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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sphingolipids, the major lipid components of the eukaryotic plasma membrane, play critical roles in many physiologically important events, such as signal transduction, membrane trafficking and cell-to-cell interaction (Degroote et al., 2004; Futerman and Hannun, 2004). Complex sphingolipids are composed of a polar head group and a hydrophobic segment, ceramide, which contains a fatty acid and a sphingoid long-chain base (LCB). The fundamental structure of sphingolipids in the yeast Saccharomyces cerevisiae is similar to that in mammalian cells; however, the characteristics of the LCB, fatty acid and polar head group of yeast sphingolipids differ in the following points: (i) yeast sphingolipids contain phytosphingosine or dihydrosphingosine, but not sphingosine, which is the desaturated LCB typically present in mammals, (ii) the fatty acid in mammalian sphingolipids can vary in chain length, degree of saturation and hydroxylation state, whereas the fatty acid in yeast sphingolipids is primarily 26 carbons long and unsaturated, and is hydroxylated or not, and (iii) mammalian sphingolipids can carry phosphocholine or hundreds of carbohydrate chains as polar head groups, whereas yeast sphingolipids can carry one of only three different types of polar head group, and therefore complex sphingolipids in yeast can be divided into three types, IPC (inositol phosphorylceramide), MIPC (mannosylinositol phosphorylceramide) and M(IP)2C (mannosyldiinositol phosphorylceramide), all of which include phosphoinositol (Fig. S1) (Dickson et al., 2006). Due to the limited molecular class of the fatty acid moiety and polar head group, the structural diversity of sphingolipids in yeast is relatively simple as compared with that in mammalian cells, making yeast a useful model for investigating the physiological significance of the structural complexity of sphingolipids.

According to its hydroxylation status, the ceramide in yeast can be classified into five types (A, B, B′, C and D) (Fig. S1). Dihydroceramide (ceramide A) contains a dihydrosphingosine and a fatty acid. Sur2p can convert dihydrosphingosine into phytosphingosine or dihydroceramide into phytoceramide (ceramide B) (Haak et al., 1997). Hydroxylation of the fatty acid of ceramide A and ceramide B at the C-2 position by Scs7p yields ceramide B′ and ceramide C respectively (Haak et al., 1997). Ceramide D is generated through further hydroxylation, at an unknown position, of the fatty acid of ceramide C (Beeler et al., 1997). CCC2, the gene encoding an intracellular Cu2+ transporter, is involved in the conversion of ceramide C to ceramide D (Beeler et al., 1997). IPC, the simplest complex sphingolipid in yeast, is formed by IPC synthase, an enzyme catalysing the transfer of the head group of phosphatidylinositol (PI) to ceramides (Nagiec et al., 1997). Subsequently, IPC is converted to MIPC by the addition of mannose, and Csg1p, Csh1p and Csg2p are involved in MIPC synthesis (Zhao et al., 1994; Beeler et al., 1997; Uemura et al., 2003). Finally, MIPC is converted to M(IP)2C by addition of another phosphoinositol, which is catalysed by Ipt1p (Dickson et al., 1997).

The genes for sphingolipid-metabolizing enzymes, SCS7, SUR2, CSG1, CSG2, CSH1 and IPT1, are basically non-essential for yeast cell growth; however, several lines of evidence indicate that the defect of each gene causes abnormal phenotypes in relation to stress responses and sensitivity to antibiotics, etc. For example, defect of conversion of IPCs to MIPCs caused by the deletion of CSG2 causes supersensitivity to Ca2+ (Zhao et al., 1994; Beeler et al., 1997). Deletion of IPT1 results in a complete loss of M(IP)2Cs and affects sensitivity to drugs, such as Dahlia merckii antimicrobial peptide 1 (DmAMP1) (Thevissen et al., 2000) and syringomycin E (Im et al., 2003). M(IP)2Cs are found primarily in the plasma membrane (Hechtberger et al., 1994) and are suggested to serve as targets for these drugs in the extracellular region (Thevissen et al., 2000). Similar to IPT1, SCS7 is also involved in determining sensitivity to some drug (Hama et al., 2000; Stock et al., 2000; Herrero et al., 2008). These mutational analyses of non-essential sphingolipid-metabolizing enzyme genes imply distinct physiological functions of each subtype of sphingolipid. In addition, mutation of ELO3, the gene encoding a fatty acid elongase that produces C26 very-long-chain fatty acids (Oh et al., 1997), causes impairments of sphingolipid- and ergosterol-enriched microdomain formation in the plasma membrane (Eisenkolb et al., 2002), the secretory pathway (David et al., 1998) and vacuolar H+-ATPase activity (Chung et al., 2003), suggesting the physiological importance of the chain length of the fatty acid in the ceramide portion of sphingolipids.

Aureobasidin A is a cyclic depsipeptide antifungal antibiotic, which inhibits IPC synthase (Endo et al., 1997; Nagiec et al., 1997). Aureobasidin A treatment results not only in reductions in complex sphingolipid levels but also in increases in ceramide levels, both of which are believed to lead to the growth defects caused by this inhibitor (Nagiec et al., 1997; Schorling et al., 2001; Cerantola et al., 2009); however, the molecular mechanisms underlying the growth defects are largely unknown. To address the mechanisms of the aureobasidin A-induced growth defect, we screened a collection of ∼4800 yeast mutant strains lacking a non-essential gene for mutants showing high sensitivity to a low concentration of this drug, and found that a lack of SAC1, the gene encoding phosphoinositides (PIPs) phosphatase (Hughes et al., 2000), causes high sensitivity to the inhibitor. Double mutation analysis involving SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that the deletion of SAC1 and a specific group of enzyme genes, i.e. SCS7, CSG1, CSG2 and IPT1, causes a synthetic lethal phenotype.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Synthetic lethal interaction between SAC1 and non-essential sphingolipid-metabolizing enzyme genes

To identify mutants highly sensitive to impaired sphingolipid metabolism, we used an IPC synthase inhibitor, aureobasidin A, for the screening. Using a yeast knockout library, we screened for mutants that show high sensitivity to 0.03 µg ml−1 aureobasidin A on a YPD plate. We identified 18 mutants highly sensitive to aureobasidin A (the details of this screening will be given elsewhere). Among them, SAC1, the gene encoding phosphoinositides (PIPs) phosphatase, attracted our attention, because it caused a strong growth defect in the presence of 0.01 and 0.03 µg ml−1 aureobasidin A (Fig. 1), and several reports have indicated a functional relationship between PIPs-mediated signalling and sphingolipids (Dickson, 2008). To investigate more precisely how the defect in sphingolipid metabolism genetically interacts with the deletion of SAC1, the sac1Δ strain was mated with each of 17 strains lacking a non-essential sphingolipid-metabolizing enzyme gene, and the resulting diploids were allowed to sporulate and then subjected to a random spore analysis for cell growth. As shown in Fig. S2, this analysis revealed that scs7Δ, csg1Δ, csg2Δ and ipt1Δ were synthetic lethal with sac1Δ, although none of the single-deleted cells showed a significant growth defect. These results suggested that the deletion of SAC1 genetically interacts with specific defects of sphingolipid biosynthesis.

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Figure 1. Deletion of SAC1 confers high sensitivity to aureobasidin A. Wild-type (BY4741) and sac1Δ cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium containing 0, 0.01, 0.03 or 0.1 µg ml−1 aureobasidin A (AbA), and then incubated at 30°C. At the indicated times, cell growth was measured with a spectrophotometer (A600). Data shown are the averages of three independent experiments.

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Growth defect on repression of SAC1 gene expression and deletion of sphingolipid-metabolizing enzymes

Since double-deletion mutants could not be used for further analyses due to the lethal phenotype, we created a mutant strain that carries the SAC1 gene under the control of a tetracycline-regulatable (Tet) promoter (tet-SAC1). To examine the effect of the Tet promoter on SAC1 expression, the 3′-end of chromosomal SAC1 was tagged with 9xmyc in tet-SAC1 cells (tet-SAC1-9xmyc). As shown in Fig. 2A, a reduction in the expression level of Sac1p-9xmyc was observed at 3.5 h after the addition of doxycycline (Dox), which represses expression of the gene under the Tet promoter. Sac1p-9xmyc was dramatically reduced at 6 h after the addition of Dox. The repression of Sac1p in Dox-treated tet-SAC1 cells was also confirmed by inositol auxotrophy, a typical phenotype of sac1 mutants (Whitters et al., 1993; Rivas et al., 1999). As shown in Fig. 2B, in the absence of Dox, the growth of tet-SAC1 cells was comparable to that of wild-type cells in inositol-free medium, but the addition of Dox resulted in a significant growth defect. The growth defect of Dox-treated tet-SAC1 cells in inositol-free medium was less severe than that of sac1Δ cells, suggesting that Sac1p did not completely disappear on Dox treatment (Fig. 2B). Figure 2C shows effects of deletion of CSG1, CSG2, IPT1 and SCS7, on the growth of Dox-treated tet-SAC1 cells. Although the growth of tet-SAC1 cells was normal on YPD plates even in the presence of Dox, tet-SAC1 csg1Δ, tet-SAC1 csg2Δ, tet-SAC1 ipt1Δ and tet-SAC1 scs7Δ cells exhibited strong growth defects in the presence of Dox (Fig. 2C). CSG1 encodes mannosyltransferases catalysing MIPC synthesis, and CSG2 is essential for the activity of Csg1p (Zhao et al., 1994; Uemura et al., 2003; 2007). Thus, we used tet-SAC1 csg1Δ, but not tet-SAC1 csg2Δ, for further analysis. The tet-SAC1 csg1Δ, tet-SAC1 ipt1Δ and tet-SAC1 scs7Δ cells are collectively termed tet-SAC1 xxxΔ cells.

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Figure 2. Synthetic growth defects caused by deletion of specific non-essential sphingolipid-metabolizing enzyme genes and repression of SAC1 expression by a tetracycline-regulatable system (tet-SAC1). A. Western blotting analysis of expression of Sac1p-9xmyc by a tetracycline-regulatable system. tet-SAC1-9xmyc cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD with or without 10 µg ml−1 doxycycline (Dox), and then incubated for 0, 3.5 or 6 h at 30°C. Yeast cell extracts were immunoblotted using anti-myc or anti-Pgk1p. B. Inositol auxotrophy of tet-SAC1 cells. Cells were cultured overnight in YPD medium at 30°C, diluted (0.7 A600 units ml−1) in SC medium lacking inositol (Whitters et al., 1993) with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 26°C. Cells were spotted onto SC plates lacking inositol but containing 0 or 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.7 A600 units ml−1. All plates were incubated at 26°C and photographed after 35 h. The same experiments were also performed by using SC medium and plates containing inositol (75 µg ml−1). C. Cells were cultured overnight in YPD medium, diluted (0.3 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were spotted onto YPD plates with or without 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.07 A600 units ml−1. All plates were incubated at 30°C and photographed after 27 h. D. Time-course of cell growth. Cells were cultured overnight in YPD medium and then diluted (0.02 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and aliquots of cell suspensions were subjected to cell density measurements (A600) at the indicated times. The results presented are for one experiment (triplicate) representative of at least three independent ones.

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Figure 2D shows the time-course of growth of tet-SAC1 xxxΔ cells in the presence or absence of Dox. tet-SAC1 cells showed a similar growth rate to wild-type cells even in the presence of Dox. However, the growth of tet-SAC1 csg1Δ, tet-SAC1 ipt1Δ and tet-SAC1 scs7Δ cells began to slow down at 7.5–9 h after the addition of Dox. Thus, to avoid non-specific effects caused by growth inhibition, we investigated the phenotype of Dox-treated tet-SAC1 xxxΔ cells at earlier time points (around 8 h after the addition of Dox).

Alterations in glycerophospholipid levels under SAC1-repressive conditions

Mutation of SAC1 causes a dramatic accumulation of PIPs due to a defect in their dephosphorylation (Hughes et al., 2000). The accumulation of PIPs was also observed in Dox-treated tet-SAC1 and tet-SAC1 xxxΔ cells when cells were pulse-radiolabelled with [3H]inositol for 60 min (Fig. 3A). It has also been reported that the mutation of SAC1 causes a reduction in phosphatidylserine (PS) level and an enhancement of phosphatidylcholine (PC) biosynthesis (Rivas et al., 1999). To confirm this, we examined the cellular levels of major phospholipids [cardiolipin (CL), phosphatidic acid (PA), phosphatidylethanolamine (PE), PS, PI and PC] in sac1Δ and Dox-treated tet-SAC1 cells by means of steady-state radiolabelling with [32P]orthophosphate. As shown in Fig. 3B, the steady-state PS levels in both sac1Δ and Dox-treated tet-SAC1 cells were reduced 40% as compared with those in wild-type and Dox-untreated tet-SAC1 cells. These reductions well coincide with the result of the pulse-radiolabelling of glycerophospholipids in sac1-22 mutant cells (Rivas et al., 1999). The reduction in PS level was also confirmed in Dox-treated tet-SAC1 xxxΔ cells by pulse-radiolabelling with [32P]orthophosphate for 60 min; that is, all mutant cells exhibited a striking (50–70%) reduction in PS level after Dox treatment (Fig. 3C). The PC level was increased to around 10% in sac1Δ cells and all SAC1-repressive conditions (Fig. 3B and C ), which may reflect the enhancement of PC biosynthesis. These results indicated that alterations of glycerophospholipid metabolism observed in sac1 mutants occur in Dox-treated tet-SAC1 and tet-SAC1 xxxΔ cells.

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Figure 3. Alteration of glycerophospholipid metabolism by the SAC1 defect. A. [3H]myo-inositol labelling of tet-SAC1 and tet-SAC1 xxxΔ cells. Cells were cultured overnight in YPD medium, diluted (0.05 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 8 h at 30°C. Cells were washed, resuspended in fresh SC/MSG medium with minimum inositol and with or without 10 µg ml−1 Dox, and then labelled with [3H]myo-inositol for 1 h at 30°C. Radiolabelled phospholipids were extracted and resolved by TLC. The bands of PIPs include PI(4)P and PI(3)P (Kihara et al., 2008). The details are given under Experimental procedures. B. Steady-state radiolabelling of wild-type, sac1Δ and tet-SAC1 cells with [32P]orthophosphate in the presence or absence of 10 µg ml−1 Dox. The radiolabelled phospholipids were extracted and resolved by TLC. Major glycerophospholipids (CL, PA, PE, PS, PI and PC) were quantified using a Bio Imaging analyser. Data shown are the averages of two independent experiments performed in duplicate. C. tet-SAC1 and tet-SAC1 xxxΔ cells cultured with or without 10 µg ml−1 Dox for 8 h were pulse-radiolabelled for 1 h with [32P]orthophosphate at 30°C. Data shown are for one experiment (triplicate) representative of at least three independent experiments. Details are given under Experimental procedures.

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Restoration of the PS level complements the growth defect of Dox-treated tet-SAC1 xxxΔ strains

We next determined whether or not the alterations of PC and PS metabolism caused by the SAC1 defect are involved in the synthetic growth defect with mutations of SAC1 and sphingolipid-metabolizing enzyme genes. It has been demonstrated that the increase in PC biosynthesis in sac1-22 mutant cells is caused by enhancement of the CDP-choline pathway; however, deletion of CKI1 encoding one of the enzymes of this pathway resulted in no significant change in the growth defect of tet-SAC1 xxxΔ cells (Fig. S3). To determine whether or not the reduction in PS level is involved in the growth defect of tet-SAC1 xxxΔ cells, PSS1, which is responsible for PS biosynthesis from CDP-diacylglycerol and serine (Letts et al., 1983), was overexpressed in tet-SAC1 and tet-SAC1 xxxΔ cells through substitution of the promoter region of chromosomal PSS1 with a strong constitutive TEF promoter (the promoter-substituted gene is named TEFp-PSS1). In the in vitro PS synthase activity assay, tet-SAC1 TEFp-PSS1 cells exhibited an over fivefold increase in activity as compared with tet-SAC1 cells (data not shown). Figure 4A shows the phospholipid compositions determined on steady-state radiolabelling of tet-SAC1 and tet-SAC1 TEFp-PSS1 cells with [32P]orthophosphate in the presence or absence of Dox. The PS level in Dox-treated tet-SAC1 TEF-PSS1 cells had recovered to normal (Fig. 4A). To determine whether or not PSS1 overexpression affects the abnormal accumulation of PIPs in cells with the SAC1 defect, tet-SAC1 and tet-SAC1 TEFp-PSS1 cells were labelled with [3H]inositol in the presence or absence of Dox. PIPs were remarkably accumulated in tet-SAC1 cells treated with Dox, and the level was not affected by the introduction of TEFp-PSS1 (Fig. 4B). These results indicated that PSS1 overexpression can complement the reduction in PS level under SAC1-repressive conditions without affecting the level of PIPs. When TEFp-PSS1 was introduced into tet-SAC1 scs7Δ cells, the growth defect caused by Dox treatment was almost completely complemented (Fig. 4C and D). The growth defect of tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells in the presence of Dox was partially complemented by the introduction of TEFp-PSS1. Taken together, these results suggested that a reduction in the PS level per se causes, at least in part, the synthetic growth defect of tet-SAC1 xxxΔ cells in the presence of Dox.

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Figure 4. Overexpression of PS synthase overcomes the growth defect of tet-SAC1 xxxΔ cells. A. Steady-state radiolabelling of tet-SAC1 and tet-SAC1 TEFp-PSS1 cells with [32P]orthophosphate in the presence or absence of Dox. Data shown are the averages of two independent experiments performed in duplicate. B. [3H]myo-inositol labelling of tet-SAC1 and tet-SAC1 TEFp-PSS1 cells. Cells were cultured overnight in YPD medium with or without 10 µg ml−1 Dox, diluted (0.5 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were washed, resuspended in fresh SC/MSG medium with minimum inositol and with or without 10 µg ml−1 Dox, and then labelled with [3H]myo-inositol for 1 h at 30°C. Radiolabelled phospholipids were extracted and resolved by TLC. C. Growth of tet-SAC1 xxxΔTEFp-PSS1 cells. Cells were cultured overnight in YPD medium, diluted (0.3 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were spotted onto YPD plates with or without 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.07 A600 units ml−1. All plates were incubated at 30°C and photographed after 27 h. D. Time-course of cell growth. Cells were cultured overnight in YPD medium and then diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and aliquots of cell suspensions were subjected to cell density measurements (A600) at the indicated times. The results presented are for one experiment (triplicate) representative of at least three independent ones. Details are given under Experimental procedures.

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Genetic interaction of PSS1 with CSG1, IPT1 and SCS7

To determine whether or not the reduction in PS level directly interacts with deletion of CSG1, IPT1 and SCS7 on yeast cell growth, we created mutant strains that carry the PSS1 gene under the control of the Tet promoter (tet-PSS1). As shown in Fig. 5A, when tet-PSS1 cells were treated with Dox, the steady-state levels of PS and PE decreased to 3% and 50%, respectively, of those in Dox-untreated cells. The combination of repression of PSS1 expression by Dox and deletion of CSG1 or SCS7 resulted in a severe growth defect (Fig. 5B). tet-PSS1 ipt1Δ cells also exhibited a significant growth defect in the presence of Dox, but the defect was less severe than that in tet-PSS1 csg1Δ or tet-PSS1 scs7Δ cells. The reduction in PE level induced by the repression of PSS1 expression seemed not to be related to these growth defects, because deletion of PSD1 or PSD2, both of which catalyse PE synthesis from PS (Birner et al., 2001), did not genetically interact with the deletion of CSG1, IPT1 or SCS7 (Fig. S4). These results suggested that PS is required for normal cell growth in the absence of specific non-essential sphingolipid-metabolizing enzyme genes.

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Figure 5. Genetic interaction of PSS1 with CSG1, IPT1 and SCS7. A. Steady-state radiolabelling of tet-PSS1 cells with [32P]orthophosphate in the presence or absence of Dox. Data shown are the averages of two independent experiments performed in duplicate. B. Growth of tet-PSS1 and tet-PSS1 xxxΔ cells. Cells were cultured overnight in YPD medium, diluted (0.3 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were spotted onto YPD plates with or without 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.07 A600 units ml−1. All plates were incubated at 30°C and photographed after 27 h. Details are given under Experimental procedures.

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Arf-GAP Age1p functions as a multicopy suppressor in tet-SAC1 xxxΔ and tet-PSS1 xxxΔ cells

To investigate how the synthetic lethal phenotypes are induced by deletion of the SAC1 and sphingolipid-metabolizing enzyme genes, we screened for multicopy suppressor gene(s) that complement the growth defect of tet-SAC1 xxxΔ cells. A pool of tet-SAC1 csg1Δ cells was transformed with a yeast genomic library in a high-copy-number vector, YEp13 (Yoshihisa and Anraku, 1989), and then plated on SC plates containing 2 µg ml−1 Dox. Although several clones were obtained, the majority of the plasmids derived from these clones contained SAC1 or CSG1. However, two plasmids (named YEp13-9 and YEp3-10) contained, instead of SAC1 and CSG1, a common region of chromosome IV (1 482 917–1 490 554 bp) harbouring the complete open reading frames of seven genes. This chromosomal fragment contained one candidate gene, AGE1, which encodes the ADP-ribosylation factor GTPase-activating protein (Arf-GAP) (Zhang et al., 1998). A DNA fragment containing AGE1 and its potential promoter and terminator regions was subcloned into a 2 µ-based vector, pRS425, and the resulting plasmid (pRS425-AGE1) was introduced into tet-SAC1 csg1Δ cells. As shown in Fig. S5, the growth defect of tet-SAC1 csg1Δ cells caused by Dox was partly suppressed by the transformation of pRS425-AGE1, as well as YEp13-9, indicating that AGE1 functions as a multicopy suppressor of tet-SAC1 csg1Δ cells. A similar result was obtained when AGE1 was overexpressed under the control of the TEF promoter (pRS415TEFp-AGE1). When tet-SAC1 scs7Δ and tet-SAC1 ipt1Δ cells were transformed with pRS415TEFp-AGE1, the growth defects caused by Dox were clearly suppressed (Fig. 6A). In contrast, deletion of AGE1 caused a more severe growth defect in Dox-treated tet-SAC1 xxxΔ cells, although it did not affect the growth of tet-SAC1 cells even in the presence of Dox (Fig. 6B). The opposite effects of AGE1 overexpression and deletion on tet-SAC1 xxxΔ cells suggested the involvement of AGE1-mediated functions in the growth defects. Since the reduction in the PS level caused by the SAC1 defect is a key factor in the growth defect of tet-SAC1 xxxΔ cells (Fig. 4), we determined whether or not AGE1 affects the reduction in the PS level by using mutant strains in which the promoter region of chromosomal AGE1 was substituted with the TEF promoter (the promoter-substituted gene was named TEFp-AGE1). The introduction of TEFp-AGE1 into tet-SAC1 xxxΔ cells complemented the cell growth in the presence of Dox, like in the case of pRS415TEFp-AGE1 (Fig. S6 and Fig. 6A). When the phospholipid compositions of tet-SAC1 and tet-SAC1 TEFp-AGE1 cells were determined by steady-state radiolabelling with [32P]orthophosphate in the presence or absence of Dox, the reduction in the PS level was found not to be complemented by the introduction of TEFp-AGE1 (Fig. 6C). In addition, the overexpression of AGE1 was able to suppress the growth defects of tet-PSS1 csg1Δ, tet-PSS1 ipt1Δ and tet-PSS1 scs7Δ cells in the presence of Dox, as well as that of tet-SAC1 xxxΔ cells (Fig. 6A and D). Thus, taken together, these results suggested that AGE1 functions downstream of the genetic interaction between the reduction in PS level and the defect of sphingolipid-metabolizing enzymes.

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Figure 6. AGE1 functions as a multicopy suppressor in tet-SAC1 xxxΔ and tet-PSS1 xxxΔ cells. A. tet-SAC1 xxxΔ cells harbouring pRS415TEFp-AGE1 or the empty plasmid were cultured overnight in SC/MSG medium lacking leucine, diluted (0.3 A600 units ml−1) in fresh SC/MSG medium lacking leucine but containing 0 or 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were spotted onto SC/MSG plates lacking leucine but containing 0 or 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.07 A600 units ml−1. All plates were incubated at 30°C and photographed after 27 h or 45 h. B. Growth of tet-SAC1 xxxΔage1Δ cells. Cells were cultured overnight in YPD medium, diluted (0.3 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 3.5 h at 30°C. Cells were spotted onto YPD plates with or without 10 µg ml−1 Dox in 10-fold serial dilutions starting with a density of 0.07 A600 units ml−1. Plates were incubated at 30°C and photographed after 27 h or 45 h. C. Steady-state radiolabelling of tet-SAC1 and tet-SAC1 TEFp-AGE1 cells with [32P]orthophosphate in the presence or absence of Dox. Data shown are the averages of two independent experiments performed in duplicate. Details are given under Experimental procedures. D. Complementation of the growth defect of tet-PSS1 xxxΔ cells on transformation with the pRS415TEFp-AGE1 plasmid. The cell growth on SC/MSG plates lacking leucine in the presence of 3 mM choline was evaluated by serial dilution of the cells as described in (A).

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Anterograde membrane-trafficking system in tet-SAC1 xxxΔ cells

Arf-GAP plays a vital role in several membrane-trafficking systems (Poon et al., 1999; 2001; Yanagisawa et al., 2002; Zhang et al., 2003; Robinson et al., 2006). Therefore, the above observation that AGE1 functions as a suppressor in tet-SAC1 xxxΔ cells prompted us to investigate the membrane-trafficking systems in Dox-treated tet-SAC1 xxxΔ cells. Thus, we investigated anterograde membrane-trafficking systems by assaying the extracellular secretion of invertase, and the transport of alkaline phosphatase (ALP) and carboxypeptidase Y (CPY) to the vacuole. The rate of extracellular secretion of invertase was not impaired in Dox-treated tet-SAC1 xxxΔ cells (Fig. S7A). ALP is transported directly from the TGN to the vacuole and is processed from a precursor form to the mature form (Klionsky and Emr, 1989). Western blotting of ALP revealed that Dox-treated tet-SAC1 xxxΔ cells did not accumulate the precursor form, suggesting that the transport of ALP to the vacuole is not impaired under steady-state conditions (Fig. S7B). The transport of CPY to the vacuole was determined by metabolic labelling of cells with [35S]methionine/cysteine, and chasing with excess non-radiolabelled methionine and cysteine. CPY is initially synthesized in the ER as a precursor form (p1 form), glycosylated in the Golgi (p2 form), and finally transported to the vacuole to give the mature form through the late endosomes (Robinson et al., 1988). In Dox-untreated tet-SAC1 xxxΔ cells, CPY had largely been converted to the mature form after a 20 min chase (Fig. 7A). However, Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells exhibited accumulation of the precursor form even after a 20 min chase, as well as a deletion mutant of VPS10, the gene encoding the sorting receptor for vacuolar hydrolases (Marcusson et al., 1994). In contrast, tet-SAC1 scs7Δ cells did not show any significant defect of CPY processing even in the presence of Dox (Fig. 7A). A previous report indicated that the deletion of SAC1 causes delayed CPY processing (Mayinger et al., 1995; Wiradjaja et al., 2007). As shown in Fig. 7B, a slight delay of CPY processing was observed in sac1Δ cells as compared with wild-type cells, but the processing was almost completed after a 60 min chase; however, in Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells, the precursor form remained even after a 60 min chase (Fig. 7B) and was secreted extracellulary (Fig. 7C), as in vps10Δ cells. These results indicated that Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells were defective in CPY sorting, which is not caused by the SAC1 defect alone. To determine whether or not AGE1 can restore the CPY-sorting defect, we used tet-SAC1 csg1ΔTEFp-AGE1 and tet-SAC1 ipt1ΔTEFp-AGE1 cells. As shown in Fig. 7D, the introduction of TEFp-AGE1 did not restore the CPY-sorting defect in Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells, indicating that this defect is independent of AGE1-mediated functions.

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Figure 7. Transport of CPY to the vacuole in tet-SAC1 xxxΔ cells. A. Cells were metabolically labelled with [35S]methionine/cysteine for 10 min, and then chased with excess non-radiolabelled methionine and cysteine at 30°C for 20 min. Then, cell extracts were immunoprecipitated with CPY antiserum and analysed by SDS-PAGE. p1, ER precursor of CPY; p2, Golgi precursor of CPY; m, vacuolar mature form of CPY. B. CPY pulse-chase experiments were performed as described in (A), except for a 10, 20 or 60 min chase. C. Secretion of CPY by Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells, and vps10Δ cells. Cells were converted to spheroplasts, which were metabolically labelled with [35S]methionine/cysteine for 10 min and then chased for 30 min. Secreted and intracellular CPY were separated into external (E) and internal (I) fractions respectively. D. CPY pulse-chase experiments as described in (A) on tet-SAC1 xxxΔ and tet-SAC1 xxxΔTEFp-AGE1 cells. Details are given under Experimental procedures.

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Aberrant vacuolar morphology in tet-SAC1 xxxΔ cells

Mutation of AGE1 causes an aberrant vacuolar morphology due to a defect of membrane-trafficking systems in the absence of ARF1 or other Arf-GAPs (Zhang et al., 2003). Thus, we next examined the vacuolar morphology in tet-SAC1 xxxΔ cells using the lipophilic dye FM4-64 (Vida and Emr, 1995). As shown in Fig. 8A, in the absence of Dox, tet-SAC1 and all tet-SAC1 xxxΔ cells showed a normal vacuolar morphology, i.e. a large and round structure. However, in Dox-treated tet-SAC1 csg1Δ, tet-SAC1 ipt1Δ and tet-SAC1 scs7Δ cells, the vacuolar morphology was significantly abnormal (Fig. 8A). In contrast, the vacuoles in Dox-treated tet-SAC1 and sac1Δ cells did not exhibit any significant abnormalities, indicating that the aberrant change was caused by the genetic interaction of SAC1 and sphingolipid-metabolizing enzyme gene defects. It has been reported that an abnormal vacuolar morphology was observed in sac1 single mutant cells, due to the development of lipid droplets surrounding the vacuole (Foti et al., 2001). This discrepancy may be caused by differences in the experimental conditions (for instance, they used temperature-sensitive sac1 cells and performed the experiment at 38°C). It has also been reported that deletion of SAC1 causes impairment of endocytic route, and thus delay of the transport of FM4-64 into vacuole occurs in sac1Δ cells (Tahirovic et al., 2005). However, the morphological defects of Dox-treated tet-SAC1 xxxΔ cells were also confirmed by observation of a yeGFP–ALP fusion protein by fluorescence microscopy (Fig. S8), indicating that these aberrant FM4-64 staining patterns were not caused by defects of internalization and transport of this dye into the vacuole from the plasma membrane. Furthermore, these morphological defects observed with FM4-64 were reversed by substitution of the promoter region of chromosomal PSS1 or AGE1 by the TEF promoter (Fig. 8B). Thus, a reduction in the PS level and AGE1-mediated cellular functions are suggested to be involved in the vacuolar abnormalities in tet-SAC1 xxxΔ cells.

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Figure 8. Vacuolar morphology in tet-SAC1 xxxΔ cells. A. Cells were cultured overnight in YPD medium, diluted (0.05 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 7 h at 30°C. Cells were stained with FM4-64 for 15 min, chased for 2 h at 30°C, and analysed by fluorescent microscopy. Details are given under Experimental procedures. B. FM4-64 staining of Dox-treated tet-SAC1 xxxΔ, tet-SAC1 xxxΔTEFp-PSS1 and tet-SAC1 xxxΔTEFp-AGE1 cells.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The present study clearly indicated that a specific group of non-essential sphingolipid-metabolizing enzyme gene, i.e. SCS7, CSG1, CSG2 and IPT1, is essential for yeast cell growth in the genetic background of a SAC1 defect, through a random spore analysis of heterozygous diploid strains and the use of tet-SAC1 xxxΔ (tet-SAC1 scs7Δ, tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ) cells. Because the repression of SAC1 expression led to a decrease in the cellular PS level, and the growth defects of Dox-treated tet-SAC1 xxxΔ strains were complemented by restoration of the PS level, the reduction in the PS level caused by the SAC1 defect was indicated to be one of the key factors for the growth defects (the schematic model was shown in Fig. 9). This notion is supported by the finding that the deletion of SCS7, CSG1 or IPT1 also caused growth defects under PSS1-repressive conditions (Fig. 5).

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Figure 9. Schematic model of the synthetic lethal interaction of specific non-essential sphingolipid-metabolizing enzyme genes with SAC1. Under SAC1-repressive conditions, deletion of each of SCS7, CSG1 and IPT1 caused a synthetic growth defect phenotype, which was complemented by PSS1 or AGE1 overexpression. The overexpression of AGE1 also complemented the growth defects of Dox-treated tet-PSS1 xxxΔ cells, but not the reduction in PS level in Dox-treated tet-SAC1 cells (Fig. 6C and D), suggesting that AGE1 affects events downstream of the genetic interaction between PS and sphingolipids. The growth defect of Dox-treated tet-SAC1 scs7Δ cells was almost completely complemented by the overexpression of PSS1 or AGE1, while the complementation was only partial in the case of Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells. This may imply some additional pathway(s) inducing the growth defect of Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells (shown by grey arrows).

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It was found that Arf-GAP AGE1 functioned as a multicopy suppressor of the growth defects of Dox-treated tet-SAC1 xxxΔ cells (Fig. 6A). Arf is a member of the Ras GTPase superfamily, which have been implicated as regulators of a large number of essential cellular functions including membrane trafficking, and its GTPase activity is regulated by four Arf-GAPs, AGE1, AGE2, GCS1 and GLO3 (Stearns et al., 1990; Zhang et al., 1998; 2003). AGE1 is the most potent multicopy suppressor of Arf-deficient cells among the four Arf-GAPs and involved in vacuolar morphogenesis by regulating the membrane-trafficking systems (Zhang et al., 1998; 2003). In the present study, it was shown that Dox-treated tet-SAC1 xxxΔ cells exhibited vacuolar morphological defects, which were complemented by the overexpression of AGE1 (Fig. 8). Thus, it is seemed that the defect in membrane-trafficking system(s) is one of the key factors for the growth defects of Dox-treated tet-SAC1 xxxΔ cells. Moreover, overexpression of AGE1 also complemented the growth defects of Dox-treated tet-PSS1 xxxΔ cells, but not the reduced PS level in Dox-treated tet-SAC1 cells (Fig. 6C and D), suggesting that AGE1 affects events downstream of the genetic interaction between PS and sphingolipids (Fig. 9).

The phenotypes of tet-SAC1 scs7Δ, tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells were basically similar, but tet-SAC1 scs7Δ was somewhat different from the other two strains in the following aspects. First, the growth defect of Dox-treated tet-SAC1 scs7Δ cells was almost completely complemented by the overexpression of PSS1, while the complementation was only partial in the case of Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells (Fig. 4C and D). Second, impaired CPY sorting, which was not complemented by the overexpression of AGE1, was found in Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells, but not in Dox-treated tet-SAC1 scs7Δ ones (Fig. 7C). These results suggested that the growth defect of Dox-treated tet-SAC1 scs7Δ cells is largely dependent on the PS level, while some additional pathway(s) inducing the growth defect of Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells may exist (Fig. 9).

The four synthetic lethal genes SCS7, CSG1, CSG2 and IPT1 contribute to the creation of a diversity of complex sphingolipids. The deletion of CSG1 or CSG2, and IPT1 causes a reduction in MIPC levels and loss of M(IP)2Cs respectively (Beeler et al., 1997; Dickson et al., 1997). Since the phenotypes of Dox-treated tet-SAC1 csg1Δ and tet-SAC1 ipt1Δ cells were similar, it is supposed that the growth defects of these cells are triggered by loss of M(IP)2Cs, which is commonly caused by each of csg1Δ and ipt1Δ. SCS7 is essential for the α-hydroxylation of sphingolipid-associated fatty acids (Haak et al., 1997), implying a requirement for α-hydroxylated sphingolipids under SAC1-repressive conditions. These results suggested that the structure of both the polar head group and ceramide portion in sphingolipids is important for cell viability under SAC1-repressive conditions. Furthermore, the combination of SCS7, CSG1, CSG2 and IPT1 raises the possibility that a more specific subtype of sphingolipids (α-hydroxylated M(IP)2C) is required under SAC1-repressive conditions. For further investigation of the structural requirements of sphingolipids, a biochemical approach (for instance, exogenous addition of specific subtypes of sphingolipids to cell cultures) is needed.

SAC1 encodes the phosphatase for PIPs, including PI(4)P, PI(3)P, PI(4,5)P2 and PI(3,5)P2, and its deletion causes a marked increase in the PI(4)P level (Hughes et al., 2000; Foti et al., 2001). PIPs regulate many basic cell biological processes such as intracellular membrane trafficking, cytoskeletal organization, cell proliferation and gene expression (D'Angelo et al., 2008; Dove et al., 2009). Several lines of evidence also indicated a functional relationship between PIPs-mediated signalling and sphingolipids in stress responses (Dickson, 2008). Very recently, it has been reported that Sac1p regulates sphingolipid biosynthesis by forming a complex with serine palmitoylatransferase (Lcb1p and Lcb2p), the first enzyme in the pathway of sphingolipid biosynthesis, and the mutation of SAC1 causes the accumulation of LCBs and a decrease in complex sphingolipid levels (Brice et al., 2009; Breslow et al., 2010). To examine whether or not the change in total sphingolipid levels relates to the deletion of each of CSG1, IPT1 and SCS7 to affect yeast cell growth, csg1Δ, ipt1Δ and scs7Δ cells were treated with myriocin, an inhibitor of serine palmitoyltransferase, or phytosphingosine, a major LCB in yeast. However, the effect of these treatments on the growth of csg1Δ, ipt1Δ and scs7Δ cells was similar to that on the growth of wild-type cells (data not shown). Thus, it is speculated that the genetic interaction between the SAC1 defect and deletion of CSG1, IPT1 or SCS7 is not induced by the alteration of sphingolipid levels caused by the SAC1 defect.

In a previous study, a reduction in the PS level was found in the sac1-22 mutant on pulse labelling of cells with [32P]orthophosphate (Rivas et al., 1999). Consistent with this finding, the reduction in the PS level in sac1Δ and Dox-treated tet-SAC1 cells was shown by both pulse and steady-state radiolabelling studies (Fig. 3). The question arises as to how the SAC1 defect affects the PS level. The protein level of PS synthase in sac1Δ cells was comparable to that in wild-type cells (our unpublished results), indicating that the reduction in the PS level is not caused by transcriptional suppression of PS synthase. On the other hand, very recently, it has been reported that phosphorylation of Pss1p by protein kinase A regulates the cellular PS level (Choi et al., 2010). Furthermore, PS synthase activity was regulated by several lipids, including glycerophospholipids, sphingoid bases, inositol and CTP, in an in vitro enzyme assay (Carman and Henry, 1999). The molecular mechanism underlying the reduction in PS level in SAC1-defective cells is currently under investigation in our laboratory.

In yeast, the physiological functions of PS remain largely obscure. PS is an intermediate metabolite in PE and PC biosynthesis via the CDP-diacylglycerol pathway (Carman and Henry, 1999). PS biosynthesis by Pss1p is not essential for cell viability if PE and PC can be synthesized through the alternative CDP-ethanolamine and CDP-choline (Kennedy) pathway utilizing exogenously supplied ethanolamine and choline (Letts et al., 1983), indicating that the existence of PS per se is not essential for yeast cell growth. However, previous reports indicated that a lack of PS biosynthesis causes high sensitivity to divalent metal ions and basic amino acids (Hamamatsu et al., 1994), vacuolar fragmentation (Hamamatsu et al., 1994) and impairment of the incorporation of extracellular tryptophan (Nakamura et al., 2000). The observation of vacuolar fragmentation in PSS1-deleted cells would be of interest for further investigation as to how vacuolar morphological defects are induced in Dox-treated tet-SAC1 xxxΔ cells. Furthermore, a mutation of PGS1, a gene involved in PG and CL biosynthesis, was shown to give a synthetic lethal phenotype with a mutation of PSS1, indicating the functional interaction of PS and other phospholipids in cell growth (Janitor et al., 1996).

Notably, several recent studies suggested that the asymmetry of aminophospholipids, including PS and PE, in lipid bilayers is important for membrane-trafficking pathways. The type 4 subfamily of P-type ATPases is implicated in the translocation of phospholipids from the external to the cytosolic leaflet. In yeast, five members of this subfamily (DRS2, NEO1, DNF1, DNF2 and DNF3) have been identified as putative PS and/or PE translocases (Catty et al., 1997; Hua et al., 2002). They are required for several membrane-trafficking pathways, including endocytic internalization at low temperatures (Pomorski et al., 2003), early endosomes-to-TGN transport (Furuta et al., 2007) and retrograde transport from the Golgi to the ER (Hua and Graham, 2003). Furthermore, mutation of each of DRS2 and CDC50, a gene encoding a protein forming a complex with Drs2p, causes synthetic lethality with that of ARF1 and Arf-GAP GCS1, respectively (Chen et al., 1999; Sakane et al., 2006), indicating the involvement of aminophospholipid translocase in Arf-mediated membrane-trafficking systems. Therefore, taking into consideration that alteration of the PS level is one of the key events in the growth defect in tet-SAC1 xxxΔ cells (Fig. 4), and overexpression of Arf-GAP AGE1 complements the growth defect of tet-SAC1 xxxΔ and tet-PSS1 xxxΔ cells (Fig. 6), the asymmetry of PS would be of interest for further investigation of the molecular mechanism underlying the growth defects.

In summary, the present study revealed a novel functional interaction between sphingolipids and glycerophospholipids, which are essential for the viability of yeast in the absence of SAC1. Further study will provide a new insight into the physiological significance of the multiple networks between sphingolipids and glycerophospholipids in cellular functions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Yeast strains and media

The S. cerevisiae strains used are listed in Table 1 (strains used in supplementary figures are in Table S1). To generate a yeast in which the expression of SAC1 or PSS1 is regulated by doxycycline (Dox), the SAC1 upstream region from −1 to −70 bp, or the PSS1 upstream region from −1 to −10 bp was replaced with a tetracycline operator cassette containing a repressor binding site [tetO2 (for PSS1) or tetO7 (for SAC1)] and a TetR-VP16 tTA transactivator, as described previously (Belli et al., 1998). Disruption of SAC1, CSG1, CSG2, IPT1 and SCS7 was performed by replacing their open reading frames with the URA3 marker from pRS406 (Christianson et al., 1992). Disruption of AGE1 was performed by replacing its open reading frame with the natMX4 marker from p4339 (pCRII-TOPO::natMX4) (Tong and Boone, 2006). For replacement of the native PSS1 and AGE1 promoter regions with a constitutive and strong TEF promoter (TEFp-PSS1 and TEFp-AGE1), a TEF promoter cassette with the natNT2 marker from pYM-N19 was introduced immediately upstream of the initiator ATG of chromosomal PSS1 and AGE1 as described (Janke et al., 2004). For tagging of the C-terminus of Sac1p with nine copies of the myc epitope (9xmyc), a 9xmyc-tag fusion cassette with the natNT2 marker from pYM21 was introduced immediately upstream of the stop codon of chromosomal SAC1 as described (Janke et al., 2004). The cells were grown in either YPD medium (1% yeast extract, 2% peptone and 2% glucose) or synthetic complete (SC/MSG) medium [0.17% yeast nitrogen base with/without amino acids and ammonium sulphate (BD Difco, Heidelberg, Germany), 0.1% l-glutamic acid sodium salt hydrate (MSG; Sigma) and 2% glucose] containing nutritional supplements (Tong and Boone, 2006).

Table 1.  Strains used in this study.
StrainGenotypeSource
BY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Brachmann et al. (1998)
Y7092MATα can1Δ::STE2pr-his5 lyp1Δhis3Δ1 leu2Δ0 met15Δ0 ura3Δ0Tong and Boone (2006)
MTY52BY4741, sac1Δ::URA3This study
MTY74Y7092, sac1Δ::URA3This study
MTY90BY4741, tetO7-SAC1::kanMX4This study
MTY95BY4741, tetO7-SAC1::kanMX4 csg1Δ::URA3This study
MTY97BY4741, tetO7-SAC1::kanMX4 scs7Δ::URA3This study
MTY119BY4741, tetO7-SAC1::kanMX4 csg2Δ::URA3This study
MTY120BY4741, tetO7-SAC1::kanMX4 ipt1Δ::URA3This study
MTY598BY4741, tetO7-SAC1::kanMX4 SAC1-9xmyc::hphNTIThis study
MTY296BY4741, tetO7-SAC1::kanMX4 TEFp-PSS1::natNT2This study
MTY298BY4741, tetO7-SAC1::kanMX4 csg1Δ::URA3 TEFp-PSS1::natNT2This study
MTY300BY4741, tetO7-SAC1::kanMX4 scs7Δ::URA3 TEFp-PSS1::natNT2This study
MTY302BY4741, tetO7-SAC1::kanMX4 ipt1Δ::URA3 TEFp-PSS1::natNT2This study
MTY304BY4741, tetO2-PSS1::kanMX4This study
MTY323BY4741, tetO2-PSS1::kanMX4 csg1Δ::URA3This study
MTY324BY4741, tetO2-PSS1::kanMX4 scs7Δ::URA3This study
MTY325BY4741, tetO2-PSS1::kanMX4 ipt1Δ::URA3This study
MTY585BY4741, tetO7-SAC1::kanMX4 age1Δ::natMX4This study
MTY586BY4741, tetO7-SAC1::kanMX4 csg1Δ::URA3 age1Δ::natMX4This study
MTY587BY4741, tetO7-SAC1::kanMX4 scs7Δ::URA3 age1Δ::natMX4This study
MTY588BY4741, tetO7-SAC1::kanMX4 ipt1Δ::URA3 age1Δ::natMX4This study
MTY491BY4741, tetO7-SAC1::kanMX4 TEFp-AGE1::natNT2This study
MTY492BY4741, tetO7-SAC1::kanMX4 csg1Δ::URA3 TEFp-AGE1::natNT2This study
MTY493BY4741, tetO7-SAC1::kanMX4 scs7Δ::URA3 TEFp-AGE1::natNT2This study
MTY494BY4741, tetO7-SAC1::kanMX4 ipt1Δ::URA3 TEFp-AGE1::natNT2This study
vps10ΔBY4741, vps10Δ::kanMX4Winzeler et al. (1999)

Plasmids

A multicopy plasmid [pRS425 (Christianson et al., 1992)] containing AGE1 and its 5′- and 3′-untranslated regions (500 and 500 bp respectively) was constructed as described below. A DNA fragment was amplified by PCR using a 5′ primer with a XhoI site (5′-CCCCTCGAGATAGAACAAGAAGAAAGACCGCTCT-3′), a 3′ primer with a NotI site (5′-AGAGCGGCCGCGTGCTATTTTCTTTTGTTGTTTAGTA-3′), and yeast genomic DNA as a template. The fragment obtained was subcloned into pRS425. The pRS415TEFp yeast vector was constructed to allow constitutive and strong expression of proteins. The TEF promoter region was amplified from the kanMX4 module of pCM225 (Belli et al., 1998) using a 5′ primer with a XhoI site (5′-CCCCTCGAGTTAATTAAGGCGCGCCAGATC-3′) and a 3′ primer with a HindIII site (5′-GATAAGCTTGGTTGTTTATGTTCGGATGTGAT-3′). The terminator region was also amplified from pCM225 using a 5′ primer with a NotI site (5′-AGAGCGGCCGCTCAGTACTGACAATAAAAAGATTCT-3′) and a 3′ primer with a SpeI site (5′-TCCACTAGTAATTCGAGCTCGTTTTCGACA-3′). These PCR fragments were subcloned into pRS415 (Christianson et al., 1992) to yield pRS415TEFp. To construct a expression plasmid for AGE1, i.e. pRS415TEFp-AGE1, a DNA fragment containing the AGE1 ORF was amplified by PCR using a 5′ primer with a SmaI site (5′-CAGCCCGGGTCAATAATGGATTTTTATACTACTGATATC-3′), a 3′ primer with a NotI site (5′-AGAGCGGCCGCTTATTTTTTGTCCTTTTTGGCCA-3′), and yeast genomic DNA as a template. The fragment obtained was subcloned into pRS415TEFp to yield pRS415TEFp-AGE1.

Random spore analysis

To observe the synthetic lethal genetic interaction between SAC1 and non-essential sphingolipid-metabolizing enzyme genes, a random spore analysis was performed according to the method of Tong and Boone (2006), using yeast MATa knockout library (BY4741) strains (Winzeler et al., 1999) and the MATα Y7092 sac1Δ::URA3 strain. The selection media used for this study were as follows: SC/MSG lacking histidine, arginine and lysine, but containing 50 µg ml−1 canavanine and 50 µg ml−1 thialysine (for haploid selection); SC/MSG lacking histidine, arginine and lysine, but containing 50 µg ml−1 canavanine, 50 µg ml−1 thialysine and 200 µg ml−1 G418 (for kanMX4 selection); SC/MSG lacking histidine, arginine, lysine and uracil, but containing 50 µg ml−1 canavanine and 50 µg ml−1 thialysine (for URA3 selection); and SC/MSG lacking histidine, arginine, lysine and uracil, but containing 50 µg ml−1 canavanine, 50 µg ml−1 thialysine and 200 µg ml−1 G418 (for kanMX4 and URA3 selection).

Vacuole labelling with FM4-64

Yeast cells grown in a YPD medium were collected by centrifugation and then resuspended in fresh YPD medium to 1 A 600 unit ml−1. FM4-64 (Molecular Probes, Eugene, OR) was added to the cells to a final concentration of 20 µM, followed by reaction at 30°C for 10 min, washing three times with YPD medium and then chasing in fresh YPD medium without FM4-64 for 120 min. Endocytosis was stopped by the addition of NaF and NaN3 to a final concentration of 10 mM. Cells were collected by centrifugation and viewed under a fluorescence microscope (Leica DMRB; Leica, Solms, Germany).

Yeast protein extraction, SDS-PAGE and Western blotting

Yeast cells grown in a YPD medium were collected by centrifugation, washed with distilled water, and then resuspended in 100 µl of 0.2 N NaOH containing 0.5% 2-mercaptoethanol. The suspension was incubated on ice for 15 min and then centrifuged, the pellet being resuspended in 100 µl of distilled water. Five microlitres of each sample was used to determine protein content by means of the bicinchoninic acid protein assay with bovine serum albumin as a standard. One millilitre of ice-cold acetone was added to the suspension, followed by incubation for 30 min at −20°C, and then proteins were precipitated by centrifugation for 10 min at 10 000 g. The pellet was resuspended in 100 µl of SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 1% 2-mercaptoethanol and 0.001% bromophenol blue). The suspension was mixed well, heated for 3 min at 95°C and then centrifuged for 5 min at 10 000 g. Then the supernatant was separated by SDS-PAGE according to the method of Laemmli (1970). For Western blotting, following separation by SDS-PAGE, proteins were electrotransferred to a nitrocellulose membrane. The membrane was blocked with PBS/0.1% Tween 20 (PBS-T) containing 3% dried milk. Proteins were identified by incubation with anti-alkaline phosphatase (diluted 1:100; Molecular Probes), anti-myc (0.24 µg ml−1; Invitrogen) or anti-Pgk1p (2 µg ml−1; Molecular Probes) in 3% dried milk/PBS-T at 4°C overnight. Secondary antibodies [horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:5000; Biosource, Camarillo, CA)] were incubated in 3% dried milk/PBS-T at room temperature for 1 h. Labelling was visualized with an ECL AdvanceTM Western Blotting Detection Kit (GE Healthcare, England).

[3H]myo-inositol labelling

Yeast cells grown in a YPD medium were collected by centrifugation and then washed with SC/MSG medium with minimum inositol (2 µg ml−1). Then the cells were resuspended in 500 µl of SC/MSG medium with minimum inositol to 2 A600 units ml−1 and labelled with [3H]myo-inositol (1 µCi per 1 A600 unit of cells; PerkinElmer Life Sciences, Norwalk, CT) for 1 h at 30°C. The cells were chilled on ice, collected by centrifugation, washed with distilled water and then suspended in 150 µl of ethanol/water/diethyl/ether/pyridine/15 N ammonia (15:15:5:1:0.018, v/v). Radioactivity was measured using a liquid scintillation system, and samples containing lipids exhibiting equal level of radioactivity were used for further study. After a 15 min incubation at 60°C, the residue was centrifuged at 10 000 g for 1 min and extracted once more in the same manner. The resulting supernatant was dried and suspended in 20 µl of chloroform/methanol/water (5:4:1, v/v). Lipids were separated on Silica Gel 60 TLC plates (Merck, Whitehouse Station, NJ) with chloroform/methanol/4.2 N ammonia (9:7:2, v/v) as the solvent system.

[32P]orthophosphate labelling

For pulse labelling, cells were cultured overnight in YPD medium, diluted (0.05 A600 units ml−1) in YPD medium with or without 10 µg ml−1 Dox, and then incubated for 8 h. The cells were collected by centrifugation, resuspended in 500 µl of fresh YPD medium with or without Dox to 2 A600 units ml−1, and then labelled with [32P]orthophosphate (1 µCi per 1 A600 unit of cells; MP Biomedicals, Morgan Irvine, CA) for 1 h at 30°C. For steady-state labelling, cells were cultured overnight in YPD medium with or without 10 µg ml−1 Dox, diluted (0.1 A600 units ml−1) in 2 ml of fresh YPD medium with or without Dox, and then labelled with 4 µCi [32P]orthophosphate for 12 h at 30°C. The cells were resuspended in 1 ml of fresh YPD medium with or without Dox to 0.3 A600 units ml−1 and then labelled with 2 µCi [32P]orthophosphate for 5 h. The radiolabelled cells were chilled on ice, collected by centrifugation, washed with distilled water and then suspended in 150 µl of 80% ethanol. After heating at 95°C for 30 min, 800 µl of chloroform/methanol (1:1, v/v) and subsequently 330 µl of PBS were added to the sample. The sample was mixed well and then centrifuged at 10 000 g for 1 min. The lower phase was collected, dried and applied to a LK5 silica gel 150 A TLC plate (Whatman, Clifton, NJ), which had been pre-washed in chloroform/methanol (1:1, v/v) and treated with 2% boric acid in ethanol. The TLC plate was developed two times with chloroform/ethanol/water/triethylamine (30:35:7:35, v/v) and then analysed with a Bio Imaging analyser FLA-2000 (Fuji Photo Film, Kanagawa, Japan).

Pulse-chase labelling of CPY with [35S]methionine/[35S]cysteine

Yeast cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 6.5 h at 30°C. They were again collected by centrifugation, washed with distilled water, resuspended in SC/MSG medium lacking methionine and cysteine, and then incubated at 30°C for 1 h. Cells were collected by centrifugation, and resuspended in 700 µl of fresh SC/MSG medium lacking methionine and cysteine to 1.4 A600 units ml−1. Then the cells were pulse-labelled with [35S]methionine/[35S]cysteine (25 µCi per 1 A600 unit of cells; EXPRESSTM protein labelling mix; 1000 Ci mmol−1; PerkinElmer Life Sciences) for 10 min, and then chased with cold methionine (final concentration, 0.5 mg ml−1) and cysteine (0.1 mg ml−1) for 20 min. After cell samples had been washed with SC/MSG medium containing 10 mM NaN3 and 10 mM NaF, pellets of the cell extracts were prepared as described under Yeast protein extraction, SDS-PAGE and Western blotting. The pellets were dissolved in 100 µl of 20 mM Tris-HCl (pH 7.5) containing 1% SDS and 1% 2-mercaptoethanol, and then heated for 3 min at 95°C. Each cell lysate was centrifuged at 10 000 g for 5 min, and the supernatant was mixed with 1 ml of 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2 mM EDTA and 1% Triton X-100 (buffer A). The mixture was incubated with 5 µl of CPY antiserum (Rockland, Gilbertsville, PA) at 4°C for 12 h. Then 5 µl of protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and the mixture was incubated at 4°C for 3 h. The precipitate was spun down by centrifugation, washed five times with buffer A and then suspended in 20 µl of SDS-sample buffer. After heating for 3 min at 95°C, the sample was subjected to SDS-PAGE. The gel was fixed, treated with Amplify Fluorographic ReagentTM (GE Healthcare), dried and then analysed with the FLA-2000. To assay internal and external CPY levels, cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium with or without 10 µg ml−1 Dox, and then incubated for 6.5 h at 30°C. They were again collected by centrifugation, washed with distilled water, resuspended in SC/MSG medium lacking methionine and cysteine with or without 10 µg ml−1 Dox, and then incubated at 30°C for 1 h. Cells were collected by centrifugation, resuspended in 700 µl of fresh SC/MSG medium lacking methionine and cysteine but containing 1.2 M sorbitol, and with or without 10 µg ml−1 Dox to 1.4 A600 units ml−1, and then incubated with Zymolyase 100T (100 µg ml−1; Seikagaku Kogyo, Tokyo, Japan) at 30°C for 20 min. The resulting spheroplasts were pulse-labelled with [35S]methionine/[35S]cysteine (25 µCi per 1 A600 unit of cells) for 10 min and then chased for 30 min. After the pulse-chase, NaN3 and NaF were added to spheroplast suspensions to a final concentration of 10 mM, and the suspensions were separated by centrifugation into spheroplasts and media as the intracellular and extracellular fractions respectively. The intracellular fraction was dissolved in 100 µl of 20 mM Tris-HCl (pH 7.5) containing 1% SDS and 1% 2-mercaptoethanol, heated for 3 min at 95°C, and then subjected to immunoprecipitation as described above. The extracellular fraction (700 µl) was mixed with 17 µl of 10% SDS, heated for 3 min at 95°C, and then mixed with 1 ml of 50 mM Tris-HCl (pH 7.5) containing 270 mM NaCl, 3.6 mM EDTA and 1.8% Triton X-100. Immunoprecipitation was performed by incubation with 10 µl of CPY antiserum for 12 h at 4°C followed by 10 µl of protein A/G agarose for 3 h at 4°C.

Multicopy suppressor screening

A S. cerevisiae genomic DNA library based on YEp13, a multicopy vector with the LEU2 marker (kindly provided by Dr Akihiko Nakano, RIKEN, Japan), was introduced into tet-SAC1 csg1Δ cells. After the transformation, cells were cultured in SC medium lacking leucine at 30°C for 3 h. Then 2 µg ml−1 Dox was added to the culture, followed by culturing for an additional 5 h. The cells were plated on SC plates lacking leucine but containing 2 µg ml−1 Dox, and then incubated at 30°C for 3 days. Colonies were re-streaked onto SC plates lacking leucine but containing 2 µg ml−1 Dox, and the growth was confirmed. Plasmids were recovered from the candidate clones and re-introduced into tet-SAC1 csg1Δ cells to verify the suppressive activity. Plasmid DNA from positive clones was isolated and amplified in Escherichia coli, and the DNA sequences were verified with an ABI PRISM® 3100 genetic analyser (Applied Biosystems, Foster, CA).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Makoto Ito (Kyushu University), Dr Kaoru Takegawa (Kyushu University), Dr Tadashi Ogishima (Kyushu University) and Dr Sakae Kitada (Kyushu University) for valuable suggestions regarding this study. This work was supported by Program for Improvement of Research Environment for Young Researchers from Special Coordination Funds for Promoting Science and Technology (SCF) commissioned by the Ministry of Education, Culture, Sports, Science, and Technology (MXST) of Japan. This study was partially funded by KAKENHI (19870017 and 21770217) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Core Research for Evolutional Science and Technology, the Japan Science and Technology Agency.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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