A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

Abstract

The function of the vacuolar H⁺-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V₀ domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ) and vma21QQ in combination with voa1Δ, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects.

THE vacuolar H⁺-ATPase (V-ATPase) is a multisubunit complex that is highly conserved across eukaryotes (Graham  et al. 2003). It functions to actively acidify cellular compartments by coupling the hydrolysis of ATP to the translocation of protons across membranes through a conserved rotary mechanism (Hirata  et al. 2003; Yokoyama  et al. 2003; Imamura  et al. 2005). Organelle acidification plays a crucial role in various cellular functions such as vesicular trafficking, endocytosis, neurotransmitter uptake, membrane fusion, and ion homeostasis (Kane 2006; Forgac 2007). Specialized isoforms of the V-ATPase complex can be found on different cellular membranes including the plasma membrane (Forgac 2007). A number of human diseases have been associated with or directly linked to defects in the V-ATPase complex: osteopetrosis (Frattini  et al. 2000), renal tubular acidosis (Karet  et al. 1999), and cancer cell migration (Martinez-Zaguilán  et al. 1999). The V-ATPase is an essential complex for all eukaryotes with the exception of some fungi.

The budding yeast Saccharomyces cerevisiae requires the V-ATPase to survive under specific environmental conditions, including alkaline conditions or otherwise toxic levels of metals (Eide  et al. 2005; Kane 2006). Yeast utilize the proton gradient created by the V-ATPase to drive sequestration of Ca²⁺ and Zn²⁺ ions within the vacuole (Klionsky  et al. 1990). A variety of proton-exchange antiporter pumps reside on the vacuolar membrane and other organelles that participate in maintaining nontoxic cytosolic levels of various ions and metals including calcium and zinc (Miseta  et al. 1999; MacDiarmid  et al. 2002). Deletion of any of the V-ATPase component proteins results in a number of specific growth and cellular phenotypes, including sensitivity to excess metals and a lack of vacuolar acidification. This makes yeast a useful model system to study the V-ATPase complex (Graham  et al. 2003; Kane 2006). Complete disruption of V-ATPase function in yeast results in a characteristic Vma^– phenotype: failure to grow on media buffered to pH 7.5 (Kane 2006). Additionally, numerous genetic screens have demonstrated that a loss of the V-ATPase renders yeast sensitive to a variety of metals (including zinc and calcium) and drugs (Eide  et al. 2005; Kane 2007). Yeast lacking the V-ATPase complex do not acidify their vacuoles as shown through a lack of quinacrine staining (Weisman  et al. 1987).

The V-ATPase enzyme in yeast contains 14 protein subunits within two domains: the V₁ portion is responsible for hydrolyzing ATP, and the V₀ portion shuttles protons across a lipid bilayer (Forgac 2007). The V₁ domain (subunits A–H) is peripherally associated, and the V₀ domain (subunits a, d, e, c, c′, and c′′) is imbedded within the membrane except subunit d, which is a peripheral membrane protein. The functional V-ATPase enzyme requires the presence of all of these subunits. Subunit a has two isoforms in yeast, the absence of one of them is not sufficient to cause a Vma^– phenotype. Yeast contain two populations of the V-ATPase complex and their localization is dictated by the incorporation of one of two isoforms of subunit a, Stv1p or Vph1p (Manolson  et al. 1992, 1994). While higher eukaryotes contain numerous isoforms for many of the different V-ATPase subunits (Marshansky and Futai 2008), Stv1p/Vph1p is the only structural difference between the two yeast enzymes. V-ATPase complexes containing the Vph1 protein are trafficked to the vacuolar membrane while Stv1p-containing V-ATPases are retained within the Golgi/endosomal network (Manolson  et al. 1994). Similarly, higher eukaryotes use different isoforms of subunit a to direct the localization of the V-ATPase to specific cellular compartments (Forgac 2007). One mechanism of V-ATPase regulation occurs through the rapid, reversible dissociation of the V₁ and V₀ domains (Kane 2006).

In the absence of any V₁ subunit, the V₀ domain is still properly assembled and targeted to the vacuole (Graham  et al. 2003). In the absence of the V₀ domain, V₁ is still assembled (Tomashek  et al. 1997). Loss of any V₀ subunit protein prevents proper V₀ assembly and ER exit (Graham  et al. 2003), and Vph1p undergoes ubiquitin-dependent ER-associated degradation (ERAD) (Hill and Cooper 2000). Assembly of the V₀ domain occurs in the ER and requires the presence of a number of additional proteins (Forgac 2007). Five ER-localized assembly factors have been identified in yeast that are required for full V-ATPase function yet are not part of the final complex: Vma21p, Vma22p, Vma12p, Pkr1p, and Voa1p (Hirata  et al. 1993; Hill and Stevens 1995; Malkus  et al. 2004; Davis-Kaplan  et al. 2006; Ryan  et al. 2008). Deletion of VMA21, VMA12, or VMA22 causes a failure of the V₀ subunits to properly assembly in the ER and a complete loss of V-ATPase function, resulting in a full Vma^– phenotype (Graham  et al. 2003). Yeast lacking PKR1 show a limited amount of V₀ assembly (Davis-Kaplan  et al. 2006) and yeast lacking VOA1 display only a slight reduction in V-ATPase enzyme activity (Ryan  et al. 2008). Consequently, pkr1Δ cells score a partial Vma^– phenotype while voa1Δ cells appear normal.

Genetic screens in S. cerevisiae have been critical in identifying the components of the V-ATPase and its associated factors (Ohya  et al. 1991; Ho  et al. 1993; Sambade  et al. 2005). However, the most recently discovered V-ATPase assembly factor, Voa1p, was identified by proteomics and voa1Δ cells have no detectable growth phenotype (Ryan  et al. 2008). Voa1p physically associates with the Vma21p-V₀ complex early in V-ATPase assembly and deletion of VOA1 displays a dramatic growth phenotype in conjunction with a specific mutant allele of the VMA21 assembly factor, vma21QQ (Ryan  et al. 2008). In yeast, it has been shown that Vma21p plays a critical role in V-ATPase assembly and chaperones the completed V₀ subcomplex out of the ER to the Golgi (Hill and Stevens 1994; Malkus  et al. 2004). Vma21p is retrieved back to the ER through a conserved, C-terminal dilysine motif and participates in multiple rounds of assembly and transport (Hill and Stevens 1994; Malkus  et al. 2004). Mutation of the dilysines to diglutamine residues, as in vma21QQ, results in mislocalization of yeast Vma21p to the vacuolar membrane and a significant loss of V-ATPase function.

The identification of V-ATPase assembly factors like Voa1p that do not display a full Vma^– are not likely to be found using traditional forward genetic screens. Also, pathways involved in promoting full V-ATPase function may act independently of V₁ and/or V₀ assembly and require a sensitized genetic background to produce a detectible growth phenotype. However, vma21QQ mutant yeast and, more so, the vma21QQ  voa1Δ double mutant are two cases where the V-ATPase is partially compromised for function (Hill and Stevens 1994; Ryan  et al. 2008). We have chosen to use these two assembly mutants in genome-wide enhancer screens to identify additional factors that assist in promoting full V-ATPase function by searching for genes that will cause an increase in calcium or zinc sensitivity when deleted.

Here we report the identification of HPH1 and ORM2 in a genome-wide search for V-ATPase effectors. We describe the characterization of these two redundant yeast gene pairs, HPH1/HPH2 and ORM1/ORM2, both of which display synthetic growth defects when deleted in combination with the vma21QQ mutant. Both sets of genes were found to have specific growth phenotypes on zinc and calcium media. Deletion of either gene pair did not affect vacuolar acidification or assembly of the V₀ domain. However, deletion of ORM1 and ORM2 results in a reduction of V-ATPase activity. The Orm proteins have very recently been shown to be negative regulators of sphingolipid synthesis (Breslow  et al. 2010; Han  et al. 2010). Consistent with these reports, we find that disruption of sphingolipid biogenesis is able to suppress Orm-related growth defects.

MATERIALS and METHODS

Plasmids and yeast strains:

Bacterial and yeast manipulations were done using standard laboratory protocols for molecular biology (Sambrook and Russel 2001). Plasmids for this study are listed in Table 1. ORM1 plus flanking sequence was amplified by polymerase chain reaction (PCR) from BY4741 (Invitrogen, Carlsbad, CA) genomic DNA using primers containing an upstream BamHI restriction site and downstream SalI restriction site. This fragment was inserted into pCR4Blunt-TOPO (Invitrogen), digested, and ligated into the BamHI and SalI sites of YEp351 to create pGF127. pGF87 was created using homologous recombination and in vivo ligation by gapping pRS415 and cotransforming a PCR fragment containing prVPH1∷VPH1∷GFP∷Sp_HIS5 (amplified from pGF06) with flanking sequence to the pRS415 vector.

TABLE 1

Plasmids used in this study

Plasmid	Description	Reference
pRS415	CEN, LEU2	Simons  et al. (1987)
pRS316	CEN, URA3	Sikorski and Hieter (1989)
YEp351	2μ, LEU2	Hill  et al. (1986)
pGF127	YEp351 ORM1	This study
pGF87	pRS415 VPH1∷GFP∷Sp_HIS5	This study
pGF06	pRS316 VPH1∷GFP∷Sp_HIS5	Ryan  et al. (2008)
pGF20	pRS316 VMA2∷mCherry∷NatR	This study

Yeast strains used in this study are listed in Table 2. GFY164 was created by PCR amplifying the hph1Δ∷Kan^R cassette plus 500 bp flanking sequence from corresponding BY4741 strains of the genome deletion collection (Open Biosystems, Huntsville, AL). It was subcloned into pCR4Blunt-TOPO, reamplified by PCR, transformed into SF838-1Dα, and selected on YEPD plus G418 (Gold Biotechnology, St. Louis, MO). Strains containing deletion cassettes other than Kan^R (Hyg^R or Nat^R) were created by PCR amplifying either the Hyg^R or Nat^R cassette from pAG32 or pAG25, respectively (Goldstein and McCuster 1999) and transforming the fragment into the corresponding Kan^R genome deletion strain to exchange drug resistance markers. Deletions in the SF838-1Dα strain (GFY164, GFY165, GFY166, GFY168, GFY169, and GFY170) were constructed by PCR amplifying the appropriate gene locus (including 500 bp of 5′-UTR and 3′-UTR flanking sequence), transforming into wild-type (WT) SF838-1Dα, and selecting for the appropriate drug resistance. GFY167, GFY171, and GFY172 were created using LGY183 as the parental strain. GFY173 was created using MRY5 as the parental strain. All deletion strains in SF838-1Dα were confirmed by diagnostic PCR from genomic DNA with primers complementary to the 5′-UTR (750–1000 bp upstream of the start codon) and internal to the drug resistance gene. A disruption cassette was created to delete TSC3 by first PCR amplifying the TSC3 open reading frame with 500 bp of flanking UTR, subcloning in pCR4Blunt-TOPO, and introducing a unique restriction site within the ORF. The TSC3 gene was subcloned into pRS316 and the entire ORF was replaced by the Nat^R cassette using homologous recombination. The deletion cassette was amplified and cloned into pCR4Blunt-TOPO for use in creating both GFY174 (using SF838-1Dα as the parental strain), GFY175 (using GFY170 as the parental strain), and GFY313 (using MRY5 as the parental strain). pGF20 was created by first swapping GFP for mCherry (Shaner  et al. 2004) in pRS316 VMA2-GFP and introducing a unique PmeI restriction site downstream of mCherry using site-directed mutagenesis. Second, in vivo ligation was used to insert the ADH1 terminator and Nat^R cassette at the 3′ end of VMA2-mCherry. A single, C-terminal mCherry (PCR amplified from pGF20 including the Nat^R cassette) was integrated at the VMA2 locus in strains GFY170, SF838-1Dα, and TASY006. This created yeast strains GFY302, GFY304, and GFY305, respectively.

TABLE 2

Strains used in this study

Strain	Genotype	Reference
SF838-1Dα	MATα ura3-52 leu2-3,112 his4-519 ade6 pep4-3 gal2	Rothman and Stevens (1986)
Y7092	MATα can1Δ∷prSTE2-Sp_HIS5 lyp1Δ ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 LYS2	Tong and Boone (2006)
GFY36	Y7092; vma21QQ∷HA∷NatR	This study
GCY2	Y7092; vma21QQ∷HA voa1Δ∷KanR	This study
GCY3	Y7092; vma21QQ∷HA voa1Δ∷NatR	This study
GFY104	Y7092; vma21QQ∷HA voa1∷HygR	This study
TASY006	SF838-1Dα; vma21Δ∷KanR	Compton  et al. (2006)
LGY183	SF838-1Dα; vma21QQ∷HA	Ryan  et al. (2008)
MRY5	SF838-1Dα; vma21QQ∷HA voa1∷HygR	Ryan  et al. (2008)
MRY14	SF838-1Dα; voa1∷HygR	Ryan  et al. (2008)
GFY163	SF838-1Dα; vma21QQ∷HA∷NatR	This study
GFY164	SF838-1Dα; hph1Δ∷KanR	This study
GFY165	SF838-1Dα; hph2Δ∷KanR	This study
BY4741	MATa  leu2Δura3Δ met15Δ his3Δ	Yeast Deletion Collection
GFY181	BY4741; hph1Δ∷KanR hph2Δ∷HygR	This study
GFY166	SF838-1Dα; hph1Δ∷KanR hph2Δ∷HygR	This study
GFY167	SF838-1Dα; vma21QQ∷HA hph1Δ∷KanR hph2Δ∷NatR	This study
GFY168	SF838-1Dα; orm1Δ∷KanR	This study
GFY169	SF838-1Dα; orm2Δ∷KanR	This study
GFY170	SF838-1Dα; orm1Δ∷HygR orm2Δ∷KanR	This study
GFY171	SF838-1Dα; vma21QQ∷HA orm2Δ∷KanR	This study
GFY172	SF838-1Dα; vma21QQ∷HA orm1Δ∷HygR orm2Δ∷KanR	This study
GFY173	SF838-1Dα; vma21QQ∷HA voa1∷HygR hph1Δ∷KanR	This study
GFY174	SF838-1Dα; tsc3Δ∷NatR	This study
GFY175	SF838-1Dα; orm1ΔHygR orm2Δ∷KanR tsc3Δ∷NatR	This study
GFY304	SF838-1Dα; VMA2∷mCherry∷NatR	This study
GFY305	SF838-1Dα; VMA2∷mCherry∷NatR vma21Δ∷KanR	This study
GFY302	SF838-1Dα; VMA2∷mCherry∷NatR orm1Δ∷HygR orm2Δ∷KanR	This study
GFY313	SF838-1Dα; vma21QQ∷HA voa1∷HygR tsc3Δ∷NatR	This study

The synthetic genetic array (SGA) query strains were created from yeast parental strain Y7092 (Tong and Boone 2006). After PCR amplifying the vma21QQ∷HA∷Nat^R locus with 500 bp of flanking sequence from GFY163 genomic DNA, the PCR product was transformed into Y7092 to create GFY36. To create GCY3, GCY2 (vma21QQ∷HA  voa1Δ∷Kan^R) was transformed with the Nat^R cassette to replace the Kan^R cassette. Both the VMA21 and VOA1 loci were PCR amplified with flanking sequence and the PCR product was transformed into Y7092. GFY104 was created by PCR amplifying both the VMA21 and VOA1 loci from MRY5 (vma21QQ∷HA voa1∷Hyg^R) with 500 bp of flanking sequence and transforming the fragment into GCY3.

Culture conditions:

Yeast were cultured in YEPD (1% yeast extract, 2% peptone, and 2% dextrose), YEPD buffered to pH 5.0 using 50 mm succinate/phosphate plus 0.01% adenine, or synthetic minimal media with dextrose (SD) and the appropriate amino acids. Growth tests were performed by culturing exponentially growing yeast in rich medium to a cell density of 1.0 OD₆₀₀, serially diluted fivefold, and spotted onto agar plates. Plates used included YEPD pH 5.0, YEPD + 4.0 mm or 5.0 mm ZnCl₂, YEPD + 100 mm CaCl₂, and YEPD + 25 mm or 50 mm CaCl₂ pH 7.5 (using 50 mm HEPES).

Synthetic genetic array screen:

A synthetic genetic enhancer screen was performed as previously described (Tong  et al. 2001; Tong and Boone 2006). Briefly, the query strains (GFY36, GCY3, and GFY104) were mated to the MATa haploid genome deletion collection (BY4741, his3Δ1  leu2Δ0, met15Δ0, ura3Δ0) and double or triple haploid mutants were selected. The final haploid mutant array was spotted onto YEPD pH 5.0, YEPD + ZnCl₂, or YEPD + CaCl₂ pH 7.5 and incubated at 30° for 2–3 days. Scans of each plate were visually scored for colony size on each plate type used; colonies were scored for increased sensitivity to calcium or zinc. The genome deletion collection was also arrayed under identical conditions and scored in the same way. Colonies that showed equivalent sensitivity to either metal as a single deletion strain and as part of the double (or triple) mutant collection were not scored as positive hits. Only mutants that displayed a synthetic growth defect that was not present (or not as strong) in either of the single mutant strains were scored as positive hits. Gene ontology (GO) analysis was performed using the Saccharomyces Genome Database (SGD) GO term finder (version 0.83) using a P-value cutoff of 0.01.

Whole cell extract preparation and immunoblotting:

Whole cell extracts were prepared as previously described (Ryan  et al. 2008). Briefly, cultures were grown overnight in SD dropout media and then diluted to 0.25 OD₆₀₀/ml in YEPD pH 5.0 and grown to a cell density of OD₆₀₀ = 1.0. A total of 10 OD₆₀₀ of the culture was centrifuged, resuspended in 0.25 ml Thorner buffer (8 m urea, 5% SDS, and 50 mm TRIS pH 6.8), and vortexed with 0.2 ml of glass beads. Following centrifugation, protein concentrations were determined using a modified Lowry protein assay (Markwell  et al. 1978). Equal amounts of protein were separated by SDS–PAGE, transferred to nitrocellulose membrane, and probed with antibodies. Antibodies used included monoclonal primary anti-Vph1p (10D7; Invitrogen), anti-Vma1p (8B1; Invitrogen), and anti-Dpm1p (5C5; Invitrogen), and secondary horseradish peroxidase-conjugated antimouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were visualized by ECL detection.

Fluorescence microscopy:

Yeast were stained with quinacrine as previously described (Flannery  et al. 2004). Briefly, cells were grown overnight in YEPD pH 5.0 plus adenine, and diluted to a cell density of 0.25 OD₆₀₀/ml in YEPD. Yeast were harvested at a density of 0.8–1.0 OD₆₀₀/ml and 1 ml of culture was placed on ice for 5 min. Cells were pelleted and resuspended in 200 μm quinacrine, 100 mm Hepes pH 7.6, and 50 μg/ml of concanavalin A tetramethylrhodamine (Invitrogen) in YEPD for 10 min at 30°. Following staining with quinacrine, cells were placed on ice and washed three times in 100 mm Hepes pH 7.6 plus 2% glucose (4°). Microscopy images were obtained using an Axioplan 2 fluorescence microscope (Carl Zeiss, Thornwood, NY). A ×100 objective was used as well as AxioVision software (Carl Zeiss).

V-ATPase activity assay:

Yeast vacuoles were isolated from wild-type (SF838-1Dα and BY4741), vma21Δ∷Kan^R (TASY006), vma21QQ∷HA (LGY183), orm1Δ∷Hyg^R  orm2Δ∷Kan^R (GFY170), vma21QQ∷HA  voa1∷Hyg^R (MRY5), vma21QQ∷HA  orm1Δ∷Hyg^R  orm2Δ∷Kan^R (GFY172), and hph1Δ∷Kan^R  hph2Δ∷Hyg^R (GFY166 and GFY181) strains as previously published (Uchida  et al. 1985). Modifications to this protocol included harvesting cells at 1.8–2.2 OD₆₀₀/ml and use of a tighter-fitting dounce homogenizer (five strokes). Fresh vacuoles were assayed by a coupled spectrophotometric assay (Conibear and Stevens 2002). In this assay system, ATP hydrolysis is coupled to NADH oxidation (340 nm) in a reaction mixture containing 50 μg/ml vacuole membrane protein, 25 mm MES, 25 mm MOPS, 25 mm KCl, 5 mm MgCl₂, 1 mm NaN₃, 0.05 mm Na₃VO₄, 2 mm phosphoenolpyruvate, 0.5 mm NADH, 30 units/ml pyruvate kinase, and 37 units/ml lactate dehydrogenase; pH 7 (KOH), with and without 1 μm concanamycin A. Reactions were initiated by adding Mg²⁺-ATP to 2 mm and thermostatted at 30°. For each mutant strain, one to three separate vacuole preparations were assayed, and the assay was repeated two to six times for each preparation. Concanamycin A-sensitive ATPase activities were determined by calculating the activity as a percentage of wild-type activity for each biological replicate. For samples with multiple vacuole preparations, these percentages were averaged and the error was presented as the standard error of the mean. For samples with only a single biological preparation, the error is presented as the standard deviations for replicate assays.

RESULTS

Genome-wide SGA screen for V-ATPase effectors:

To identify new genes that assist in promoting full V-ATPase function, we performed three SGA Vma^– enhancer screens using the S. cerevisiae haploid deletion mutant collection (4741 mutants). Since a loss of VOA1 only displayed a synthetic growth defect upon combination with the vma21QQ mutation, additional factors might only be identified in a sensitized genetic background. The three query mutants used were vma21QQ∷Nat^R, vma21QQ  voa1Δ∷Nat^R, and vma21QQ  voa1∷Hyg^R (subsequent experiments were all performed with the voa1∷Hyg^R allele, designated as voa1Δ). The VMA21 and VOA1 loci are tightly linked; only 146 bp separate their open reading frames. We chose to use both mutants containing the VOA1 disruption as it has been shown that a complete deletion of this open reading frame results in a decrease in the steady-state levels of Vma21p, whereas the voa1∷Hyg^R allele does not lower Vma21p levels (Ryan  et al. 2008). Therefore, the voa1Δ∷Nat^R allele served as an additional sensitized genetic background.

Yeast that contain Vma21p having the mutated retrieval/retention signal, vma21QQ, show a partial growth defect on elevated calcium media buffered to pH 7.5 and have reduced V-ATPase activity (Hill and Stevens 1994; Ryan  et al. 2008). Additionally, vma21QQ  voa1Δ yeast are also compromised for V-ATPase function yet do not display a full Vma^– phenotype (Ryan  et al. 2008).

The haploid double or triple mutant yeast were plated onto rich media and media containing either zinc (2.75 mm or 7.0 mm) or calcium (50 mm or 100 mm) buffered to pH 7.5 in quadruplicate. Yeast that displayed increased sensitivity to these conditions were scored as positive hits. Genes were identified from a diverse set of cellular processes such as protein modification, metabolism, chromatin remodeling, and transcriptional regulation (Figure 1 and supporting information, Table S1). A comprehensive GO analysis was performed for categories of genes that were enriched in our SGA screens (Table S2). Some of the most highly enriched categories included vacuolar transport (P-value of 4.94 × 10⁻¹⁷), vesicle-mediated transport (P-value of 3.48 × 10⁻¹⁵), and intracellular transport (P-value of 9.46 × 10⁻¹⁰). Genes identified by the three SGA screens that correspond to elements of protein trafficking, vacuolar morphology, and the V-ATPase complex are listed in Table 3 and several were chosen for further study.

Figure 1.—

Molecular function of 144 genes identified in at least two of three SGA screens for an enhanced Vma⁻ phenotype. Genes were categorized according to their presumed molecular function. The miscellaneous category includes lipids/sphingolipids, cellular morphogenesis, nuclear import, and several other processes, and also genes with no molecular characterization. A comprehensive list of all genes identified can be found in Table S1 and a comprehensive GO analysis can be found in Table S2.

TABLE 3

Genes identified by genome-wide SGA screens for V-ATPase effectors that are trafficking related or whose protein products are ER localized

	Trafficking-related genes
AP-3 (4)	apl5Δ, apl6Δ, apm3Δ, aps3Δ
Vesicle formation (5)	arf1Δ, cdc50Δ, drs2Δ, vps1Δ, yap1801Δ
Rabs/vesicle targeting factors (5)	gyp1Δ, sro7Δ, vps21Δ, vps9Δ, ypt7Δ
TGN trafficking (11)	age2Δ, arl1Δ, arl3Δ, cog5Δ, cog6Δ, cog7Δ, cog8Δ, coy1Δ, gyp36Δ, sys1Δ, vps13Δ
ESCRT/MVB sorting (13)	vps2Δ, vps22Δ, vps23Δ, vps24Δ, vps25Δ, vps27Δ, vps36Δ, vps37Δ, vps4Δ, vps46Δ, vps55Δ, vps60Δ, vta1Δ
Vacuole inheritance (2)	cla4Δ, vac8Δ
HOPS/CORVET (4)	vam6Δ, vps3Δ, vps33Δ, vps41Δ
Endocytosis/exocytosis (6)	chs5Δ, chs6Δ, gsf2Δ, lst4Δ, rcy1Δ, drs2Δ
ER-Golgi trafficking (12)	bst1Δ, erd1Δ, erp3Δ, erv46Δ, gcs1Δ, get1Δ, get2Δ, get3Δ, gsg1Δ, pho86Δ, sec28Δ, ubp3Δ
Autophagy (5)	atg15Δ, atg21Δ, atg27Δ, atg8Δ, vps62Δ
GARP (2)	vps52Δ, vps53Δ
SNAREs and fusion (8)	gos1Δ, pep12Δ, sec22Δ, snx4Δ, swf1Δ, tlg2Δ, vam10Δ, vam7Δ
Phosphatidylinositol synthesis (3)	inp53Δ, sac1Δ, vac14Δ
Retromer (5)	vps17Δ, vps26Δ, vps29Δ, vps35Δ, vps5Δ
H+-V-ATPase (7)	pkr1Δ, rav1Δ, rav2Δ, stv1Δ, vph1Δ, vtc1Δ, vtc4Δ
Miscellaneous (5)	apm1Δ, ccz1Δ, mon1Δ, vps19Δ, yck3Δ
	Genes whose protein products are ER localized
Protein degradation (2)	cue1Δ, ubc7Δ
Protein import and maturation (5)	cne1Δ, cwh41Δ, emc1Δ, scj1Δ, sec66Δ
Miscellaneous (18)	alg6Δ, alg8Δ, bsd2Δ, csg2Δ, erg3Δ, erg6Δ, flc2Δ, hph1Δ, ice2Δ, ilm1Δ, mga2Δ, orm2Δ, ost3Δ, ost4Δ, per1Δ, scs2Δ, spf1Δ, sur4Δ

Genes found in our screens that are involved in either protein trafficking or are ER localized are listed above. A comprehensive list of all genes identified in all three screens can be found in Table S1. Screens were performed with query strains vma21QQ voa1∷Hyg^R (GFY104), vma21QQ voa1ΔNat^R (GCY3), and vma21QQ-Nat^R (GFY36). Colonies from the final double or triple mutant strains were analyzed for fitness defects on rich media plus 2.75 mm or 7.0 mm ZnCl₂ or rich media buffered to pH 7.5 plus 50 mm or 100 mm CaCl₂. The haploid deletion library was also tested and scored under identical media conditions. Fitness defects of single knockout strains were noted and considered when determining synthetic growth effects. HPH1 and ORM2 (shown in boldface type) were chosen for further study. A comprehensive gene ontology (GO) analysis for enriched categories of genes can be found in Table S2.

Since we were searching for genes that showed increased sensitivity to zinc or calcium when deleted in combination with vma21QQ or vma21QQ  voa1Δ, we did not identify any of the essential V-ATPase subunits or assembly factors (those with a VMA designation) as expected. Also, VOA1 was not identified in the vma21QQ SGA screen because it is genetically linked to the VMA21 locus. For the voa1Δ locus to be paired with the vma21QQ mutation during the SGA protocol, a cross-over event would be required between these two loci. However, we did identify V-ATPase subunits VPH1, STV1, the assembly factor PKR1, and genes within the vacuolar transporter chaperone (VTC) and regulator of vacuolar and endosomal membrane (RAVE) complexes.

In addition, genes involved in ER-resident processes including protein folding and degradation were identified (Table 3). A number of genes were found that have been poorly characterized according to the SGD and these were most interesting to us. We chose to examine Orm2p for further study because it was an ER-localized, integral membrane protein. Also, Hph1p has been identified as an ER-localized binding partner of calcineurin (Heath  et al. 2004). Due to the genetic link between calcineurin and the V-ATPase (Tanida  et al. 1995), we also chose HPH1 for further study.

HPH1/HPH2 or ORM1/ORM2 null mutants cause synthetic growth defects in vma21QQ yeast:

The hph1Δ mutation was moved to the SF838-1Dα genetic background and carefully tested by serial dilution in comparison to Vma^– and Vma-compromised strains. The growth phenotypes of vma21QQ  voa1Δ yeast deleted for HPH1 were tested on media containing 50 mm calcium buffered to pH 7.5 (Figure 2A). Whereas wild-type yeast were able to grow under these conditions, yeast deleted for VMA21 were unable to grow as they lack functional V-ATPase complexes. vma21QQ  voa1Δ yeast showed a compromised level of growth under these conditions and a deletion of HPH1 in this strain caused a further increase in sensitivity (Figure 2A). HPH1 has a homolog in S. cerevisiae, HPH2, and both Hph1p and Hph2p reside in the ER membrane (Heath  et al. 2004). It has been reported that deletions in HPH1 and HPH2 display a synthetic growth defect under alkaline conditions of pH 8.8 (Heath  et al. 2004). Since a reduction in V-ATPase function results in increased sensitivity to calcium or zinc, we determined whether a loss of the HPH genes results in a metal-specific phenotype. Loss of either HPH1 or HPH2 did not result in any sensitivity to excess zinc yet deletion of both HPH genes caused a dramatic growth defect on 5.0 mm ZnCl₂ (Figure 2B). Both HPH1 and HPH2 were also deleted in a strain containing the vma21QQ mutation and tested on the less stringent conditions of 4.0 mm ZnCl₂. Both vma21QQ and hph1Δ  hph2Δ yeast grew comparable to WT but the vma21QQ  hph1Δ  hph2Δ mutant was fully sensitive under these conditions and unable to grow (Figure 2C). Interestingly, the hph1Δ  hph2Δ double mutant did not show any sensitivity to 4.0 mm ZnCl₂ but displayed a dramatic shift in sensitivity between 4.0 mm and 5.0 mm ZnCl₂.

Figure 2.—

HPH1 and HPH2 display synthetic growth defects with V₀ assembly mutants. (A) Exponentially growing cultures of wild type (WT; SF838-1Dα), vma21Δ (21Δ; TASY006), vma21QQ voa∷Hyg^R (21QQ voa1Δ; MRY5), and vma21QQ voa1∷Hyg^R hph1Δ (21QQ voa1Δ hph1Δ; GFY173) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media buffered to pH 7.5 plus 50 mm CaCl₂. (B) Exponentially growing cultures of wild type, hph1Δ (hph1Δ; GFY164), hph2Δ (hph2Δ; GFY165), and hph1Δ hph2Δ (hph1Δ hph2Δ; GFY166) were spotted onto rich media or rich media plus 5.0 mm ZnCl₂. (C) Cultures of wild type, vma21Δ, vma21QQ (21QQ; LGY183), hph1Δ hph2Δ, and vma21QQ hph1Δ hph2Δ (21QQ hph1Δ hph2Δ; GFY167) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media plus 4.0 mm ZnCl₂.

The SGA screens also identified cells lacking ORM2 as an enhancer of the assembly factor mutant strains vma21QQ and vma21QQ  voa1Δ on media containing either zinc or calcium. The orm2Δ was recreated in SF838-1Dα cells expressing vma21QQ and tested on media containing 25 mm calcium buffered to pH 7.5. Loss of ORM2 in vma21QQ yeast caused a slight increase in the sensitivity of this strain compared to vma21QQ yeast (Figure 3A). ORM2 has a homolog in S. cerevisiae, ORM1, and the double orm1Δ  orm2Δ mutant shows synthetic growth defects under various environmental stress conditions including elevated mercury or the reducing agent DTT (Hjelmqvist  et al. 2002). We tested whether a loss of both ORM1 and ORM2 caused an increase in sensitivity to 25 mm calcium at pH 7.5. While deletion of ORM1 did not result in any growth defect, yeast deleted for ORM2 were partially sensitive under these conditions (Figure 3B). However, the orm1Δ  orm2Δ double mutant displayed a synthetic growth defect under these conditions. Due to the high similarity between ORM1 and ORM2 (∼67% identical), we tested whether ORM1 and ORM2 are functionally redundant. Overexpression of Orm1p was able to fully rescue the growth defect of an orm2Δ strain (Figure 3C). These data suggest that ORM1 and ORM2 are a functionally redundant gene pair; we chose to examine the effect of a loss of both Orm1p and Orm2p on the V-ATPase.

Figure 3.—

ORM1 and ORM2 display synthetic growth effects in cells expressing vma21QQ. (A) Exponentially growing cultures of wild type (WT; SF838-1Dα), vma21Δ (21Δ; TASY006), vma21QQ (21QQ; LGY183), and vma21QQ orm2Δ (21QQ orm2Δ; GFY171) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media buffered to pH 7.5 plus 25 mm CaCl₂. (B) Cultures of wild type, orm1Δ (orm1Δ; GFY168), orm2Δ (orm2Δ; GFY169), and orm1Δ orm2Δ (orm1Δ orm2Δ; GFY170) spotted onto rich media and rich media buffered to pH 7.5 plus 25 mm CaCl₂. (C) Cultures of wild type, orm2Δ, and orm2Δ transformed with a high-copy vector expressing ORM1 were spotted onto rich media and rich media buffered to pH 7.5 plus 50 mm CaCl₂. (D) Exponentially growing cultures of wild type, vma21Δ, vma21QQ, orm1Δ orm2Δ, and vma21QQ orm1Δ orm2Δ (21QQ orm1Δ orm2Δ; GFY172) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media plus 100 mm CaCl₂.

Finally, we compared the growth of vma21QQ and orm1Δ  orm2Δ yeast to the triple vma21QQ  orm1Δ  orm2Δ mutant using less stringent conditions of unbuffered 100 mm CaCl₂. Since a loss of both ORM1 and ORM2 was only sensitive under stringent growth conditions, we tested whether reducing V-ATPase function using the vma21QQ mutant would result in a more dramatic growth phenotype. Similar to the HPH1/2 genes, a loss of both ORM genes in vma21QQ yeast caused a severe growth defect (Figure 3D). This suggests that the ORM genes are required when V-ATPase activity is reduced.

Vacuolar acidification, V₀ assembly, and V-ATPase localization are normal in HPH and ORM mutants:

Yeast disrupted for V-ATPase function show decreases in vacuolar acidification (Davis-Kaplan  et al. 2006; Ryan  et al. 2008). To determine whether the growth defects seen with both the HPH and ORM mutants result from a loss of V-ATPase function, we assayed vacuolar acidification by fluorescent staining with quinacrine (Figure 4). Wild-type yeast displayed accumulation of quinacrine within the acidified vacuole while vma21Δ yeast showed no quinacrine staining. As previously shown, yeast mutant for either vma21QQ or voa1Δ displayed wild-type levels of quinacrine staining and vacuolar acidification (Ryan  et al. 2008; Figure 4). As expected, the vma21QQ  voa1Δ double mutant accumulated a very low level of quinacrine (Figure 4). Surprisingly, both the double mutants (hph1Δ  hph2Δ and orm1Δ  orm2Δ) and the triple mutant (vma21QQ  hph1Δ  hph2Δ) had fully acidified vacuoles (Figure 4). Only the vma21QQ  orm1Δ  orm2Δ mutant displayed a partial loss of quinacrine staining, indicating reduced V-ATPase function.

Figure 4.—

Loss of HPH1/HPH2 or ORM1/ORM2 does not result in a loss of vacuolar acidification. Exponentially growing cultures of wild type (WT; SF838-1Dα), vma21Δ (21Δ; TASY006), vma21QQ (21QQ; LGY183), voa1∷H (voa1Δ; MRY14), vma21QQ voa1∷H (21QQ voa1Δ; MRY5), hph1Δ hph2Δ (hph1Δ hph2Δ; GFY166), and vma21QQ hph1Δ hph2Δ (21QQ hph1Δ hph2Δ; GFY167), orm1Δ orm1Δ (orm1Δ orm2Δ; GFY170), and vma21QQ  orm1Δ orm2Δ (21QQ orm1Δ orm2Δ; GFY172) were stained with quinacrine (green) and concanavalin A-tetramethylrhodamine (red) and viewed by fluorescent and DIC microscopy.

While there was no detectable difference in vacuolar acidification, it is possible that the HPH and ORM mutants have slightly reduced levels of the V-ATPase present on the vacuole that might not be apparent using fluorescent microscopy but still be consistent with the observed growth phenotypes. We assayed the levels of Vph1p within these mutant strains by Western blotting to examine any defects in V₀ assembly (Figure 5). In wild-type yeast, Vph1 protein is extremely stable (>4-hr half-life; Graham  et al. 1998; Hill and Cooper 2000) and incorporated into the V₀ subcomplex. However, Vph1p is rapidly degraded (25–30 min half-life) if there is a V₀ assembly defect in the ER (Graham  et al. 1998; Hill and Cooper 2000). As predicted, the levels of Vph1p in vma21Δ yeast were greatly reduced compared to wild-type levels. In contrast, voa1Δ, hph1Δ  hph2Δ, and orm1Δ  orm2Δ yeast all displayed wild-type levels of Vph1p. This result indicates there is no V₀ assembly defect in these strains that would result in increased turnover of Vph1p. The vma21QQ mutant showed a slight decrease in Vph1p levels. Careful analysis has shown that this decrease was mirrored in HPH and ORM mutant strains also containing the vma21QQ mutation (vma21QQ  hph1Δ  hph2Δ and vma21QQ  orm1Δ  orm2Δ). Only vma21QQ  voa1Δ yeast showed a clear reduction in Vph1p that was greater than that seen in the vma21QQ mutant. The ER-resident protein Dpm1p was probed as a loading control. The steady-state levels of the V₁ subunit Vma1p did not change in any of the queried mutants.

Figure 5.—

Vph1p levels are not reduced in strains lacking either HPH1/HPH2 or ORM1/ORM2. Whole cell extracts were prepared from wild type (WT; SF838-1Dα), vma21Δ (21Δ; TASY006), vma21QQ (21QQ; LGY183), voa1∷H (voa1Δ; MRY14), vma21QQ voa1∷H (21QQ voa1Δ; MRY5), hph1Δ hph2Δ (hph1Δ hph2Δ; GFY166), and vma21QQ hph1Δ hph2Δ (21QQ hph1Δ hph2Δ; GFY167), orm1Δ orm1Δ (orm1Δ orm2Δ; GFY170), and vma21QQ orm1Δ orm2Δ (21QQ orm1Δ orm2Δ; GFY172). Proteins were separated by SDS–PAGE and probed with anti-Vph1p and anti-Vma1p antibodies. Anti-Dpm1p antibody was used as a loading control. The molecular mass (kilodaltons) of the nearest marker is shown on the left.

The localization of the V-ATPase was also examined in both the ORM and HPH mutant strains. Yeast expressing both the V₀ subunit Vph1p-GFP and the V₁ subunit Vma2p-mCherry were visualized by fluorescent microscopy. In wild-type yeast, Vph1p-GFP localized to the limiting membrane of the vacuole and colocalized with Vma2p-mCherry (Figure 6). There was also a pool of Vma2p-mCherry staining within the cytosol in a diffuse pattern. In vma21Δ yeast, Vph1p-GFP was found in both cortical and perinuclear ER structures and Vma2p-mCherry was only localized within the cytosol. Yeast mutant for orm1Δ  orm2Δ showed both V₀ and V₁ localized to the vacuolar membrane similar to wild-type cells (Figure 6). Strains mutant for hph1Δ  hph2Δ localized Vph1p-GFP to the vacuole (data not shown).

Figure 6.—

V₁ and V₀ are localized to the vacuole membrane in Orm mutant yeast. A single mCherry was integrated at the VMA2 locus in the following strains: wild type (WT; GFY304), vma21Δ (21Δ; GFY305), and orm1Δ orm2Δ (orm1Δ orm2Δ; GFY302). Yeast strains also contained a vector expressing Vph1p-GFP (pGF87). Exponentially growing cells were visualized by fluorescent and DIC microscopy.

Loss of the ORM genes (but not the HPH genes) results in reduced V-ATPase enzyme activity:

Since it is possible that the growth defects seen in the HPH and ORM strains could result from defects in V-ATPase enzyme function (rather than from assembly defects), we performed V-ATPase activity assays on isolated vacuole membranes in these mutant strains. We measured vacuole membranes from the hph1Δ  hph2Δ strain to have 88% V-ATPase activity, whereas the orm1Δ  orm2Δ mutant had 67% activity relative to wild-type yeast (Table 4). We also tested V-ATPase enzyme activity for the hph1Δ  hph2Δ mutant in a separate genetic background (BY4741) and found no difference from wild-type vacuole membranes (116% of WT; Table 4). We determined the vma21QQ mutant activity to be 22% of wild-type yeast despite the appearance of fully acidified vacuoles. The double vma21QQ  voa1Δ mutant showed a dramatic decrease to 9%. The vma21QQ  orm1Δ  orm2Δ triple mutant had a comparable reduction of V-ATPase activity to 8% relative to wild-type yeast. These results indicate that the Hph proteins do not affect the activity of the V-ATPase and that the Orm proteins are required for full V-ATPase function.

TABLE 4

V-ATPase activity and quinacrine staining of mutants

Strain	% wild-type ATPase activity	Quinacrine
Wild type (SF838-1Dα)	100	++++
Wild type (BY4741)	100	++++a
vma21Δ	1 ± 0.3 (1)d	—
hph1Δ hph2Δ (SF838-1Dα)	88 ± 6 (4)b	++++
hph1Δ hph2Δ (BY4741)	116 ± 0.7 (1)d	++++a
orm1Δ orm2Δ	67 ± 6 (3)	++++
voa1Δ	75c	++++
vma21QQ	22 ± 1 (3)	++++
vma21QQ voa1Δ	9 ± 0.5 (2)	+
vma21QQ orm1Δ orm2Δ	8 ± 0.5 (2)	++

Loss of both ORM1 and ORM2 results in a decrease in V-ATPase activity. Activity assays were performed for wild-type strains (WT; SF838-1Dα and WT; BY4741), vma21Δ (vma21Δ; TASY006), vma21QQ (vma21QQ; LGY183), voa1∷H (voa1Δ; MRY14), orm1Δ orm1Δ (orm1Δ orm2Δ; GFY170), vma21QQ voa1∷H (vma21QQ voa1Δ; MRY5), hph1Δ hph2Δ SF838-1Dα (hph1Δ hph2Δ; GFY166), hph1Δ hph2Δ BY4741 (hph1Δ hph2Δ; GFY181), and vma21QQ orm1Δ orm2Δ (vma21QQ  orm1Δ orm2Δ; GFY172). A continuous, coupled spectrophotometric assay (Conibear and Stevens 2002) was used to assay freshly prepared vacuole membranes for concanamycin A-sensitive ATPase activity. The wild-type strain SF838-1Dα had an average specific activity of 0.817 μmol min⁻¹ mg⁻¹ (average of n = 7 independent vacuole isolations) and the wild-type strain BY4741 has a specific activity of 0.836 μmol min⁻¹ mg⁻¹ (1 vacuole isolation). The specific activity of mutant samples was divided by the wild-type specific activity measurement for each independent vacuolar preparation to produce a relative percentage. For samples prepared more than once (biological replicates indicated in parentheses), the different percentages were averaged to produce the percentage of wild-type activity measurements ± the standard error of the mean. Quinacrine staining is derived from Figure 3.

a

Data not shown.

b

Three of four measurements for hph1Δ hph2Δ (SF838-1Dα) averaged 97% of wild type.

c

From Ryan  et al. (2008).

d

For strains with only a single biological preparation, the error is expressed as the standard deviation of technical replicates.

The Orm proteins function in sphingolipid regulation:

Two reports have recently demonstrated that Orm1p and Orm2p are negative regulators of the serine palmitoyltransferase (SPT) complex responsible for the first and rate-limiting enzymatic step of sphingolipid synthesis (Breslow  et al. 2010; Han  et al. 2010). Since loss of Orm1p and Orm2p has been shown to result in increased sphingolipid production (Breslow  et al. 2010; Han  et al. 2010), we tested whether inhibition of the SPT complex alleviated the defects seen in an orm1Δ  orm2Δ mutant. Tsc3p is a small protein that associates with the SPT enzyme and is required for full activity of this complex (Gable  et al. 2000). We deleted TSC3 in the orm1Δ  orm2Δ mutant and tested the triple mutant strain on media containing elevated calcium and buffered to pH 7.5 (Figure 7A). Yeast lacking TSC3 did not display any growth defect under these conditions. However, loss of TSC3 allowed for increased growth of the orm1Δ  orm2Δ strain. Additionally, suppression by deletion of TSC3 was specific to the orm1Δ  orm2Δ mutant, as loss of this regulator did not suppress the calcium sensitivity of the vma21QQ  voa1Δ mutant (Figure 7B). These results suggest that the growth defects seen in the orm1Δ  orm2Δ mutant strain are due to perturbation of sphingolipid synthesis.

Figure 7.—

Orm sensitivity to buffered calcium media can be suppressed by inhibition of sphingolipid biogenesis. (A) Cultures of wild type (WT; SF838-1Dα), vma21Δ (21Δ; TASY006), tsc3Δ (tsc3Δ; GFY174), orm1Δ orm2Δ (orm1Δ orm2Δ; GFY170), and orm1Δ orm2Δ tsc3Δ (orm1Δ orm2Δ tsc3Δ; GFY175) were spotted onto rich media and media containing 25 mm CaCl₂ buffered to pH 7.5. (B) Wild type, vma21QQ voa1Δ (21QQ voa1Δ; MRY5), tsc3Δ, and vma21QQ voa1Δ tsc3Δ (21QQ voa1Δ tsc3Δ; GFY313) yeast were spotted onto rich media and media containing 100 mm CaCl₂.

DISCUSSION

The goal of this study was to identify additional factors that contribute to V-ATPase function that may have been missed by previous forward genetic screens. The power of enhancer and suppressor screens is evident from work in both Drosophila melanogaster and Caenorhabditis elegans where it is often necessary to use a sensitized background to uncover new genetic pathways (Jorgensen and Mango 2002; St  Johnston 2002). In the case of the yeast V-ATPase, parallel genetic pathways are most likely not the main obstacles for identifying subtle effectors of this complex. Instead, the V-ATPase enzyme within the cell requires a dramatic decrease in enzyme function or assembly before cellular growth phenotypes become evident (Ryan  et al. 2008). The discovery of the fourth and fifth factors that participate in V₀ assembly (Pkr1p and Voa1p) demonstrates the complexity of the assembly processes required for the V-ATPase enzyme complex. While Voa1p has been shown to physically associate with the V₀ subcomplex in the ER (Ryan  et al. 2008), no physical association has been characterized for Pkr1p despite a strong genetic link to the assembly factor Vma21p (Graham and Stevens 1998; Davis-Kaplan  et al. 2006; data not shown). Growth phenotypes associated with perturbation of the V-ATPase are only evident upon a significant reduction in enzyme activity to ∼20% of wild type as in the case of the mutant assembly factor allele, vma21QQ (Hill and Stevens 1994). This retrieval-defective mutant allele of the highly conserved assembly factor, Vma21p, is a unique scenario to serve as a genetic tool for enhancer (and suppressor) screens because (i) the levels of functional V-ATPase are sufficiently low to allow for phenotypic scoring, and (ii) the V-ATPase is not compromised for enzyme function, but rather, is defective for assembly due to the limited supply of Vma21p in the ER.

In performing genome-wide enhancer screens with the vma21QQ and vma21QQ  voa1Δ alleles, a large number of genes involved in vesicular trafficking pathways, endosomal sorting complex required for transport (ESCRT) machinery, vesicle formation, and vacuolar morphology were identified. Deletion of some trafficking-related genes has been shown to result in sensitivity to zinc, calcium, or alkaline conditions (Serrano  et al. 2004; Sambade  et al. 2005; Pagani  et al. 2007). Our strategy for screening effectively detected reduced V-ATPase activity levels to ∼10% of wild-type yeast (scored as <50% of activity in the vma21QQ mutant strain). It is therefore not surprising that we have identified additional genes involved in trafficking pathways that were not previously found in forward genetic screens. For instance, many of the vacuolar protein sorting (VPS) genes have not been identified in previous genome-wide screens for Vma^– phenotypes (Sambade  et al. 2005). The genetic relationship between these sorting pathways and vacuolar acidification most likely results from aberrant sorting of the V-ATPase. There are many ways by which disruption of vesicular trafficking can result in mislocalization of the V-ATPase enzyme. For example, loss of the AAA-ATPase Vps4p results in an aberrant multivesicular body that traps vacuole-bound cargo in this compartment (Raymond  et al. 1992). In addition, loss of the syntaxin Pep12p (another class of trafficking mutants) results in mislocalization of Vph1p (Gerrard  et al. 2000). If the V-ATPase is not targeted to the vacuolar membrane, the pH gradient necessary to drive the sequestration of excess metals is not established, and the result is an increased sensitivity in our screen. Due to the complexity of protein sorting from the Golgi to the vacuole, there are many components that are required for proper transport of the V-ATPase to the vacuole membrane (Bowers and Stevens 2005). It will be of interest to determine whether disruption of other trafficking pathways is able to affect metal sensitivity without perturbation of V-ATPase localization and function.

We chose to characterize two gene families, HPH1 and ORM2, whose protein products had been previously reported to localize to the ER; both have homologs within budding yeast, HPH2 and ORM1, respectively (Hjelmqvist  et al. 2002; Heath  et al. 2004). HPH1 and HPH2 have been characterized as new components of calcineurin signaling (Heath  et al. 2004). Genetic screens have also found that deletion of any of the essential subunits of the V-ATPase is synthetic lethal with a loss of calcineurin (Tanida  et al. 1995; Parsons  et al. 2004). We therefore investigated the involvement of the Hph proteins in the function and/or assembly of the V-ATPase complex in the ER.

HPH1 and HPH2 have been previously reported to be functionally redundant and sensitive to high salinity or alkaline conditions (Heath  et al. 2004). We have found a unique set of growth phenotypes associated with a loss of HPH1 and HPH2 on media containing excess metals. The hph1Δ  hph2Δ mutant did not display any sensitivity to elevated calcium (data not shown) yet displayed a nonlinear shift in zinc sensitivity. This was unusual, as our previously observed V-ATPase mutants with a partial reduction in function, pkr1Δ, vma21QQ, and vma21QQ  voa1Δ (data not shown), exhibit growth defects on both zinc and calcium to varying degrees and have a gradual response to increasing concentrations of ZnCl₂. Preliminary work has also demonstrated that the zinc sensitivity of the hph1Δ  hph2Δ mutant was not completely dependent on calcineurin (data not shown). On the basis of our results, we propose that the growth defect of hph1Δ  hph2Δ yeast on ZnCl₂ likely results from a V-ATPase independent mechanism. It is unclear whether the zinc sensitivity in Hph mutants results from an effect on vacuole-localized Zn²⁺ transportation or some other mechanism.

Genome-wide screens for zinc or calcium (at pH 7.5) sensitivity have found genes that do not directly contribute to V-ATPase function yet show sensitivity to excess metals (Sambade  et al. 2005; Pagani  et al. 2007). An example is deletion of the vacuolar zinc transporter Zrc1p, which confers zinc sensitivity even though vacuolar acidification is normal (data not shown). Also, a loss of the serine protease Kex2p results in both calcium and zinc sensitivity yet does not cause any defect in vacuolar acidification (Sambade  et al. 2005). Identifying which genetic pathways are directly linked to metal sensitivity independent of V-ATPase function will require further study.

The Orm proteins are also functionally redundant, integral membrane proteins that localize to the ER in yeast (data not shown). Since loss of both ORM genes caused a reduction in vacuolar acidification and V-ATPase enzyme activity in the context of the vma21QQ mutation, we propose that Orm1p and Orm2p are necessary for full V-ATPase function. However, the phenotype associated with disruption of only the ORM genes does not completely phenocopy a loss of other assembly factors (such as PKR1 or the combination vma21QQ  voa1Δ). Key differences highlight the potential mechanism through which the Orm proteins may impact the V-ATPase including the lack of an apparent V₀ assembly defect. It is unlikely that the Orm proteins transiently participate in V₀ assembly of the V-ATPase, as we were unable to determine any physical association of Orm2p with the Vma21p-V₀ subcomplex (data not shown).

We also examined whether a variety of cargo proteins (including both subdomains of the V-ATPase) were aberrantly targeted upon a loss of the Orm proteins. Resident ER (Vma21p), Golgi (Vps10p), plasma membrane (Pma1p, Ste3p, and Snf3p), and vacuolar proteins (Sna3p, Zrt3p, Vcx1p, Pho8p, and Cps1p) did not display changes in localization patterns compared to wild-type yeast (data not shown). Consistent with these data, we found normal V₁V₀ localization of the V-ATPase to the vacuole membrane, suggesting an indirect involvement of Orm1p and Orm2p. A more likely scenario involves perturbation of V-ATPase function through other cellular pathways.

Recently, two studies have characterized Orm1p and Orm2p as negative regulators of sphingolipid synthesis (Breslow  et al. 2010; Han  et al. 2010). Both groups have reported that the Orm proteins physically associate with and regulate the SPT complex in the ER. Interestingly, inhibition of the SPT enzyme complex was found to alleviate phenotypes associated with a loss of ORM1 and ORM2, including cold sensitivity and sensitivity to tunicamycin (Han  et al. 2010). Our data are consistent with these findings and it is likely that ORM1 and ORM2 indirectly affect V-ATPase function through perturbation of sphingolipid production. This interpretation is in agreement with the effect of the lipid environment on the assembly, transport, or function of various enzymes including the amino acid permease Gap1p (Lauwers  et al. 2007), uracil permease Fur4p (Hearn  et al. 2003), and plasma membrane H⁺-ATPase Pma1p (Wang and Chang 2002; Gaigg  et al. 2006).

Also, deletion of two components of the fatty acid elongation pathway required for sphingolipid C26 acyl group synthesis (fen1Δ and sur4Δ) resulted in perturbation of vacuolar acidification, a decrease in V-ATPase enzyme activity, and a functionally compromised V₁ domain (Chung  et al. 2003). This previous study reported destabilization of the V-ATPase; specifically, a portion of the V₁ subdomain dissociates from the membrane during vacuole membrane preparation of the sur4Δ mutant. Since loss of the Orm proteins results in a similar change in the lipidome as sur4Δ yeast (Breslow  et al. 2010), loss of the V₁ subdomain during vacuolar preparation could explain why vma21QQ  orm1Δ  orm2Δ cells display levels of V-ATPase activity similar to vma21Q  voa1Δ yeast but higher vacuolar acidification in vivo. Since they are negative regulators, deletion of ORM1 and ORM2 results in an increase in the sphingolipid composition of the cell (Breslow  et al. 2010). Loss of the Orm proteins results in a subtle decrease in V-ATPase function and this differs from both the sur4Δ and fen1Δ mutations. However, this connection between sphingolipid regulation and V-ATPase function would have been missed in previous forward genetic screens. Overproduction of sphingolipids likely results in an altered lipid environment for the V-ATPase as sphingolipids play crucial roles for many cellular functions (Hanada 2003; Cowart and Obeid 2007).

The use of sensitized genetic backgrounds to identify factors that have subtle effects on V-ATPase function has revealed genes involved in a variety of cellular pathways. We have identified the Orm proteins and implicated sphingolipid regulation as important contributors to full V-ATPase enzyme activity. Future genome-wide screens that specifically assay V-ATPase function will aid in separating genes, like HPH1 and HPH2, which do not directly affect enzyme activity, from those that are dedicated effectors of V-ATPase assembly, transport, or enzyme function. Finally, further characterization of the precise mechanism by which alteration of sphingolipids and the cellular lipid composition affect function of the V-ATPase will be of great interest and provide a more complete understanding of this crucial molecular machine.

Footnotes

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.125567/DC1.

Footnotes

Communicating editor: M. D. Rose

Acknowledgements

We thank Laurie Graham and Emily Coonrod for reading the manuscript and members of the Stevens lab for discussions. This work was supported by the National Institutes of Health grant GM-38006 (to T.H.S.) and training grant S T32 GM-007257 (to G.C.F.).

Note added in proof: A very recent study implicated HPH1/HPH2 in V-ATPase biogenesis (Piña  et al., Euk. Cell, 10: 63–71, 2011). Piña  et al. report that Vph1p is degraded equally rapidly in vma21Δ and hph1Δ hph2Δ mutants (Piña  et al. 2011; Figure 3D), in contrast to our findings that Vph1p levels are normal in hph1Δ hph2Δ mutants, but significantly reduced in vma21Δ mutants (our Figure 5). Rapid turnover of Vph1p in hph1Δ hph2Δ yeast is inconsistent with near-normal growth in the presence of 250 mM Ca²⁺ (Piña  et al. 2011; Figure 3A) and 4 mM Zn²⁺ (our Figure 2C). Additionally, the stability of Vph1p in wild-type yeast determined by Piña  et al. is inconsistent with previously published measurements (>5 hours; Graham  et al., 1998). Finally and most importantly, our study reports that vacuoles from hph1Δ hph2Δ yeast (in two different genetic backgrounds) have 100% of wild-type V-ATPase activity, whereas vma21Δ cells have no measureable V-ATPase activity (our Table 4). In contrast to Piña  et al., based on our data, we conclude that the HPH1/HPH2 genes do not play a role in V-ATPase biogenesis.