INTRODUCTION

Sphingolipids along with cholesterol and phosphoglycerolipids are the three major types of lipids found in biological membranes. The sphingolipids in Saccharomyces cerevisiae are primarily located in the plasma membrane (1, 2), where they account for 7-8% of the total mass of the membrane (30% of the plasma membrane phospholipids; Ref. 1). At present, we have only a general outline of the steps in S. cerevisiae sphingolipid biosynthesis (Fig. 1) and know nothing about how the concentration of the intermediates and complex sphingolipids is regulated. Further understanding of the pathway requires identification of the biosynthetic genes and purification and characterization of the cognate enzymes, none of which have been purified. Only three S. cerevisiae genes necessary for sphingolipid synthesis, LCB1 (3), LCB2 (3-5), and AUR1 (6), have been identified. Completion of the S. cerevisiae genome sequence (7) should facilitate identification of the remaining genes. By using the S. cerevisiae genome sequence data base, we have now identified another sphingolipid biosynthetic gene, YDR072c, and renamed IPT1 (inositolphosphotransferase 1). This gene is necessary for synthesis of mannose-(inositol-P)₂-ceramide (M(IP)₂C¹; Ref. 8), the terminal and most abundant S. cerevisiae sphingolipid (Fig. 1).

[View Larger Version of this Image (21K GIF file)]

Sphingolipid synthesis in S. cerevisiae begins with the condensation of serine and palmitoyl-CoA to yield 3-ketosphinganine. This essentially irreversible reaction is catalyzed by serine palmitoyltransferase (3-ketosphinganine synthase (EC 2.3.1.50); for review, see Ref. 9), a pyridoxal phosphate-containing enzyme. Further reactions convert 3-ketosphinganine to the long chain base sphinganine (dihydrosphingosine), which is N-fatty-acylated to yield dihydroceramide. Dehydrogenation of dihydroceramide in animals yields ceramide with the long chain base sphingosine, which is rapidly converted to sphingolipids by the addition of polar components to the 1-hydroxyl group. Most ceramides in fungi and plants contain N--hydroxyfattyacylphytosphingosine (8), formed by undefined hydroxylation reactions. Phytosphingosine lacks the 4,5-double bond found in sphingosine and has instead an hydroxyl group at the 4-position. Molecular species with the same head groups have been recognized (8) that differ in hydroxylation of the long chain base and the fatty acid. The most abundant species, designated 3, e.g. M(IP)₂C-3, contains phytosphingosine and OH-hexacosanoic acid.

A common modification to phytoceramide in S. cerevisiae and other fungi is addition of myoinositol phosphate to the 1-hydroxyl to form IPC, which is then mannosylated to yield mannose-inositol-P-ceramide (MIPC). The terminal step in S. cerevisiae sphingolipid synthesis is addition of inositol-P to MIPC to yield the major sphingolipid M(IP)₂C. The later steps in sphingolipid synthesis in S. cerevisiae (Fig. 1) are tentative because the enzymes have not been purified, the reaction requirements are poorly defined, and the stoichiometry has not been determined (reviewed in Ref. 8).

We recently identified a gene, AUR1, necessary for phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase) activity (6). The gene most likely encodes the IPC synthase enzyme or a subunit of the enzyme whose subunit structure is unknown. IPC synthase appears to catalyze a reaction very similar to the one catalyzed by phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase (M(IP)₂C synthase), since both transfer inositol-P from phosphatidylinositol to ceramide or a ceramide-containing compound. This similarity suggested that there might be a protein in S. cerevisiae with similarity to the Aur1 protein. Using the Aur1 protein as a query, we found a protein in the S. cerevisiae genome data base, encoded by IPT1, that appeared to be related to the Aur1 protein. We show here that IPT1 is indeed necessary for M(IP)₂C synthase activity and for synthesis of M(IP)₂C.

EXPERIMENTAL PROCEDURES

Strain RCD113 (MATa ura3-52 lys2-801^amber ade2-101^ochre trp1-1 his3-200 leu2-1 ipt1-1) was derived from strain YPH250 (10) by gene transplacement of the YDR072c (IPT1) open reading frame with the ipt1-1 allele, made by replacing the coding region of IPT1 with the HIS3 gene using the polymerase chain reaction as described by Baudin et al. (11). Correct gene transplacement was verified by Southern blot analysis (data not shown).

The composition of defined and complex media has been described previously (12). Cultures were grown in shaker flasks at 30 °C unless indicated otherwise in the text.

Cells were cultured for 21 h (initial A₆₅₀ = 0.1) at 30 °C in a medium consisting of 1% peptone, 1% yeast extract, 0.05% Tergitol, 1% KH₂PO₄, 0.05 M sodium succinate, pH 5.5, 1 µg/ml inositol, 4% glucose, and 20 µCi/ml [2-³H]myoinositol (American Radiochemicals, Inc., 20 Ci/mmol). Cells (5 ml) were treated with trichloroacetic acid to a final concentration of 5% and chilled for 15 min on ice. After twice washing with water, cells were extracted with 2 ml of Solvent E, diethylether/95% ethanol/water/pyridine/concentrated ammonia (5:15:15:1:0.018), v/v) for 60 min at 60 °C (13). A 0.5-ml portion of the Solvent E lipid extract was dried under nitrogen and deacylated by treatment with 0.5 ml of monomethylamine reagent (14) for 30 min at 50 °C, followed by evaporation to dryness. The dried sample was resuspended in 0.5 ml of chloroform/methanol/water (16:16:5), and 10-µl samples of the neat and deacylated lipid extracts were subjected to high performance TLC on 20-cm Whatman HP-K plates developed with chloroform/methanol/4.2 N ammonia (9:7:2); each lane also contained a mixture of yeast sphingolipids, IPC-3, IPC-4, MIPC-3 (2 µg each), and 1.2 µg of M(IP)₂C-3 (8). Radioactivity was detected by using a BioScan apparatus, and the sphingolipid standards were detected after spraying the TLC plate with 10% CuSO₄·5H₂O in 8% phosphoric acid, followed by charring at 160 °C (15). The sphingolipids in the deacylated lipid extract were also detected after thin layer chromatography of larger samples (125 µl) on 20-cm Whatman K5 plates developed with the same solvent as above. The plate was first treated with orcinol reagent (16) to detect the carbohydrate-containing sphingolipids, MIPC and M(IP)₂C, and then treated with the CuSO₄/phosphoric acid reagent as above.

Membranes were prepared as described previously (6). Lipid additions, [³H]phosphatidylinositol (2 × 10⁶ dpm), MIPC-3 (50 nmol), and aureobasidin A were dried in a tube and treated in a sonic water bath after addition of the following (final concentrations): CHAPS (2 mM), KPO₄ (50 mM, pH 7.0), and water. Membranes (0-120 µg of protein) were added, and the mixture (final volume, 100 µl) was incubated at 30 °C. The reaction was terminated with 10 µl of 50 mM disodium EDTA, 0.64 ml of chloroform/methanol (1:1), and 2.25 ml of chloroform/methanol/water (16:16:5). After centrifugation, the supernatant fluid was added to a 1-ml column of AG4-X4 resin (200-400-mesh, Bio-Rad), packed using water in a Pasteur pipette, washed sequentially with six volumes of 3 N acetic acid, five volumes of water, and then with methanol followed by equilibration with chloroform/methanol/water (16:16:5). After sample addition, the column was washed to remove the bulk of the phosphatidylinositol with 1 ml of chloroform/methanol/water (16:16:5), 3 ml of 0.005 M ammonium acetate in chloroform/methanol/water (16:16:5), 1 ml of chloroform/methanol/water (16:16:5). The M(IP)₂C as well as some remaining phosphatidylinositol were eluted with 3 ml of 0.4 M ammonium hydroxide in chloroform/methanol/water (16:16:5), dried under nitrogen, deacylated with 0.5 ml monomethylamine reagent (14) for 30 min at 50 °C, dried under nitrogen, and dissolved in 200 µl of chloroform/methanol/water (16:16:5). For quantification, samples were chromatographed on EDTA-treated silica gel-impregnated paper (Whatman SG81) by using chloroform/methanol/4.2 N ammonia (9:7:2, v/v) (17). M(IP)₂C migrated to an R_F of 0.3, and the water-soluble products, mainly glycerophosphoinositol, remained near the origin. Each lane was cut into 1-cm zones, and radioactivity was measured by scintillation counting. For qualitative analysis, samples were chromatographed on high performance silica gel plates (Whatman HP-K) with the solvent chloroform/methanol/4.2 N ammonia (9:7:2, v/v). Radioactivity was determined by using a Bioscan apparatus. Each sample contained approximately 1 µg of M(IP)₂C-3, which was detected by charring the plate at 160 °C (15).

RESULTS

Structural homologs of the Aur1 protein were searched for in the S. cerevisiae genome data base by using the FASTA algorithm (18). Only one putative homolog, Ipt1 (YDR072c, GenBank accession no. Z46796x5), was identified. No function had been attributed to the predicted Ipt1 protein, which shows 27% amino acid identity to the Aur1 protein over a region of 365 amino acids (Fig. 2).

[View Larger Version of this Image (50K GIF file)]

To determine if IPT1 is necessary for synthesis of M(IP)₂C, sphingolipids were radiolabeled by growing cells long term in the presence of [³H]myoinositol. Extracted radioactive sphingolipids were either subjected to or not subjected to alkali-catalyzed deacylation, separated by high performance TLC, and located by using a BioScan apparatus. Comparison of the scans (Fig. 3) to the authentic S. cerevisiae sphingolipid internal standards shows that the ipt1-deletion strain RCD113 lacks the radioactive fraction representing the alkali-stable M(IP)₂C species present in the scans of the lipids isolated from wild type YPH250 cells. Mutant strain RCD113 shows an increase in radiolabeled MIPC-3 (Fig. 3), indicating that this species of sphingolipid accumulates.

[View Larger Version of this Image (19K GIF file)]

Mutant cells carrying the SLC1-1 suppressor gene and unable to make sphingolipids when a long chain base is withheld from the culture medium, accumulate inositol-containing lipids in which the ceramide is replaced with diacylglycerols containing a C₂₆-fatty acid (19, 20). No such lipids are evident in RCD113 cells (Fig. 3, bottom).

To verify the absence of M(IP)₂C and an increase in MIPC-3 in strain RCD113, non-radioactive deacylated lipid extracts were again analyzed by TLC and the plate was treated with the orcinol reagent to detect carbohydrates (mannose) and then charred to detect all the carbon-containing polar head groups. This analysis shows that M(IP)₂C is not present in strain RCD113 and that the concentration of MIPC is elevated (Fig. 4). The identity of these lipids was confirmed by their reaction with the orcinol reagent.

[View Larger Version of this Image (69K GIF file)]

M(IP)₂C synthase activity was measured in membranes prepared from YPH250 and ipt1-mutant RCD113 cells by the incorporation of radiolabeled inositol into M(IP)₂C from [³H-inositol]phosphatidylinositol. The reaction catalyzed by M(IP)₂C synthase is not well characterized (8) but is believed to use MIPC and phosphatidylinositol as substrates. The [³H]M(IP)₂C produced in the reaction was first analyzed by thin layer chromatography to verify that the correct product was being made. Membranes prepared from YPH250 but not from the mutant RCD113 cells make [³H]M(IP)₂C, as judged by its co-migration with the internal M(IP)₂C standard (Fig. 5). Reactions conditions were then examined in more detail. Membranes from YPH250 cells produced [³H]M(IP)₂C linearly with time and with the protein concentrations examined. No production of [³H]M(IP)₂C was observed with membranes from RCD113 cells (Fig. 6). Omission of exogenous MIPC from the reaction mixture (Table I) reduces product formation by about 65%, consistent with this lipid being a substrate for the enzyme. We conclude from these data that RCD113 cells lack M(IP)₂C synthase activity.

[View Larger Version of this Image (28K GIF file)]

[View Larger Version of this Image (23K GIF file)]

Table I. In vitro M(IP)2C synthesis: MIPC dependence, inhibition by aureobasidin A Synthesis of radioactive M(IP)2C by membranes (101 µg of protein) prepared from YPH250 cells in the presence of [3H-inositol]phosphatidylinositol was measured as described under "Experimental Procedures" over a 2-h incubation period. The data represent the average of duplicates. Changes in basal assay conditions [3H]M(IP)2C formed pmol None 40.5  ± 6 Minus MIPC 14.2  ± 1 Plus aureobasidin A (0.1 µM) 40.4  ± 2.6 Plus aureobasidin A (0.5 µM) 23.0  ± 6 Plus aureobasidin A (1.0 µM) 21.2  ± 2.1 Plus aureobasidin A (5.0 µM) 11.9  ± 3.8

Table II. Growth inhibition by calcium Cells were grown in PYED medium containing the indicated concentration of calcium chloride to an A600 nm of 0.6, diluted into 2 ml of fresh medium to give 105 cells/ml and grown 17 h with aeration at 30 °C. The final cell density at A600 nm is shown. Calcium A600 nm YPH250 RCD113 (ipt1-1) mM   0 1.66 2.1  10 1.67 2.3  50 0.32 1.22 100 0.096 0.31 200 0.023 0.083

IPC synthase activity, presumably encoded by AUR1, is strongly inhibited by the antifungal drug aureobasidin A (6). Since the Ipt1 protein is related to the Aur1 protein and each is required for expression of the IPC and M(IP)₂C synthases, respectively, and since both enzymes utilize phosphatidylinositol as one of their substrates, catalyzing a similar phosphoinositol transfer, it was logical to examine the inhibition of M(IP)₂C synthase by aureobasidin A. The data presented in Table I show that aureobasidin A strongly inhibits M(IP)₂C synthase in the micromolar range, with 50% inhibition (IC₅₀) at 0.5-1 µM.

To begin to understand the function of M(IP)₂C, we compared the behavior of RCD113 mutant and wild type YPH250 cells grown under a variety of conditions. Examination of mutant and wild type cells in log and stationary phase growth by light microscopy showed no difference in their size or morphology. Growth in a complex medium at 30 °C showed that RCD113 mutant cells grew as rapidly as parental YPH250 cells and to the same density during the course of this experiment (Fig. 7), indicating that the lack of M(IP)₂C had no adverse effect upon growth rate during log phase and early stationary phase growth nor upon the ability of cells to grow to a fairly high density.

[View Larger Version of this Image (17K GIF file)]

We had shown previously (21) that one or more sphingolipids are necessary for S. cerevisiae cells to grow under stress conditions including incubation at 37 °C, in the presence of 0.75 M NaCl, and at low pH. To determine if M(IP)₂C is necessary for growth under these stress conditions, RCD113 mutant cells were tested for growth on PYED complex medium plates incubated at 37 °C, containing 0.75 M NaCl and incubated at 30 °C, or having a pH of 4.1 and incubated at 30 °C (12). RCD113 cells grew as well as wild type cells under all conditions (data not shown). Thus, M(IP)₂C is not necessary for responding to these stresses.

We have also observed that mutant yeast cells lacking sphingolipids (21) are unable to be induced for what has been termed induced thermotolerance, in which survival at an elevated temperature (greater than about 45 °C) can be enhanced by preincubation at an intermediate temperature, generally 37 °C (reviewed in Ref. 22). To determine if M(IP)₂C lipids play a role in induced thermotolerance, early log phase RCD113 and YPH250 cells were incubated at 25 °C or 37 °C for 30 min and transferred to 52 °C, and surviving cells were measured at 0, 20, and 40 min. Survival was the same for both strains, and both survived the 52 °C treatment better after preincubation at 37 °C, indicating that thermotolerance had been induced (data not shown). We conclude from these data that induction of thermotolerance does not require M(IP)₂C lipids.

Sphingolipids are believed to reach the plasma membrane by the protein secretory pathway. Most sphingolipids would initially be in the outer leaflet of the plasma membrane with their polar head group pointed toward the yeast cell wall. To determine if the absence of M(IP)₂C in the outer leaflet of the plasma membrane and the consequent loss of negative charge affected the cell wall, we compared the sensitivity of mutant RCD 113 and wild type cells to lysis after digestion with a glucanase as described previously (12). This assay has been shown to be a measure of the integrity of the mannoprotein layer on the exterior of the cell wall (23). RCD113 mutant cells were found to be as resistant to digestion as wild type cells (data not shown), indicating that the outer mannoprotein layer of the wall was not altered by the absence of M(IP)₂C.

Cells carrying a mutation in either the csg1 or csg2 gene do not make either MIPC or M(IP)₂C and are inhibited for growth by high concentrations of calcium (5). Based upon these data, it seemed possible that RCD113 cells might be more sensitive to calcium ions than wild type cells. Contrary to our expectation, RCD113 cells were slightly more resistant to growth inhibition by calcium than were wild type cells (Table II).

Haploid RCD113 cells were able to mate with cells of the opposite mating type (W303-1B) at a frequency of about 50% (24), which is the same frequency we found for parental YPH250 cells, indicating that M(IP)₂C lipids are not necessary for mating. Diploid RCD113 and YPH250 cells, made by transformation of haploid cells with pHO12 (25) and selection of large, diploid-like cells, sporulated at about the same frequency (20% for RCD113 and 25% for YPH250), indicating that M(IP)₂C lipids are not essential for sporulation.

DISCUSSION

Here, we present evidence that the IPT1 gene is necessary for synthesis of M(IP)₂C, the major and terminal sphingolipid in S. cerevisiae. This assignment is based upon that fact that ipt1-deleted RCD113 cells fail to make M(IP)₂C and, instead, make increased amounts of MIPC (Figs. 3 and 4), the precursor to M(IP)₂C (Fig. 1). In addition, membranes prepared from RCD113 cells lack M(IP)₂C synthase activity since they fail to make [³H]M(IP)₂C from [³H-inositol]phosphatidylinositol (Figs. 4 and 5).

The observation that mutant RCD113 cells compensate for the loss of M(IP)₂C lipids and make a nearly equal amount of MIPC lipids indicates that S. cerevisiae cells have a mechanism for sensing their total sphingolipid content. MIPC and M(IP)₂C may be alternate fates of the ceramide available for sphingolipid synthesis.

Attempts to identify a function for M(IP)₂C lipids were inconclusive since RCD113 mutant cells grew as well as non-mutant cells in both complex and defined media and under a variety of stress conditions that are known to inhibit growth of cells that lack sphingolipids (21). RCD113 cells may not exhibit obvious phenotypes because the elevated level of MIPC may compensate for some functions of M(IP)₂C.

The limited available data indicate that, as long as S. cerevisiae cells make at least one type of sphingolipid, they are viable under non-stressful laboratory conditions. For example, mutant cells lacking mannosylated sphingolipids (5), but containing at least one species of IPC, grow well if given 10 mM calcium. However, these mutants have not been extensively tested for mutant phenotypes. S. cerevisiae cells lacking any sphingolipids grow under non-stressful conditions but are killed by stresses, indicating a role(s) for sphingolipids in stress resistance (21). Finally, no wild type fungus has been reported to exist without making sphingolipids. In addition to S. cerevisiae (26), Cryptococcus neoformans (27) and Candida albicans (28) are known to contain M(IP)₂C, whereas Neurospora crassa contains a major lipid with the composition (inositol-P)₂-ceramide (28). Histoplasma capsulatum makes several mannosylated sphingolipids but not M(IP)₂C when growing in the yeast phase (29, 30).

RCD113 cells were slightly more resistant to the growth inhibitory effect of calcium ions than were non-mutant YPH250 cells (Table II). Mutations in seven complementation groups, SCS1-7, give resistance to calcium-inhibited growth of a strain mutated in the csg2 gene (5). The scs mutant strains all have altered sphingolipid metabolism, and one complementation group, SCS1, is identical to the LCB2 gene (4), necessary for the first step in sphingolipid synthesis (Fig. 1). Based upon these results, it has been suggested that sphingolipid metabolism in S. cerevisiae is either regulated by Ca²⁺ and/or is required for Ca²⁺ homeostasis (5). The calcium resistance phenotype of RCD113 cells is consistent with these possibilities. Finally, because their synthesis has been conserved and because of their abundance, the M(IP)₂C lipids must perform an important function(s) that awaits identification.

M(IP)₂C synthase activity is found in membranes (33). Analysis of the Ipt1 protein sequence indicates seven predicted membrane-spanning domains (34) (Fig. 2). Several of these transmembrane domains correspond to predicted transmembrane domains in the Aur1 protein (Fig. 2), suggesting that these proteins have related membrane topologies. However, the predicted inside-outside orientation of some of the transmembrane domains are opposite in the two proteins, and experimental analysis will be required to verify the correct topology and existence of the transmembrane domains.

IPC synthase activity is strongly inhibited by the antifungal drug aureobasidin A, with 50% inhibition seen at a concentration of 0.2 nM (6). Because M(IP)₂C synthase and IPC synthase both use phosphatidylinositol as a substrate (Fig. 1) and because of their amino acid sequence similarity (Fig. 2), we examined M(IP)₂C synthase for inhibition by aureobasidin A. Our data demonstrate that M(IP)₂C synthase activity is strongly inhibited by the drug (Table I), but the enzyme is 2500-5000-fold less sensitive to inhibition than is IPC synthase activity. Further kinetic experiments with both enzymes, native and mutant forms (see below), along with analogues of aureobasidin, could be fruitful in pinpointing the catalytic sites in these enzymes.

Mutations in the AUR1 gene are known to give resistance to aureobasidin A. One resistant strain has Leu-137 replaced by Phe and His-157 replaced by Tyr (35), whereas another resistant strain has Phe-158 replaced by Tyr (36). Residues 157 and 158 are particularly interesting because they lie in a region whose amino acid sequence has been well conserved between the Aur1 and Ipt1 proteins (Fig. 2). This region is predicted to be a membrane-spanning domain and since there would be no strong evolutionary pressure to conserve the amino acid sequence simply to maintain the membrane-spanning function, the conservation of residues suggests some other shared function. We speculate, based upon the amino acid conservation, the predicted transmembrane domains, and the location of the aureobasidin A-resistant mutations, that the region of Aur1p from around residue 146 (residue 186 in Ipt1p) to around 259 (residue 302 in Ipt1p) is the catalytic core of the two enzymes that interacts with the hydrophobic portion of one or both lipid substrates. Furthermore, we speculate that the conserved non-membrane-bound region from around residue 300 in Aur1p (residue 323 in Ipt1p) to the C terminus of the protein (residue 409 in Ipt1p) recognizes the polar head-group in phosphatidylinositol. The corresponding regions in Ipt1p would perform similar functions.

The availability of the complete S. cerevisiae genomic sequence provides the first opportunity to identify all genes and proteins necessary for sphingolipid synthesis in any organism. Knowledge of the genes should enable characterization of the cognate proteins and enzymes and this information should expedite the task of understanding how sphingolipid synthesis is regulated in S. cerevisiae. Since it is not known how sphingolipid synthesis is regulated in any organism, insight gained from S. cerevisiae may provide clues to regulation in other eucaryotes. In addition, the level of sphingolipid metabolic intermediates, dihydrosphingosine, phytosphingosine and ceramide, thought to be signaling molecules that regulate expression of at least some of the many yeast genes whose promoter contains an STRE (stress response element), increases during heat stress by an unknown mechanism.² Knowledge of sphingolipid biosynthetic genes and proteins should facilitate studies to understand how heat stress regulates the level of these intermediates.