not have pH-related growth defects. Alkaline sensitivity was compared in isogenic BY4742 (WT), pmr1 and vma5 strains cultured overnight in minimal media buffered to the indicated pH with 50 mM 2-(N-morpholino)ethanesulfonic acid/Tris (A) or with added CaCl2 in media buffered to pH 5 or 5.5 (B). Growth was measured as OD600 and expressed as percentage of pH 5 (A) or no CaCl2 (B). Data are the average of two replicates. In C, iron deficiency was evaluated by measuring growth in liquid SC medium supplemented with the Fe-specific chelator BPS (graph) or by drop tests in YPD medium supplemented with 80 µM BPS where indicated (inset). The graph shows the average of quadruplicate determinations and the inset shows serial dilutions of 1:5.

View larger version (40K): [in this window] [in a new window]   Figure 5. Quinacrine staining of vacuoles is defective in vma5 but not pmr1. Cells were incubated with quinacrine for 5 min, as described under Materials and Methods. Micrographs of cell fluorescence (left) and phase contrast (right) show normal pH-dependent uptake of quinacrine in vacuoles of wild-type (WT) and pmr1 yeast, whereas vma5 vacuoles fail to accumulate the dye.

View larger version (16K): [in this window] [in a new window]   Figure 6. Aberrant morphology of vacuolar compartments in pmr1 can be corrected by Ca2+ influx. Fluorescence micrographs show FM4-64 staining of WT (BY4742) and pmr1 cells. Vacuoles in pmr1 have a fragmented appearance relative to wild type. Incubation with 40 µM AMD or 5 mM CaCl2 for 5 min elicits fusion of the smaller compartments into a single large vacuole with some FM4-64 staining fragments remaining.

Synthetic Growth Defect of pmr1 and vma Mutants Point to Parallel, Additive Roles
Genes that share multiple knockout phenotypes with pmr1 may lie within the same pathway, or they may function in parallel pathways that achieve the same cellular outcome. A straightforward approach to differentiate between these two possibilities is to test for synthetic fitness defects in double null mutants. We show that disruption of both PMR1 and VMA genes leads to severe synthetic fitness defects. Although all spores derived from tetrads in these crosses were able to germinate, the double null mutants of PMR1 and either VMA2 or VMA5 were much smaller in size (Supplemental Figure 2) and grew poorly (Figure 7, A and B). In contrast, a double null mutant of PMR1 and SNF6, a component of the chromatin remodeling complex, showed no synthetic growth defect (Figure 7C). These genetic data corroborate our biochemical and phenotypic findings to show that Pmr1 and V-ATPase work in parallel toward a common cellular function.

View larger version (91K): [in this window] [in a new window]   Figure 7. Synthetic fitness defect of pmr1 vma double mutants. Double null mutants of PMR1 with VMA2, VMA5 and SNF6 were generated using a NAT replacement protocol. After sporulation, mutants were plated on YPD for comparison of growth fitness. Compared with the individual null mutants, pmr1snf6 showed no synthetic growth defect, whereas both pmr1vma5 and pmr1vma2 grew poorly. Data are representative of haploid progenies dissected from 20 tetrads for each cross.

View larger version (32K): [in this window] [in a new window]   Figure 8. Shared pmr1 and vma phenotypes are enriched for Golgi-localized functions. Venn diagram representing overlap between pmr1 phenocopy screen (PC; this work), functional screens for secretion of CPY (Bonangelino et al., 2002) and low dye binding of Alcian Blue (LDB; Corbacho et al., 2005). The 22 genes common to all three data sets are listed to the right and include VMA and PMR1 genes, HOC1 that encodes a mannosyltransferase of the Golgi, and several VPS genes involved in Golgi traffic.

View larger version (38K): [in this window] [in a new window]   Figure 9. Novel pmr1 phenotypes of calcofluor white and zinc sensitivity are shared with vma5. (A) Growth of yeast strains in the presence of calcofluor white. BY4742 (WT), and isogenic pmr1 and vma5 were grown overnight in minimal media and adjusted to 1 OD/ml. Two microliters of fivefold serial dilutions of each strain were spotted on YPD plates supplemented with DMSO as solvent control (YPD), 20 or 50 µg/ml calcofluor white (CW 20 and CW 50, respectively). Growth at 30°C was monitored after 2 d. (B) Zn2+ sensitivity of yeast strains. One-milliliter cultures of SC medium supplemented with indicated amounts of ZnCl2 were seeded with OD0.025 of cells and incubated at 30°C overnight. Background subtracted growth (OD600) was an average of quadruplicates and is expressed as a percentage of control (no ZnCl2). (C) Calcofluor white sensitivity of strain K616, plated as in A, is complemented by both WT (PMR1) and mutant Q783A. (D) Zn2+ sensitivity of K616, monitored as in B, is complemented by both WT (PMR1) and mutant Q783A.

View larger version (18K): [in this window] [in a new window]   Figure 10. Gene interactions between ion (H+, Ca2+) transporters and calcineurin. Heavy lines indicate synthetic sick or lethal interactions, and dotted lines indicate absence of any synthetic growth effects between null alleles in standard medium (i.e., absence of added calcium stress). Arrows represent biochemical activation; thus, the V-ATPase provides the H+ gradient that drives Ca2+ uptake by Vcx1, and activated calcineurin mediates transcriptional induction of PMR1 and PMC1 genes. See text for details and references.

In mammalian cells, there is experimental evidence for the trafficking and function of V-ATPase along secretory and endocytic pathways to and from the plasma membrane (Breton and Brown, 2007). Although yeast Vma has not been detected at the cell surface, vma mutants show markedly reduced rates of endocytosis (Perzov et al., 2002). Moreover, there is evidence for a distinct, Golgi-localized isoform of the 100-kDa a subunit of the V-ATPase, which contains the Stv1 subunit in place of Vph1, resulting in distinct functional and regulatory properties (Manolson et al., 1994; Kawasaki-Nishi et al., 2001). In contrast to the multiplicity of subunit isoforms in mammals, Stv1 and Vph1 represent the only V-ATPase subunit isoforms found in fungi, consistent with the importance of distinct Golgi and vacuolar functions of the V-ATPase. Because of the ability of these two subunit isoforms to partially compensate for each other, it has been difficult to separate Golgi-specific phenotypes of the V-ATPase from its vacuolar functions. The phenomic approach used in this study reveals distinct Vma functions that overlap with the Golgi Ca²⁺, Mn²⁺-ATPase Pmr1. We suggest that these shared phenotypes provide evidence for secretory pathway and prevacuolar functions of the V-ATPase. Given the excellent conservation of basic ion homeostasis mechanisms from yeast to human (for review, see Ton and Rao, 2005), our findings may be extrapolated to mammalian models. There are examples of defects in tissue-specific isoforms of V-ATPase or Ca²⁺-ATPase that are known or suspected to give rise to a similar disease phenotype, such as deafness and male sterility (Prasad et al., 2004; Breton and Brown, 2007). Calcium and proton homeostasis are also closely linked in normal and pathophysiological conditions; a case in point is osteopetrorickets (Kaplan et al., 1993), where defective acidification by V-ATPase leads to osteopetrosis, abnormally high body calcium, and paradoxically, poor calcium incorporation into bone. In conclusion, the overlap of calcium and proton homeostasis pathways, particularly in Golgi and prevacuolar traffic, revealed by a phenomics approach in yeast, may help in understanding and treatment of complex phenotypes associated with disease.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health grant GM-62142 (to R.R.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-11-1049) on February 21, 2007.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Rajini Rao (rrao{at}jhmi.edu)

Abbreviations used: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BPS, bathophenanthroline disulfonate; CPY, carboxypeptidase Y; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide; VPS, vacuolar protein sorting.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
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