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Eukaryotic Cell, November 2007, p. 2092-2101, Vol. 6, No. 11
1535-9778/07/$08.00+0     doi:10.1128/EC.00300-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Fission Yeast Phosphatidylserine Synthase Gene, pps1, Reveals Novel Cellular Functions for Phosphatidylserine

Yasuhiro Matsuo,¹ Edward Fisher,² Jana Patton-Vogt,² and Stevan Marcus^1*

Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487,¹ Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282²

Received 15 August 2007/ Accepted 18 September 2007

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To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1 mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1 cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1 mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1 cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.

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Phosphatidylserine (PS) is a quantitatively minor phospholipid in eukaryotic cells, typically comprising from 2 to 10% of total membrane phospholipids in any given organism (35). In the budding yeast Saccharomyces cerevisiae, PS is generated by a reaction between CDP-diacylglycerol (CDP-DAG) and serine, which is catalyzed by the single identified PS synthase enzyme in this organism, Cho1p (5, 19, 22, 26). Synthesis of PS from CDP-DAG has not been demonstrated in mammalian cells, nor have mammalian genes been identified that encode proteins structurally related to budding yeast Cho1p (35). However, two mammalian PS synthase enzymes, PS synthase 1 and PS synthase 2, have been discovered that catalyze the formation of PS through serine exchange reactions with phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively (21, 30, 31, 35). These mammalian PS synthases share significant structural homology with each other but lack homology to any proteins encoded by the S. cerevisiae genome, including Cho1p (35). In both budding yeast and mammalian cells, PS is a major precursor of PE, which is produced through reactions catalyzed by PS decarboxylases (35).

Insights into the diversity of biological functions of PS in eukaryotic organisms have been gained by previous studies in budding yeast and mammalian cells. S. cerevisiae cho1 mutants do not produce detectable PS and cannot grow on synthetic minimal medium unless it is supplemented with either ethanolamine or choline, which are converted to PE and PC, respectively, by enzymes of the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway (22, 35). Although exhibiting a number of phenotypic defects, including abnormalities in vacuole structure and function and mitochondrial stability, S. cerevisiae cho1 mutants grow at rates similar to wild-type cells in medium supplemented with ethanolamine or choline (3, 4, 16, 22), thus demonstrating that PS is not required for either the formation of functional membranes or for cell growth in this organism.

In several types of mammalian cells, it has been shown that PS, which is normally localized primarily to the cytosolic surface of the plasma membrane, becomes externalized to the cell surface during the apoptotic response (29). The resulting exposure of PS at the cell surface is believed to play a central role in the clearance of apoptotic cells by phagocytes. PS externalization has also been demonstrated to occur during platelet activation in the blood coagulation process and is required for the conversion of prothrombin to thrombin, a critical step in the blood-clotting cascade (6, 39). Interestingly, externalization of PS has also been demonstrated to occur during apoptosis-like cell death in budding yeast (23), indicating that the mechanisms regulating PS externalization likely represent a conserved and evolutionarily ancient process.

A number of recent studies have provided compelling evidence supporting roles for PS in signal transduction. Finkielstein and coworkers recently showed that the Rho family GTPases Rac1p and Cdc42p interact preferentially with PS-containing lipid bilayers (14). Indeed, these investigators showed not only that PS-enriched liposomes can induce Rac1p GTP loading and translocation to the plasma membrane in Chinese hamster ovary cells, but also that they induce membrane ruffling and the formation of filopodia, as well as activation of Cdc42p and mitogen-activated protein kinases. In another study, the membrane distribution of PS in T lymphocytes was shown to be regulated by the transmembrane tyrosine phosphatase CD45 in response to activation of the ATP receptor P2X₇ (12). The activities of several membrane proteins, including the cation channel activity of P2X₇, were shown in this study to be modulated by changes in PS distribution. PS has also been implicated in the regulation of signal transduction pathways from studies showing that protein kinase C as well as the serine/threonine kinase Raf-1 each bind with high specificity to PS and that this interaction promotes the membrane translocation that is required for activation of each kinase (7, 15).

Further insights into the biological roles and mechanisms of function of PS and PS-derived lipid products will undoubtedly be gained from studies in genetically tractable model organisms. To this end, in this paper we describe the cloning and characterization of the gene pps1, encoding the single PS synthase homolog in the fission yeast Schizosaccharomyces pombe. Our results demonstrate novel roles for PS in the regulation of cellular morphogenesis, cytokinesis, and cell survival in this model eukaryote.

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Yeast strains, manipulations, and analysis. S. pombe strains used in this study were SP870 (h⁹⁰ ade6-210 leu1-32 ura4-D18) (from D. Beach), SP870D (h⁹⁰/h⁹⁰ ade6-210/ade6-210 leu1-32/leu1-32 ura4-D18/ura4-D18) (24), CHP428 (h⁺ ade6-210 leu1-32 ura4-D18 his7-366) (from C. Hoffman), SPPPS1U (h⁹⁰ ade6-210 leu1-32 ura4-D18 pps1::ura4) (this study), and SPPPS1-GFP (h⁹⁰ ade6-210 leu1-32 ura4-D18 pps1-GFP-Kan^R) (this study). Standard yeast culture media and genetic methods were used (1, 28). S. pombe cultures were grown in either YEAU (0.5% yeast extract, 3% dextrose, 250 mg/liter adenine, 250 mg/liter uracil) or synthetic minimal medium (EMM) with appropriate auxotrophic supplements (1).

The pps1::ura4 deletion cassette was constructed using the recombinant PCR approach described by Krawchuk et al. (20). DNA fragments of 0.75 and 0.6 kb corresponding to the 5' and 3' regions, respectively, flanking the pps1 open reading frame were amplified by PCR using the oligonucleotide primers 5'-GTGCATACCAAAGTACTGTTC plus 5'-GGGGATCCGTCGACCTGCAGCGTACGACTTCCAAAATTGGCAGTTTC and 5'-GTTTAAACGAGCTCGAATTCATCGATACAGTACAGCGATCTGGGTA plus 5'-GGGACTTGGTTCTGAAAACC, respectively. The amplified fragments were attached to the ends of a ura4 gene cassette by PCR (20). SP870D was transformed with the resulting pps1::ura4 fragment, and transformants were isolated by plating on EMM lacking uracil. Diploid transformants carrying a single disrupted and single wild-type copy of the pps1 gene were identified by colony PCR (see below). pps1::ura4 haploid strains were isolated by tetrad dissection on YEAU containing 1 mM ethanolamine.

The pps1-GFP strain, SPPPS1-GFP, in which the C-terminal coding end of the chromosomal copy of the pps1 gene is fused to the green fluorescent protein (GFP) coding sequence, was constructed as follows. The pps1-GFP(S65T)-kanMX6 cassette was constructed using recombinant PCR as described elsewhere (20). DNA fragments of 0.5 and 0.6 kb corresponding to the 5' and 3' regions, respectively, of the pps1 gene were amplified by PCR using the oligonucleotide primers 5'-GTCGGTTTGTTCAATGAGTC plus 5'-GGGGATCCGTCGACCTGCAGCGTACGAAACATATTTGAGCGAATGTA and 5'-GTTTAAACGAGCTCGAATTCATCGATACAGTACAGCGATCTGGGTA plus 5'-GGGACTTGGTTCTGAAAACC, respectively. The amplified fragments were attached to the ends of a GFP(S65T)-kanMX6 cassette by PCR (20). SP870D was transformed with the resulting pps1-GFP(S65T)-kanMX6 cassette, and transformants were isolated on YEAU containing G418. Diploid transformants carrying a single GFP-tagged and single wild-type copy of the pps1 gene were identified by colony PCR. pps1-GFP haploid strains were isolated by tetrad dissection on YEAU containing 1 mM ethanolamine.

Plasmids. To construct pREP3XPps1, the oligonucleotide primers 5'-GGAAGGTCGACATGGTTAGATCACGAGTTTC and 5'-CATTGGATCCCTAAACATATTTGAGCGAATG were used to amplify a 1.1-kb fragment of the complete pps1 protein coding sequence from S. pombe genomic DNA. The amplified pps1 gene fragment was digested with SalI and BamHI and ligated to the corresponding sites of pREP3X to generate the plasmid pREP3XPps1. To induce the overexpression of pps1, pREP3XPps1 transformants were grown to early log phase in EMM containing 15 µM thiamine, washed three times in water, resuspended in EMM lacking thiamine, and incubated at 28°C for 48 h with subculturing (1:20 dilution) performed as needed to maintain the cultures at <10⁷ cells/ml.

To construct pREP41GFPPps1, the oligonucleotide primers 5'-GGAAGGTCGACCATGGTTAGATCACGAGTTTC and 5'-CATTGGATCCCTAAACATATTTGAGCGAATG were used to amplify a 1.1-kb fragment of the complete pps1 protein coding sequence from S. pombe genomic DNA. The amplified pps1 gene fragment was digested with SalI and BamHI and ligated to the corresponding sites of pREP41GFP to generate the plasmid pREP41GFPPps1.

Colony PCR. S. pombe colonies were suspended in 5 µl of water. The cell suspensions were added to PCR tubes containing 14.8 µl water, 2.5 µl of PCR buffer (TaKaRa), 2 µl of 2.5 mM deoxynucleoside triphosphate, 0.3 µl of each oligonucleotide primer pair (final concentration of 0.6 pmol/µl), and 0.1 µl of Ex-Taq polymerase (TaKaRa). The reaction mixtures were initially heated at 94°C for 2 min followed by 36 cycles of PCR (denaturation, 94°C for 30 s; annealing, 48°C for 40 s; extension, 72°C for 2 min). The final 72°C cycle was extended by 10 min.

Radiolabeling and analysis of S. pombe phospholipids. For phospholipid composition analysis, cells grown to uniform labeling in YEAU medium containing 5 µCi/ml [³²P]orthophosphate/ml were harvested at an optical density at 600 nm (OD₆₀₀) of 0.5 (1 x 10⁷ cells/ml). Where indicated, medium also contained 1 mM ethanolamine. Lipids were extracted (4), and the individual phospholipids were resolved using one-dimensional silica gel high-performance thin-layer chromatography plates (Merck) developed in chloroform-ethyl acetate-acetone-isopropanol-ethanol-methanol-water-acetic acid (30:6:6:6:16:28:6:2) (36). Phospholipids were detected and quantitated with a PhosphorImager (Bio-Rad GS-525).

PS synthase assay. Cultures were grown at 30°C in 1 liter of YEAU medium. Cells were harvested by centrifugation at 6,000 x g for 20 min, washed, and suspended in 0.1 M Tris-Cl pH 7.5, 5 mM ß-mercaptoethanol (BME), 10% glycerol (1 ml/g [wet weight]). Cells were lysed using a French press (three passes at 13,000 lb/in²). The homogenate was centrifuged at 3,000 x g for 5 min at 4°C to clear the unbroken cells. The supernatant was centrifuged at 27,000 x g for 10 min at 4°C, and the resulting pellet was suspended in 0.1 M Tris pH 7.5, 5 mM BME, 10% glycerol. The 27,000 x g supernatant was centrifuged at 100,000 x g for 1 h at 4°C, and the resulting pellet was suspended in 0.1 M Tris pH 7.5, 5 mM BME, 10% glycerol. The protein concentration was determined for all fractions in a bicinchoninic acid protein assay. PS synthase activity was measured at 30°C for 30 min by monitoring the incorporation of 0.5 mM L-[³H]serine into chloroform-soluble material, essentially as described previously (8). The optimal assay mixture contained 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 0.5 mM MnCl₂, 0.1 mM CDP-DAG (dipalmitoyl) added as a suspension in 1% Triton X-100, and 1 mg membrane protein, in a total volume of 0.1 ml. The reaction was terminated by the addition of 2 ml CHCl₃-MeOH (2:1). Following a low-speed spin to sediment membranous material, the supernatant was removed to a fresh tube and 400 µl of 0.9% NaCl was added. The resulting chloroform phase was transferred to a fresh tube and washed with 1 ml of CHCl₃-MeOH-0.9% NaCl (3:48:47). Finally, the chloroform phase was transferred into scintillation vials, dried under N₂, suspended in scintillation fluid, and counted.

Glucanase sensitivity assay. Cell wall integrity was measured essentially as described elsewhere (32). Briefly, S. pombe cells were grown at 28°C in YEAU with 1 mM ethanolamine, washed three times in YEAU, resuspended in YEAU and YEAU with 1 mM ethanolamine, and incubated overnight at 28°C to mid-log phase. The cells were collected by centrifugation and resuspended at a concentration of 10⁷ cells/ml in Tris-EDTA (TE). ß-Glucanase (Zymolyase 20T [ICN]) was treated at a concentration of 100 µg/ml at 28°C. The degree of cell lysis was evaluated by measuring the optical density at 600 nm.

Actin staining. To visualize F-actin, rhodamine-phalloidin staining was performed by using the method of Chang et al. (9), with some modifications. Cells grown in 10 ml YEAU with or without EA were fixed by 4.5 ml formaldehyde-PM (3 ml of 16% electron microscopy-grade methanol-free formaldehyde [Polyscience] and 1.5 ml PM [35 mM KH₂PO₄, pH 6.8, 0.5 mM MgSO₄]) for 5 min at 28°C. Cells were washed three times with PM, permeabilized with PM with 1% Triton X-100 for 2 min, and washed three times with PM. Three hundred units of rhodamine-phalloidin (Molecular Probes) was resuspended in 1.5 ml methanol and stored at –20°C. Before use, 5 µl rhodamine-phalloidin was evaporated in a speed vacuum and resuspended in 15 µl PM. For staining, cell pellets were incubated in rhodamine-phalloidin for 30 min at room temperature in the dark on a rotary inverter. One µl of stained cells was dried on a coverslip and mounted in 1 µl of 1-mg/ml p-phenylenediamine as an antifade agent.

Localization of Pps1-GFP and GFP-Pps1p fusion proteins. S. pombe pps1-GFP cultures were incubated at 28°C in YEAU containing 1 mM ethanolamine to mid-log phase prior to fluorescence photomicroscopy to detect GFP fusion protein localization. S. pombe cells transformed with pREP41GFPPps1 were grown to early log phase in EMM containing 15 µM thiamine, washed three times in EMM, resuspended in EMM containing 0.15 µM thiamine, and incubated at 28°C for 24 h to mid-log phase prior to visualization by fluorescence microscopy.

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Molecular cloning of the fission yeast PS synthase gene. In order to identify genes encoding potential PS synthase enzymes in fission yeast, we conducted TBLASTN and BLASTP searches of the S. pombe nucleic acid and protein sequence databases, respectively, using S. cerevisiae Cho1p (22) and mammalian PS synthase 1 protein (PSS-1p) and PSS-2p (30) as the search query sequences. BLAST searches using mammalian PSS-1p or PSS-2p as query sequences revealed no predicted protein-coding sequences in S. pombe exhibiting significant homology to the mammalian PS synthases. However, both TBLASTN and BLASTP searches identified a single predicted open reading frame (GenBank accession number AL031966) in S. pombe encoding a protein that shares a high degree of sequence homology with the S. cerevisiae PS synthase Cho1p (E value of 4 x 10^–60). Since the name pss1 was previously assigned to a gene encoding a protein unrelated to PS synthase (10), we named the putative PS synthase-encoding gene identified from our BLAST searches pps1, for S. pombe phosphatidylserine synthase 1. The full-length pps1 gene contains two introns and encodes a 250-amino-acid protein that exhibits 52% identity with S. cerevisiae Cho1p and 59% identity with a PS synthase enzyme, TaPss1p, from the bread wheat Triticum aestivum (11) (Fig. 1A). The predicted Pps1p protein contains the conserved CDP-alcohol phosphotransferase motif, DGX₂ARX₈GX₃DX₃D, found in Cho1p and TaPss1p as well as other phospholipid synthases that catalyze phosphodiester bond formation using a CDP-alcohol and a second alcohol as substrates (37) (Fig. 1B).

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FIG. 1. Homology of S. pombe Pps1p to PS synthase enzymes from budding yeast and bread wheat. (A) Amino acids 36 to 240 of the 250-amino-acid Pps1p protein are aligned with the corresponding sequences of S. cerevisiae Cho1p (Sc Cho1p) and Triticum aestivum Pss1p (Ta Pss1p). (B) Alignment of the conserved CDP-alcohol phosphotransferase motif, DGX2ARX8GX3DX3D, in Pps1p with analogous sequences of S. cerevisiae Cho1p, Ept1p (GenPept accession number AAA63572), Cpt1p (GenPept accession number AAA63571), Pis1p (GenPept accession number AAA34876), and wheat TaPss1p. Alignments were generated using the MegAlign program of Lasergene (DNAStar). Identical amino acids are indicated by black boxes.

The pps1 gene is essential for normal growth of S. pombe cells. To determine the phenotypes resulting from deletion of the pps1 gene, we used PCR to construct a gene knockout cassette, pps1::ura4, in which the entire pps1 protein-coding sequence is replaced by the S. pombe ura4 gene (see Materials and Methods). The pps1::ura4 cassette was transformed into the wild-type S. pombe diploid strain SP870D, and heterozygous pps1⁺/pps1::ura4 transformants were identified by colony PCR screening (see Materials and Methods). Two independent pps1⁺/pps1::ura4 diploids were induced to sporulate and subjected to tetrad analysis. Since S. cerevisiae mutants carrying a deletion of the PS synthase gene CHO1 require either choline or ethanolamine for growth (22), spores from pps1⁺/pps1::ura4 tetrads were dissected on nutrient-rich medium (YEAU) as well as YEAU medium supplemented with either 1 mM choline (YEAU+CH) or 1 mM ethanolamine (YEAU+EA). No more than two colony-forming spores were obtained from tetrads grown on YEAU or YEAU+CH (Fig. 2A), all of which were Ura^–. By contrast, the majority of tetrads grown on YEAU+EA produced four colony-forming spores (Fig. 2A), two of which were Ura⁺ and two that were Ura^–. Several Ura⁺ progeny obtained from YEAU+EA plates were analyzed by colony PCR, which confirmed that they carried the pps1::ura4 null mutation (data not shown).

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FIG. 2. The growth defect of pps1 cells is rescued by ethanolamine but not choline. (A) A pps1+/pps1::ura4 diploid strain was induced to sporulate and subjected to tetrad analysis. Tetrad spores were dissected onto YEAU or YEAU containing either 1 mM ethanolamine (YEAU+EA) or 1 mM choline (YEAU+CH) as indicated and incubated for 4 days at 30°C. (B) Wild-type and pps1 cells were grown in YEAU+EA to mid-log phase, harvested by centrifugation, and resuspended in water at 107 cells/ml. The cell suspensions were serially diluted (1:10), and 4 µl of each dilution was spotted onto YEAU, YEAU+EA, and YEAU+CH as indicated and incubated for 3 days at 30°C. (C) Culture dilutions described in panel B were spotted onto EMM, EMM+EA, and EMM+CH as indicated and incubated for 5 days at 30°C. (D) Serially diluted suspensions of wild-type and pps1 cells prepared as described for panel B were spotted onto YEAU containing 0.1 mM, 1 mM, or 5 mM ethanolamine as indicated and incubated for 3 days at 30°C. Additional concentrations of EA were also tested for effects on the growth of wild-type and pps1 strains on both YEAU and EMM, as summarized in Table 1.

The pps1 mutant was analyzed in greater detail for its ability to grow on YEAU and YEAU supplemented with either choline or ethanolamine. In addition, pps1 cells were tested for growth on synthetic minimal medium (EMM) with or without the same supplements. We found that pps1 mutants grew very slowly on nonsupplemented YEAU, whereas on YEAU containing 1 mM ethanolamine they exhibited growth similar to wild-type S. pombe cells (Fig. 2B). Consistent with the above-described analysis of pps1⁺/pps1::ura4 tetrads, we observed that pps1 cells were unable to grow at all on YEAU supplemented with choline, even at concentrations as high as 100 mM (Fig. 2B; Table 1). This result indicates not only that choline supplementation fails to rescue the growth-defective phenotype of pps1 mutants, but also that it is inhibitory to the growth of these mutants. Interestingly, we determined that growth of pps1 mutants on minimal medium was completely dependent on 1 mM ethanolamine (Fig. 2C). Furthermore, whereas ethanolamine provided very strong suppression of the growth defect of pps1 cells on YEAU medium, it only weakly suppressed the growth defect of the mutant on EMM. Similar to results observed on YEAU medium, growth of pps1 mutants was not supported to any extent by supplementation of EMM minimal medium with choline (Fig. 2C).

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TABLE 1. Effects of EA and CH on the growth of wild-type and pps1 strains

Interestingly, we found that pps1 cells grew optimally on YEAU medium containing 0.5 or 1 mM ethanolamine (Fig. 2D; Table 1). Indeed, concentrations of ethanolamine higher than 1 mM were increasingly inhibitory to the growth of both wild-type and pps1 strains, each of which was strongly inhibited for growth by 5 mM ethanolamine on YEAU and by 10 mM ethanolamine on EMM (Table 1).

S. pombe pps1 cells do not produce detectable PS and possess negligible in vitro PS synthase activity. To determine the effects of the pps1 mutation on the relative compositions of the major phospholipids in S. pombe cells, wild-type and pps1 S. pombe strains were metabolically labeled to uniformity with [³²P]orthophosphate in YEAU medium that was either supplemented (wild type and pps1) or not (wild type) with 1 mM ethanolamine. Following extraction of lipids from the radiolabeled cells, individual phospholipids were resolved by high-performance thin-layer chromatography and quantified using a phosphorimager (see Materials and Methods). As shown in Table 2, pps1 cells contained no detectable PS, providing evidence that the pps1 gene not only encodes a PS synthase but, further, that it encodes the primary PS synthase activity in S. pombe. pps1 cells also had lower levels of PE relative to wild-type cells, suggesting that PS is likely to be a precursor for PE in S. pombe, as is the case in budding yeast and mammalian cells (35). Interestingly, we found that pps1 cells contained higher levels of phosphatidylinositol (PI) than wild-type S. pombe cells, suggesting that in the absence of PS synthase, CDP-DAG might be more readily converted to PI. The other notable difference in phospholipid composition between the two strains was that pps1 cells contained more PC relative to wild-type cells.

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TABLE 2. Phospholipid composition of wild-type and pps1 S. pombe cellsa

To determine whether pps1 cells possess measurable PS synthase activity, lysates prepared from wild-type and pps1 S. pombe cells were fractionated and assayed for the presence of PS synthase enzyme activity. In preliminary experiments, the 27,000 x g pellet of wild-type cell lysates was found to contain the majority of PS synthase activity and was therefore used as the protein source for subsequent PS synthase assays. As shown in Table 3, negligible PS synthase activity was detected in the pps1 mutant compared to wild-type S. pombe cells. Furthermore, PS synthase activity in the wild-type strain was stimulated by MnCl₂ and by a substrate of the reaction, CDP-DAG, as has been reported for the S. cerevisiae PS synthase Cho1p (8). Taken together, the results of our metabolic labeling experiments and in vitro assays indicate that pps1 is the structural gene for the major PS synthase enzyme in S. pombe.

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TABLE 3. In vitro PS synthase activity in wild-type and pps1 cells

pps1 is required for proper regulation of morphology, cytokinesis, and cell integrity in S. pombe. To determine the effects of loss of pps1 function on the morphology of normally rod-shaped fission yeast cells, we cultured wild-type and pps1 S. pombe strains in YEAU and YEAU+EA liquid media and then examined the cultures microscopically. In pps1 cultures incubated in YEAU for 8 h, we observed that 15 to 20% of cells were severely necrotic in appearance, suggesting that pps1 cells have defects in cell integrity. Within the nonnecrotic cell population, we found that about 20% of cells exhibited severe morphological abnormalities, including cells that were bent, bulbous, branched, and/or ovoid in appearance (Fig. 3B). In addition, we observed that 15 to 20% of septated pps1 cells contained two or more septa, a phenotype indicative of a defect in cell separation at the end of cytokinesis (Fig. 3B). These observations demonstrate that PS synthase is required for proper regulation of cellular morphogenesis and cytokinesis in S. pombe. pps1 cultures, although viable, grew very poorly in YEAU liquid medium (generation time, >12 h), and in cultures incubated longer than 24 h (2 generations), we observed very high frequencies of necrotic cells (data not shown). As was shown above for the growth-defective phenotype of the pps1 mutant on YEAU agar plates, we observed that the morphology-, cytokinesis-, and cell integrity-defective phenotypes exhibited by this mutant in YEAU liquid medium were substantially suppressed by 1 mM ethanolamine (Fig. 3C).

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FIG. 3. pps1 cells exhibit morphology- and cytokinesis-defective phenotypes. Wild-type (A) and pps1 (B and C) cells were grown in YEAU (A and B) or YEAU+EA (C) to mid-log phase and then observed microscopically. pps1 cultures grown in YEAU (B) exhibited high frequencies of morphologically aberrant cells, including bent, bulbous, and branched cells. Lower frequencies of pps1 cells were also multiseptated (note the cell marked by an arrow), indicating a defect in cell separation at the end of cytokinesis. pps1 cells grown in YEAU+EA were similar to wild-type cells in morphology, demonstrating that ethanolamine rescues the morphology-defective phenotypes of pps1 cells (C).

pps1 overexpression induces morphology- and cytokinesis-defective phenotypes. To determine the effects of pps1 overexpression on S. pombe growth and morphology, we constructed the plasmid pREP3XPps1, which allows for overexpression of the pps1 protein-coding sequence from the S. pombe thiamine-repressible nmt1 promoter (25). This plasmid restored normal growth to pps1 cells on minimal medium, indicating that it expresses a functional Pps1p protein (Fig. 4A). When incubated on EMM lacking thiamine, pREP3XPps1-transformed wild-type and pps1 cells grew similarly to wild-type cells transformed with a control plasmid, indicating that pps1 overexpression is not inhibitory to cell growth (data not shown). Wild-type S. pombe cells transformed with pREP3XPps1 or the control plasmid, pREP3X, were incubated in EMM containing 15 µM thiamine (EMM+T) and then subcultured into EMM lacking thiamine to allow for controlled derepression of pps1 expression. After approximately 48 h of growth in EMM, we observed that pREP3XPps1 transformant cultures contained a substantial frequency of bent, branched, and multiseptated cells, which were not observed in the pREP3X transformant cultures (Fig. 4B). Taken together, our results showing that both loss and gain of pps1 function leads to defects in cell morphogenesis and cytokinesis in S. pombe suggest that the pps1 gene product is a dosage-dependent regulator of these processes.

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FIG. 4. Overexpression of pps1 leads to defects in cell morphology. (A) S. pombe pps1 cells were transformed with the control plasmid pREP3X or with the plasmid pREP3XPps1, which allows for overexpression of pps1 from the thiamine-repressible nmt1 promoter. The transformants were streaked onto EMM or EMM+EA as indicated and incubated for 4 days at 30°C. (B) Wild-type S. pombe pREP3XPps1 transformants were cultured in EMM containing 15 µM thiamine to mid-log phase and then washed, resuspended in EMM lacking thiamine, and incubated for 48 h prior to microscopic observations. pREP3XPps1 transformant cultures contained high frequencies of morphologically aberrant cells, including bent, branched, and multiseptated cells.

pps1 mutants exhibit severe defects in cell wall remodeling and integrity. As noted above, pps1 mutants grow very slowly on synthetic minimal medium agar plates supplemented with ethanolamine and do not grow at all on minimal medium lacking ethanolamine. We determined further that pps1 cells were unable to grow at all in liquid minimal medium whether it was supplemented with ethanolamine or not. The nature of the growth defect of pps1 cells in minimal medium became apparent upon microscopic inspection of the cultures, which, unlike cultures of wild-type S. pombe cells (Fig. 5A), were found to contain high frequencies of severely necrotic and/or lysed cells (Fig. 5B). Since necrotic cells were also detected in pps1 cultures grown in unsupplemented YEAU liquid medium, we carried out experiments to investigate whether pps1 mutants exhibit cell wall-defective phenotypes.

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FIG. 5. The pps1 mutant exhibits defects in cell wall remodeling and integrity. (A and B) Wild-type (A) and pps1 cells (B) were grown to mid-log phase in YEAU+EA, washed with water, and then resuspended in EMM+EA and incubated for 8 h at 28°C prior to observing microscopically. Whereas wild-type cells were normal in appearance (A), pps1 cells appeared severely necrotic and/or lysed (B). (C to E) Wild-type cells grown in YEAU (C) and pps1 cells grown in YEAU (D) or YEAU+EA (E) cultures were incubated at 28°C to mid-log phase, stained with calcofluor, and visualized by fluorescence microscopy. (F) Serially diluted (1:5) suspensions of wild-type and pps1 cells prepared as described for Fig. 2B but resuspended at 106 cells/ml were spotted onto YEAU+EA containing (+Calc) or not (-Calc) 0.5 mg/ml Calcofluor as indicated and incubated for 3 days at 30°C. (G) Wild-type and pps1 cells were grown in YEAU+EA to mid-log phase, washed three times in YEAU, resuspended in YEAU or YEAU+EA, and incubated for 18 h to mid-log phase at 28°C. The cells were collected by centrifugation and resuspended at 107 cells/ml in TE. Zymolyase 20T was added to a concentration of 100 µg/ml. The degree of cell lysis was evaluated by measuring the OD600. The OD at 0 min was set at 100%.

To determine whether pps1 cells exhibit defects in cell wall organization, pps1 cultures grown to mid-log phase in YEAU or YEAU+EA were stained with the fluorescent cell wall binding compound calcofluor and observed by fluorescence microscopy. Intriguingly, we observed that pps1 cultures grown in YEAU lacking ethanolamine contained substantial frequencies of cells with abnormal cell wall-enriched aggregates (Fig. 5D, left panel). These aggregates, which were not detected in wild-type S. pombe cells (Fig. 5C), were most frequently observed at or near cell tips and/or septa. In some cells, heavy concentrations of cell wall material were retained at the cell tips (Fig. 5D, right panel). Like the growth and morphology defects of the pps1 mutant, this abnormality in cell wall deposition was strongly suppressed by the addition of 1 mM ethanolamine to YEAU medium (Fig. 5E). These results indicate that pps1 is required for proper regulation of cell wall remodeling in S. pombe.

We next carried out two assays to determine whether pps1 cells are impaired for cell wall integrity. First, we compared the sensitivities of wild-type and pps1 S. pombe strains to calcofluor, which has been shown to be inhibitory to the growth of cell wall integrity-defective S. pombe mutants (2). We found that even in YEAU supplemented with 1 mM ethanolamine, pps1 mutants were significantly more sensitive to calcofluor than wild-type S. pombe cells (Fig. 5F). As a more direct measure of cell wall integrity, we compared the rates at which the cell walls of wild-type and pps1 cells were hydrolyzed by the cell wall-digesting enzyme zymolyase (32). As shown in Fig. 5G, the cell walls of pps1 cells were significantly less resistant to zymolyase digestion than wild-type S. pombe cells. Similar to results obtained from calcofluor sensitivity assays, we determined that the zymolyase-hypersensitive phenotype of pps1 cells was only partially suppressed by 1 mM ethanolamine.

pps1 cells exhibit severe actin cytoskeletal defects. Given the severe morphology-defective phenotypes exhibited in pps1 cultures grown in YEAU lacking ethanolamine, we carried out experiments to examine whether loss of pps1 affects actin cytoskeletal organization. To do this, wild-type and pps1 cultures were grown to mid-log phase in YEAU liquid medium supplemented or not with 1 mM ethanolamine. The cells were harvested, fixed with formaldehyde, stained with the florescent F-actin binding compound rhodamine-phalloidin, and observed by fluorescence microscopy. In cultures of wild-type S. pombe cells (Fig. 6A) and in cultures of pps1 cells grown in YEAU supplemented with ethanolamine (Fig. 6C), we observed normal patterns of F-actin localization, namely, small dots at the tips of interphase cells and equatorial rings or patches in dividing cells. By contrast, in pps1 cultures grown in YEAU medium lacking ethanolamine, we observed a high incidence of cells containing large actin aggregates, most frequently in close proximity to cell tips and septa, as well as thick, rod-shaped cytoplasmic F-actin structures (Fig. 6B). These results demonstrate that pps1 is required for normal actin cytoskeletal organization in S. pombe.

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FIG. 6. The pps1 mutant displays actin cytoskeletal defects. Wild-type cells grown in YEAU (A) and pps1 cells grown in YEAU (B) or YEAU+EA (C) cultures were incubated at 30°C to mid-log phase, stained with the fluorescent F-actin stain rhodamine phalloidin, and visualized by fluorescence microscopy.

Subcellular localization of Pps1p. Experiments were next carried out to determine the subcellular localization of Pps1p. We first constructed an S. pombe strain, pps1-GFP, in which the protein-coding sequence of GFP is fused to the C-terminal coding end of the pps1 protein-coding sequence. However, we found that the pps1-GFP strain required ethanolamine for growth (Fig. 7A), suggesting that the GFP moiety is inhibitory to Pps1p function when fused to the C terminus of Pps1p. Fluorescence microscopic analyses revealed that Pps1-GFP fusion proteins were concentrated to the cell and nuclear peripheries of S. pombe cells as well as to presumptive endoplasmic reticulum (ER) membranes (Fig. 7B), based on the similarity of Pps1-GFP localization to other ER resident proteins, such as BiP (Fig. 7B) (27). Diffuse cytoplasmic fluorescence was also detected in pps1-GFP cells. Since Pps1-GFP fusion proteins are lacking in functionality, as an alternative approach for detecting the subcellular localization of Pps1p we constructed the plasmid pREP41GFP-Pps1, in which the GFP coding sequence is fused to the N-terminal coding end of the pps1 protein coding sequence. In contrast to the pps1-GFP strain, pREP41GFP-Pps1-transformed pps1 cells were capable of growing on EMM lacking ethanolamine, indicating that the GFP-Pps1p fusion protein, unlike Pps1-GFP, is functional (Fig. 7C). Fluorescence microscopic analyses revealed that GFP-Pps1p exhibited a pattern of localization indistinguishable from Pps1-GFP under normal growth conditions (Fig. 7D).

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FIG. 7. Subcellular localization of Pps1p. (A) Wild-type, pps1, and pps1-GFP S. pombe strains were streaked onto YEAU plates and incubated for 3 days at 30°C. (B) Wild-type and pps1-GFP S. pombe strains as well as wild-type cells expressing GFP-BiP (see Materials and Methods) were incubated in YEAU+EA (wild type and pps1-GFP) or EMM to mid-log phase and then visualized by fluorescence microscopy. (C) pps1 S. pombe cells transformed with pREP41GFP or pREP41GFP-Pps1 were streaked onto EMM lacking EA and incubated for 5 days at 30°C. (D) Wild-type cells transformed with pREP41GFP and pps1 cells transformed with pREP41GFP-Pps1 were incubated in EMM containing 0.15 µM thiamine to mid-log phase and then visualized by fluorescence microscopy. (E) pps1 cells transformed with pREP41GFP-Pps1 were cultured in EMM containing 0.15 µM thiamine to stationary phase and then visualized by fluorescence microscopy.

Intriguingly, in pREP41GFP-Pps1 transformant cultures grown to stationary phase, we observed that in the majority of cells, GFP-Pps1p fusion proteins were concentrated to unusual cytoplasmic structures, with less confluent cell cortex localization than observed in log-phase cultures and no obvious localization to the nuclear periphery (Fig. 7E). The dramatic redistribution of GFP-Pps1p fusion proteins observed in stationary-phase cultures was not detected under the same conditions in the pps1-GFP strain (data not shown), suggesting that the C-terminal end of Pps1p is required for stationary-phase-induced redistribution of the protein.

The pps1 gene is essential for stationary-phase survival in S. pombe. In light of our finding that Pps1p undergoes a dramatic subcellular redistribution during stationary-phase growth, we sought to determine whether the protein might be required for viability of stationary-phase cultures of S. pombe. To address this question, wild-type and pps1 cultures were grown to stationary phase in YEAU (wild type and pps1) or YEAU+EA (pps1) and tested for viability after 4 days of incubation at 28°C. Whereas wild-type S. pombe cultures showed little loss of viability after 4 days of stationary-phase growth, pps1 cultures retained virtually no viability (Fig. 8). Importantly, we found that ethanolamine did not rescue to any extent the stationary-phase survival defect of pps1 mutants (Fig. 8). This finding suggests that PS plays a critical role in the physiological adaptations required for S. pombe cells to maintain viability during stationary-phase growth.

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FIG. 8. The pps1 gene is essential for survival of stationary-phase S. pombe cells. Wild-type and pps1 cells were cultured in YEAU or YEAU+EA as indicated to mid-log phase, washed three times with H2O, and resuspended at 5 x 106 cells/ml in YEAU or YEAU plus 0.5 mM EA. The cells were incubated at 28°C to stationary phase and then incubated an additional 96 h at 28°C. Additional EA (to 0.5 mM) was added to YEAU+EA pps1 every 24 h. After 96 h, the cultures were harvested by centrifugation and resuspended in water at 106 cells/ml. The cell suspensions were serially diluted (1:10), and 4 µl of each dilution was spotted onto YEAU+EA plates. The cultures were then incubated for 3 days at 30°C.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this paper, we have described the cloning and functional characterization of pps1, the single gene in the fission yeast S. pombe encoding a protein sharing significant structural homology with eukaryotic PS synthases. S. pombe pps1 mutants do not produce detectable PS in vivo, and extracts prepared from pps1 cells have negligible in vitro PS synthase activity. We therefore conclude that pps1 encodes the major PS synthase activity in S. pombe. We found that pps1 mutants exhibit severe defects in cell growth, morphology, cytokinesis, actin cytoskeletal organization, cell wall remodeling and integrity, and stationary-phase survival. All of these phenotypes, with the exception of the stationary-phase survival defect, can be suppressed to some extent by ethanolamine, suggesting that they are at least partially attributable to PE deficiency. BLAST searches of the S. pombe nucleic acid and protein sequence databases indicate that genes encoding enzymes corresponding to the CDP-ethanolamine and CDP-choline branches of the S. cerevisiae Kennedy pathways are conserved in S. pombe (Y. Matsuo and S. Marcus, unpublished results). This fact, together with the findings presented in this report showing that S. pombe PS synthase mutants can produce PE when cultured in medium supplemented with ethanolamine and results of previous studies by other investigators showing that S. pombe mutants defective in enzymes that convert PE to PC can be rescued by choline supplementation (18), strongly suggests that the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway are conserved in S. pombe.

Importantly, we found that in synthetic minimal medium, ethanolamine provided only a very modest suppression of the growth-defective phenotype of pps1 cells and only on semisolid medium. When cultured in liquid minimal medium, whether supplemented with ethanolamine or not, pps1 cells were severely necrotic and/or lysed in appearance, indicating that ethanolamine only weakly suppresses the cell integrity defect(s) of this mutant under nutrient-limited conditions. In preliminary studies, we have begun characterizing S. pombe mutants carrying deletions in three previously undescribed genes, tentatively designated psd1-3, encoding proteins homologous to the S. cerevisiae PS decarboxylase enzymes Psd1p and Psd2p, that convert PS to PE (33, 34). Interestingly, we have found that S. pombe psd triple mutants, while absolutely dependent on ethanolamine for growth, do not display cell integrity-defective phenotypes, even when cultured in synthetic medium lacking ethanolamine (Matsuo and Marcus, unpublished). Taken together, these findings suggest that under nutrient-limited conditions, PS is likely to have PE-independent functions required for the maintenance of cell integrity in S. pombe.

Our results demonstrating that both deletion and overexpression of the pps1 gene result in severe defects in morphology and cytokinesis in S. pombe suggest that PS is a dosage-dependent regulator of these processes. However, it remains to be determined whether PS has PE-independent functions in regulating S. pombe morphology and cytokinesis or if, once produced, its primary role is that of being a precursor for PE. Recent studies have shown that PE is localized to the cell tips and cell division site in S. pombe cells (17), and studies in mammalian cells have provided evidence of an essential role for PE in cytokinesis (13). Consistent with a role for PE in the regulation of cell morphogenesis and cytokinesis in S. pombe, we have determined in preliminary studies that S. pombe PS decarboxylase mutants exhibit severe defects in cell morphology and cytokinesis resembling those observed in pps1 cultures and that these phenotypes are strongly suppressed by ethanolamine (Matsuo and Marcus, unpublished results). Further characterization of S. pombe mutants defective in PE synthesis, including analysis of genetic interactions with PS synthase mutants, will help clarify the relative contributions of PS and PE in regulating cell morphogenesis and cytokinesis in S. pombe.

Consistent with functions for PS in processes required for proper regulation of cell morphology and cell wall remodeling, we determined that during log-phase growth, GFP-Pps1p fusion proteins are concentrated at the cell cortex in S. pombe cells, in addition to being concentrated to the nuclear periphery and probable ER membranes. Intriguingly, we found that GFP-Pps1p undergoes a dramatic redistribution to unusual cytoplasmic structures during stationary-phase growth. This finding prompted us to investigate whether pps1 is required for viability of stationary-phase S. pombe cultures. Indeed, we found that stationary-phase pps1 cells retain virtually no viability relative to wild-type S. pombe cells. Furthermore, we determined that this phenotype is not rescued to any extent by ethanolamine. Cumulatively, these findings suggest that PS has a critical, PE-independent role in the physiological adaptations required for S. pombe cells to retain viability during stationary-phase growth.

It is worth noting that in the budding yeast S. cerevisiae, growth-defective phenotypes resulting from deletion of the PS synthase gene, CHO1, can be rescued by the addition of either ethanolamine or choline to the growth medium (22). Moreover, S. cerevisiae cho1 mutants, unlike S. pombe pps1 mutants, do not appear to have obvious cell morphology-, cytokinesis-, or cell wall integrity-defective phenotypes (16, 22). The fact that S. pombe PS synthase mutants cannot be rescued by choline, whereas in budding yeast the opposite is true, suggests that PE plays a more important role in S. pombe cell growth than it does in S. cerevisiae.

Interestingly, we found that pps1 cells had higher relative levels of PI than wild-type S. pombe cells. S. cerevisiae PS synthase-defective (cho1) mutants were likewise shown to have higher relative levels of PI in comparison to wild-type cells in a previous study by Atkinson and coworkers (3). One possible explanation for the increased relative abundance of PI in both yeasts is that in the absence of PS synthase, CDP-DAG might be more readily converted to PI. An alternative explanation put forth by Atkinson et al. is that the increased synthesis of PI in S. cerevisiae cho1 mutant cells might reflect regulation of net lipid charge in the absence of phosphatidylserine, since phosphatidylserine and phosphatidylinositol carry the same net charge (3). This is obviously an equally plausible explanation for the increased levels of PI detected in S. pombe pps1 cells in the present study.

Previous studies have implicated PS involvement in the regulation of diverse cellular processes in eukaryotic organisms (35). However, the underlying mechanisms of action of this ubiquitous phospholipid remain largely unknown. The results of this study demonstrate the value of the genetically tractable fission yeast as a model organism for gaining new insights into potentially conserved cellular and molecular functions of PS in eukaryotic organisms. In this regard, it is worth noting that a predicted gene encoding a protein sharing significant structural homology with mammalian PS receptors (38) is conserved in S. pombe but not in its evolutionarily distant cousin, S. cerevisiae (Matsuo and Marcus, unpublished). Thus, S. pombe may prove to be uniquely useful as a model organism for shedding insights into the currently controversial functional relationship(s) of PS and PS receptor-like proteins in higher eukaryotes.

 
We thank Peirong Yang for technical assistance, Eric Chang, Makoto Kawamukai, and Katsunori Tanaka for plasmid reagents, and Brittney McInnis for editorial assistance.

This study was funded by National Institutes of Health grants R01GM59817 (to J.P.-V.) and R01GM068685 (to S.M.).

 
* Corresponding author. Mailing address: Dept. of Biological Sciences, The University of Alabama, 343A Shelby Hall, 250 Hackberry Lane, Box 870336, Tuscaloosa, AL 35487. Phone: (205) 348-8094. Fax: (205) 348-9104. E-mail: smarcus{at}bama.ua.edu

Published ahead of print on 28 September 2007.

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Eukaryotic Cell, November 2007, p. 2092-2101, Vol. 6, No. 11
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