phytosphingosine as a specific inhibitor of growth and nutrient

Phytosphingosine as a specific inhibitor of growth and nutrientimport in Saccharomyces cerevisiae

Running Title: Specificity of Yeast Sphingolipids

Namjin Chung†*, Cungui Mao§, Joseph Heitman†‡, Yusuf A. Hannun¶, and Lina M. Obeid§||

Departments of †Pharmacology & Cancer Biology, ‡Genetics, Microbiology, and Medicine, andthe Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710

Departments of §Medicine, and ¶Biochemistry and Molecular Biology, Medical University ofSouth Carolina, Charleston, SC 29425

*Present address: Department of Biology, Massachusetts Institute of Technology, 77Massachusetts Ave., Cambridge, MA 02139

||To whom correspondence should be addressed: E-mail: [email protected]; Phone: (843) 876-5169; Fax: (843) 876-5172

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 23, 2001 as Manuscript M105653200 by guest on A

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SUMMARY

In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids

in the heat stress response through inhibition of nutrient import (1). In this study, we used a

combination of pharmacological and genetic approaches to determine which endogenous

sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous

phytosphingosine (PHS, 20 µM) or structurally-similar or metabolically-related molecules

including 3–ketodihydrosphingosine (KDS), dihydrosphingosine (DHS), C2-phytoceramide

(PHC), and stearylamine (STA), only PHS inhibited growth. Also, PHS was shown to inhibit

uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS.

Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude

that growth inhibition was caused by PHS itself. Using mutant yeast strains defective in various

steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2 strain,

which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of

ceramides and to be hypersensitive to PHS (5 µM), suggesting that PHS does not need to be

converted to PHC. The lcb4 lcb5 strain is defective in the conversion of PHS to PHS 1-

phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2 mutant strain, was

defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high

concentrations of DHS (40 µM) that inhibited the growth of an isogenic wild-type strain,

demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data

demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely

related and yet unidentified metabolite.

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INTRODUCTION

Certain sphingolipid metabolites including ceramide, sphingosine, and sphingosine 1-

phosphate have pleiotropic effects on cellular growth and proliferation. The yeast

Saccharomyces cerevisiae has emerged as an excellent model system for studying sphingolipid-

mediated signal transduction. First, compared to over 300 different kinds of sphingolipids found

in mammalian cells, there is only a limited number of sphingolipid species in the yeast, which

simplifies lipid analysis (2,3). Moreover, the basic structure, biosynthesis, and metabolism of

sphingolipids are well conserved between mammalian and yeast systems. Second, many yeast

genes in the sphingolipid biosynthetic and metabolic pathways have been cloned, providing

opportunities for studying the effects of endogenous sphingolipids using genetics tools. Lastly,

although this is not exclusive to sphingolipid studies, yeast genetics provides excellent tools to

identify and characterize components in signal transduction pathways (4-6).

Evidence for conservation of the sphingolipid signaling pathway in yeast comes from

several studies. These include demonstrating that D-erythro ceramide inhibited yeast cell growth

in liquid culture and activated a protein phosphatase 2A that could be inhibited by okadaic acid

(7). Later, Nickels et al. showed that ceramide inhibited yeast cell growth by arresting cell cycle

at G1 phase, and that ceramide-activated protein phosphatase (CAPP) is composed of three

protein phosphatase 2A components encoded by the TPD3, CDC55, and SIT4 genes (8).

More recent studies showed that upon heat stress, cellular levels of DHS and PHS rapidly

increase several fold within 10 to 20 minutes, then slowly return to basal levels over 30 to 60

minutes (9,10). The levels of ceramide also increased several fold but with slow kinetics

corresponding to 60 – 120 minutes. On the other hand, the levels of complex sphingolipids show


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little if any change in response to heat stress. This increase in sphingoid bases derives primarily

from de novo synthesis initiated by serine palmitoyltransferase. More recently, DHS and PHS

were shown to inhibit yeast growth by inhibiting tryptophan import (11). These studies suggest

important signaling and regulatory functions for sphingoid bases, their phosphates and/or

ceramide, but they do not provide insight into which specific sphingolipids are involved in what

specific cellular functions.

In this report, we set out to determine which sphingolipid (among various sphingolipid

species including 3-KDS, DHS, PHS, PHC, and PHS 1-phosphate) is the primary inducer of

growth inhibition. Through pharmacological and genetic approaches, we found that DHS

inhibits growth, but it needs to be first converted to PHS to do so. PHS, on the other hand, does

not need to be converted to PHS 1-phosphate or PHC, and PHS itself is sufficient to inhibit

growth. Our data demonstrate that PHS inhibits growth in a specific manner, suggesting that this

sphingoid base may play a specific role in growth regulation of Saccharomyces cerevisiae and

that it targets a specific pathway responsive only to PHS and not to any of its known precursors

or subsequent metabolites.


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EXPERIMENTAL PROCEDURES

Strains––All the yeast strains used in this study are isogenic to a normal lab strain JK9-3d

(MATa trp1 his4 leu2-3,112 ura3-52 rme1 HMLa; 12): NC58-1a (TRP1), NC55 (syr2 ::G418R),

NC75 (elo2 ::G418R), NC75-3 (elo2 ::G418R SEL1-1), NC78 (TRP1 elo2 ::G418R), NC127-

1b (lcb4 ::G418R), NC128-1c (lcb5 ::G418R), NC129 (lcb4 ::G418R lcb5 ::HygR), CM1

(ysr2 ::G418R ysr3 ::URA3; 13), and JS16 (bst1 ::G418R; 14).

Media and Genetic Manipulations––Recipes for media and yeast genetic methods followed

standard protocols (15). Yeast transformation followed the protocol developed by Gietz et al.

(16). Gene disruptions were carried out as described previously (17,18). In short, open reading

frames were replaced by the PCR products consisting of the G418R or HygR gene cassettes

flanked by 43-bp sequence homology to targeted genes on both sides of an open reading frame.

Each gene disruption was confirmed by PCR, which was designed to amplify the specific

chimeric junction of the target gene and the G418R or HygR cassette.

Preparation of Sphingolipid Derivatives––PHS and STA were purchased from Sigma, and

DHS from Biomol. KDS and C2-PHC were kind gifts from Dr. Alicja Bielawska (Medical

University of South Carolina, Charleston, SC). The quality of these sphingolipid derivatives was

evaluated by thin-layer chromatography. These lipids were dissolved in ethanol as 20 mM stock

solutions and stored at –20 ˚C in the dark as previously described (19). They were warmed to

30˚C before use. For solid medium, sphingolipid derivatives were added to the medium that had

been autoclaved and cooled down to 50 ˚C, together with 0.05% Tergitol (Type NP-40; Sigma)

to help even distribution of lipids in solid agar (20). For liquid medium, warmed-up stock

solutions of the lipids were directly added to medium, vigorously shaken, and equilibriated


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before use. Tergitol did not affect the biological activities of sphingolipid derivatives in liquid

culture, and the results shown here represent experiments with and without the use of Tergitol.

Measurement of Yeast Growth––Measurement of yeast growth was carried out as described

previously (19). Briefly, in liquid culture an overnight culture of cells at exponential growth

phase was diluted into fresh medium containing indicated lipids or ethanol (0.1% at the final

concentration) as a control. While incubating at 30 ˚C, with vigorous shaking, growth was

monitored at a given time by measuring absorbance at 600 nm (A600), and the numbers were

converted into cell density (cells/ml), using a pre-configured conversion table, when it is

necessary. On solid medium, a small amount of cells from a single colony were streaked by

three successive uses of toothpicks, or exponential-phase cells in liquid culture were plated onto

medium. Plates were incubated at 30 ˚C for 2 days and photographed for record.

Nutrient Import Assay––Uptake of amino acids or uracil was measured as previously

described (21), with the following modifications. Cells were grown to early-to-mid log phase

(A600=0.3-0.5) in YPD media, harvested, and resuspended in a modified buffer (10 mM sodium

citrate, pH 4.5 plus 2% glucose), omitting 20 mM ammonium sulfate from the original recipe.

Cell density was adjusted to A600=0.3-0.4, for which the amount of substrates uptaken by cells at

any given sampling does not exceed 10% of total available substrates to avoid possible saturation

of uptake reactions. Sphingolipid derivatives or 0.1% ethanol were added, and the uptake was

initiated with the addition of radiolabeled amino acid or uracil at the final activity of 1 µCi/ml.

Two milliliters of assay cultures were withdrawn at the indicated times, and placed immediately

into ice-chilled tubes to stop uptake reactions. One milliliter was measured for absorbance at

600 nm; another was filtered through a pre-equilibrated, 0.45-µm Durapore membrane filter

(Millipore), extensively washed three times with two volumes of the wash buffer (10 mM


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sodium citrate, pH 4.5 plus 2 mM corresponding substrate), air-dried, and quantified by liquid

scintillation counting. Counted values were normalized to cell density, and expressed as

cpm/A600 for total uptake activity.

The reason for omitting ammonium sulfate from the original assay buffer is as follows.

In the assay buffer containing ammonium sulfate, 20 µM PHS failed to show specific inhibition

of nutrient uptake, and it required several fold higher concentrations of PHS to inhibit uptake, as

shown in a previous study (11). However, this inhibition was non-specific because other

structural homologs, including STA, also inhibited uptake activities at such high concentrations.

When ammonium sulfate was omitted from the assay buffer, low concentrations of PHS (20 µM

or lower) inhibited uptake activities in a specific manner.

Thin-layer Chromatography (TLC)–– For Fig. 4A, cells grown to log phase were harvested

and resuspended in synthetic complete (SC) medium at 3 x 107 cells/ml. Cells were pretreated

with 150 µM fumonisin B1, or mock-treated with water for 1 h before being labeled with with D-

erythro-[4,5-3H] dihydrosphingosine (5 µCi/ml) for 30 min. For Fig. 5A, cells were resuspended

in SC-Ser medium supplemented with [3H] serine (American Radiochemicals; 20 µCi/ml) and

incubated for 6 h. Sphingolipids were extracted and resolved by TLC as described previously

(22). In Fig. 5A, extracted lipids were subjected to base hydrolysis to remove non-sphingolipid

serine-labeled molecules before TLC analysis. The bands of PHS, DHS, and KDS were

identified by comparing their Rf values to known standards in several different solvent systems.

Radioactive bands of sphingolipids and their derivatives were visualized by a PhosphorImager

(Molecular Dynamics) after exposure to a tritium screen.

Immunoblotting—The wild-type JK9-3d strain was transformed with a low-copy plasmid

containing either the GAP1 or the TAT2 gene that is tagged with the hemagglutinin epitope at its


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carboxy-terminus. Transformed cells were grown to an early-to-mid log phase, treated with

indicated lipids for 2 hours, and total proteins were extracted, quantitated, and immunoblotting

was performed as described previously (1). The permease proteins were detected by using

mouse monoclonal antibody against hemagglutinin (Corvance, 1:1,000).


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RESULTS

Specificity of Growth Inhibition by PHS––Previous studies suggested that growth inhibition

by sphingolipids and their derivatives is conserved in S. cerevisiae as well as in mammalian cells

(7,14), and later it was shown in liquid culture that PHS inhibits yeast growth (11,23). An

additional study showed that yeast sphingolipids are necessary for heat-induced down regulation

of nutrient permeases (1). However, these studies did not resolve questions regarding the

specificity of growth inhibition by sphingolipids, and what are the endogenous mediators of

growth suppression. This is further complicated by the interconversion of these lipids. For

example, PHS can be further converted to other sphingolipid species such as PHC, PHS 1-

phosphate, or complex sphingolipid species including IPC, MIPC, and M(IP)2C (Fig. 1).

Therefore, even when cells are treated with PHS, it is difficult to determine whether growth

inhibition is caused by PHS or by other sphingolipid species that are converted from PHS (or

precursors of the pathway). Moreover, DHS was claimed to have an equal or similar degree of

growth inhibitory potential as PHS (11). We therefore set out to determine whether PHS itself or

other sphingolipid species inhibits growth.

We first confirmed and extended previous observations by showing that PHS inhibits

growth of a normal lab strain JK9-3d in both liquid and solid media (Fig. 2A). In fact, cells in

liquid medium were more sensitive to PHS than those in solid medium, such that 15 to 20 µM

PHS was required to inhibit growth in solid medium whereas 10 to 15 µM PHS was sufficient to

attain a similar degree of growth inhibition in liquid medium (data not shown). This range of

concentrations was comparable to the ranges of sphingosine and ceramide concentrations used

for mammalian studies. On YPD medium containing 20 µM PHS, plating of more than 104 cells


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per 9-cm plate failed to yield any viable colonies; in liquid medium, more than 107 cells/ml still

showed immediate growth inhibition in response to 20 µM PHS (data not shown). This

demonstration of growth inhibition by PHS in diverse experimental conditions enabled us to use

multidisciplinary approaches, especially the genetic approach, to determine the specificity of

PHS.

PHS can be converted to PHC by ceramide synthase in vivo (24), raising a possibility that

apparent growth inhibition by PHS could be actually due to PHC. Also, in mammalian cells,

ceramide plays an important role in cell cycle regulation, apoptosis, and cellular senescence

(25,26). Therefore, we decided to test whether PHC has an equivalent function in yeast cells

and, if so, whether PHC has a comparable potency to PHS. For this purpose, we used C2-PHC

because we reasoned C26-PHC, a natural PHC to yeast, will not be efficiently delivered into cells

due to strong hydrophobicity, and short-chain ceramides such as C2- and C6-ceramide have been

commonly used in mammalian studies. C2-PHC (20 µM) had little effect on yeast growth (Fig.

2B).

We then tested other sphingolipid derivatives that are structurally similar and/or

metabolically related. At concentrations up to 100 µM, KDS and STA did not inhibit growth

(Fig. 2B). It was noteworthy that DHS, at the 20 micromolar concentration, showed weak but

some degree of growth inhibitory effects (see below).

Specificity of Nutrient Import Inhibition by PHS––In a previous study, PHS was shown to

inhibit the growth of tryptophan-auxotrophic yeast strains, and to inhibit tryptophan import (11).

Thus, it was hypothesized that a primary cause of growth inhibition by PHS is inhibition of

tryptophan import activities. Again, it was not shown whether inhibition of tryptophan import

was specific for PHS; to the contrary, both PHS and DHS were suggested to be equally active in


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inhibiting tryptophan import (11). We believe this is an important issue that needs to be resolved

because it could help determine whether such inhibition involves specific mechanisms.

Therefore, we measured tryptophan import activity in the presence of PHS or various

analogs that were used for studying the specificity of growth inhibition. In this test, only PHS

inhibited tryptophan import whereas other analogs including DHS, KDS, C2-PHC, and STA (20

µM each) did not inhibit tryptophan import (Fig. 3A, left panel). The inhibition of tryptophan

uptake activity by PHS primarily reflected the decrease in the levels of the general amino acid

permease (Gap1) rather than a tryptophan-specific permease (Tat2; Fig. 3B, right panel). Again,

the decrease in the permease protein was specific for PHS, compatible with the decrease in the

uracil permease levels by PHS (1).

While experimenting with various different auxotrophic strains for PHS sensitivity, we

found that, in addition to tryptophan-auxotrophic strains (trp1), certain other auxotrophs are also

sensitive to PHS. At 20 µM PHS, any tryptophan-prototrophic strains (TRP1), regardless of

other auxotrophic status, grew as well as they did in control medium (Fig. 3B). Leucine-

prototrophic strains (LEU2) were also found to be somewhat resistant to PHS. At 60 µM PHS,

the TRP1 strains became partially sensitive and the LEU2 strains became as sensitive as

auxotrophic strains. The TRP1 LEU2 strains were more resistant to PHS than the TRP1 leu2 and

trp1 LEU2 strains. However, all auxotrophic strains showed some degree of sensitivity to PHS,

and only the TRP1 LEU2 HIS4 fully prototrophic strains showed full resistance to 60 µM PHS.

From these observations, we concluded that the greater the auxotrophic requirement of a strain

is, the more sensitive it is to PHS.


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These results also suggested that PHS inhibits the import of multiple nutrients. To test this

idea, we measured nutrient import activities for leucine, tryptophan, histidine, and uracil in the

presence of 20 µM PHS (Fig. 3C). PHS inhibited the import of all four nutrients.

DHS Needs to Be Converted to PHS to Inhibit Growth––To assess the physiological

significance of the above pharmacological studies, we next examined the specificity of growth

inhibition by genetically modulating the levels of cellular PHS. As shown in Figure 1,

exogenous DHS could be converted to PHS, PHS to PHC, and PHC to PHS. The possibilities of

such interconversions raise the question as to which endogenous sphingolipid derivative

mediates the growth-inhibitory effects of exogenous PHS.

Because we saw that at higher concentrations (40-60µM), DHS inhibited growth to some

extent, we first tried to resolve whether DHS is a bona fide inhibitor of yeast growth, or whether

its conversion to PHS is necessary for growth inhibition. To answer this question, we used a

mutant strain that is defective in the conversion of DHS to PHS. In previous studies, the syr2

mutant strain was shown to be defective in the C-4 hydroxylation of DHS and dihydroceramide

(DHC) to PHS and PHC, respectively, and the SYR2 gene was proposed to encode a lipid

hydroxylase (27,28). Therefore, we first determined whether the syr2 mutant strain shows

defects in the conversion of DHS to PHS in the JK9-3d strain background. When we tried to

analyze sphingolipid profiles of the syr2 and isogenic wild-type strain using TLC after [3H]-

DHS labeling, we could hardly detect differences between the wild-type and the syr2 strains

(Fig. 4A, lanes 1 and 3). The levels of DHS in the syr2 strain seemed to be higher than in the

wild-type strain. In these studies, we could not detect free PHS, probably due to its rapid

conversion to PHC or due to lack of direct hydroxylation of DHS. Therefore, we attempted to

resolve this issue and trap PHS by utilizing fumonisin B1, an inhibitor of ceramide synthase


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(24,29). Under these conditions, the wild-type strain was capable of converting DHS to PHS and

accumulated PHS in the presence of fumonisin B1, whereas the syr2 strain failed to accumulate

PHS. In other words, the use of fumonisin B1 enabled us to detect the accumulation of PHS in

the wild-type strain, but not in the syr2 strain (Figs. 1 and 4A, lanes 2 and 4). Thus, syr2 is

defective in hydroxylation of exogenous DHS to PHS.

If DHS is by itself sufficient for growth inhibition, this should be the case regardless of the

status of the SYR2 gene. On the other hand, if DHS needs to be first converted to PHS to inhibit

growth, DHS would inhibit the growth of only wild-type cells but not syr2 mutant cells.

Indeed, whereas PHS (20 and 40 µM) inhibited both the wild-type and syr2 mutant strains,

DHS (40 µM) only inhibited growth of the wild-type strain and failed to inhibit growth of the

syr2 strain (Fig. 4B). We have found that DHS is as efficiently as or better taken up than PHS

by yeast cells and comparably metabolized into complex sphingolipids (data not shown). It is

therefore unlikely that the requirement for high concentration of DHS for growth inhibition was

due to slow internalization of DHS compared to PHS. Tetrad analysis of a syr2 /SYR2

heterozygous diploid strain showed co-segregation of the syr2 allele with resistance to 40 µM

DHS. Also, when the syr2 mutant strain was restored with a single copy of the wild-type SYR2

gene, it became as sensitive to DHS as the original wild-type strain (data not shown). In

conclusion, DHS does not by itself inhibit growth and requires conversion to PHS by Syr2p.

PHS Does Not Need to Be Converted to PHC and Is Sufficient for Growth

Inhibition––Next we attempted to distinguish between PHS and PHC. PHS can be converted to

PHC by ceramide synthase, and PHC can be reverted to PHS by ceramidase (24,30). Treating

cells with an excessive amount of PHS could shift the equilibrium toward PHC making it

difficult to distinguish the effects of PHS from those of PHC. In the above section, we showed


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that C2-PHC only weakly inhibited growth, but this data was not conclusive since C2-PHC may

not be an adequate substitute for the long chain natural PHC.

To avoid using natural C26-PHC, which might cause solubility and permeability problems,

we looked for other ways to differentiate the effects of PHS from those of PHC. The production

of PHC requires two substrates: PHS and C24-or C26-very-long-chain fatty acid (VLCFA; Fig. 1).

Therefore, if the supply of VLCFA is blocked, there will be less production of PHC from PHS

even when PHS is present in excess. VLCFA is the result of serial addition of acetyl groups to

more commonly found, normal-length fatty acids like palmitic acid (C16). The key steps in this

process involve the conversion of C22- to C24- VLCFA by the ELO2 gene product, and C24- to

C26- VLCFA by the ELO3 gene product (Fig. 1; 22). When we labeled the elo2 mutant strain

with [3H]-serine and analyzed by TLC, we could indeed observe the increase in the levels of

PHS and its upstream precursors including DHS and KDS and the decrease in the levels of PHC

and complex sphingolipids (Fig. 5A)

If PHS is by itself capable of inhibiting growth, then the growth of elo2 mutant cells will

be inhibited by PHS treatment. On the other hand, if PHS needs to be converted to PHC to

inhibit growth, then elo2 mutant cells will be resistant to PHS. In fact, the elo2 mutant strain

was hypersensitive to PHS such that its growth was inhibited by only 5 µM PHS, a concentration

at which the growth of wild-type cells was unaffected (Fig. 5B). Notably, the elo2 mutant

strain grew slowly even without PHS treatment (data not shown), probably due to the

accumulation of endogenous PHS.

The sensitivity of the elo2 mutant strain to PHS was tightly linked to the mutant allele of

the TRP1 gene (trp1): the elo2 trp1 strain was sensitive to PHS but the elo2 TRP1 strain was

resistant (Fig. 6B). Because these two elo2 strains showed essentially identical TLC profiles


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including PHS levels, and their only difference was the status of TRP1 allele (TRP1 vs. trp1), we

concluded that the wild-type TRP1 allele enabled the elo2 mutant strain to overcome

deleterious effects of PHS accumulation. Also, when the elo2 trp1 strain was grown on

medium containing 5 µM PHS plus excess tryptophan, it became as resistant to PHS as the elo2

TRP1 strain (Fig. 5B). These data confirm that PHS inhibits growth of the trp1 mutant strain by

inhibiting tryptophan import and further support the hypothesis that the hypersensitivity of elo2

cells is due to accumulation of endogenous PHS since it was reversed by excess tryptophan,

indicating a similar mechanism as wild type cells.

Interestingly and probably due to selective pressure stemming from growth defects,

spontaneous suppressor mutations frequently arose in the elo2 trp1 strain. One of these

suppressors (SEL1-1+ for suppressor of elo2 ) was a dominant mutant that restores the levels of

PHS and complex sphingolipids to normal levels (Fig. 5A) and cure PHS hypersensitivity (Fig.

5B). The data therefore suggest that the reduction in the levels of endogenous PHS to a normal

level in the SEL1-1+ suppressor mutant relieved the PHS hypersensitivity resulting from the

elo2 mutation.

PHS 1-phosphate Does Not Inhibit Growth––The conclusion from a series of observations

suggests that sphingosine 1-phosphate, but not sphingosine itself, inhibit yeast growth. First, the

dpl1 mutant strain accumulates sphingosine 1-phosphate and shows growth inhibition when

treated with sphingosine (14). Second, the overexpression of the YSR2 gene, which encodes for

sphingosine 1-phosphate phosphatase, in the dpl1 mutant strain reverses sphingosine 1-

phosphate accumulation and restores wild-type growth (13). Last, the lcb4 dpl1 double

mutant strain does not accumulate sphingosine 1-phosphate and is resistant to sphingosine (31).

A consensus that can be drawn from these data is that any strain that allows the accumulation of


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sphingosine 1-phosphate is sensitive to exogenous sphingosine. One may assume that PHS, too,

needs to be converted to PHS 1–phosphate to inhibit growth.

We therefore set out to determine the differences between PHS and PHS 1-phosphate.

When the SYR2 gene is deleted, DHS can still be converted to DHS 1-phosphate. Because the

syr2 mutant strain was resistant to DHS, DHS 1-phosphate does not appear to inhibit growth.

Does PHS then inhibit growth via PHS 1-phosphate? We next tested if PHS 1-phosphate

mediates the effects of PHS in two independent experiments, using mutant yeast strains defective

in two enzymes involved in PHS metabolism: sphingoid base kinase and phosphorylated

sphingoid base lyase. There are two sphingoid base kinase isoenzymes encoded by two highly

homologous genes, LCB4 and LCB5 (31). The lcb4 lcb5 double mutant strain, which cannot

convert PHS to PHS 1-phosphate, was as sensitive to PHS as a wild-type strain (Table I). This

demonstrates that PHS does not need to be converted to PHS 1-phosphate to inhibit growth. In a

second experiment, we tested PHS sensitivity of the dpl1 mutant strain, which lacks

phosphorylated sphingoid base lyase. The dpl1 mutant strain did not show hypersensitivity to

PHS, suggesting that the accumulation of PHS 1-phosphate does not lead to growth inhibition.

In short, unlike the case with sphingosine, PHS does not need to be converted to PHS 1-

phosphate and by itself inhibits growth.


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DISCUSSION

In this report, we demonstrated specificity of growth inhibition by PHS through several

approaches. PHS inhibited yeast growth at a low micromolar concentration range. It was

specific to PHS, in that other metabolically and structurally related compounds did not inhibit

growth. Using various mutants involved in sphingolipid biosynthesis and metabolism, we

demonstrated that DHS needs to be converted to PHS, and PHS does not need to be converted to

PHC or PHS 1-phosphate to inhibit growth. Therefore, PHS is a likely bona fide growth-

inhibitory sphingolipid derivative.

In addition to the above conclusion, the data presented in this report also suggest that de

novo synthesis of PHS is important in growth inhibition. The gene products of both SYR2 and

ELO2 are involved in de novo sphingolipid synthesis, and the data drawn from the mutant strains

defective in these genes support the conclusion that the accumulation of PHS via de novo

synthesis results in growth inhibition. Previously, it was suggested that heat stress signaling be

also mediated via de novo synthesis of sphingoid bases (9,10). Also in mammalian cells, de

novo synthesis of ceramide has been suggested to be important in apoptosis (32-35). Despite our

data, we cannot rule out the possibility that the generation of PHS by hydrolysis of other

sphingolipids such as PHC, IPC, and others may also play a role in growth inhibition. In our

data, C2-PHC did not inhibit growth. In addition, labeled C2-DHC in S. cerevisiae was rapidly

internalized, metabolized, and incorporated into complex sphingolipids (G. Jenkins and Y.

Hannun; unpublished observations). Thus, C2-PHC is also probably internalized, converted to

PHS by ceramidases (36,37), and incorporated into complex sphingolipids. Therefore C2-PHC

does not likely cause accumulation of PHS and consequently does not play a role in PHS-


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mediated growth inhibition. On the contrary, as in mammalian cells, both biosynthetic and

catabolic pathways to generate PHS may be important for growth inhibition, differing in

temporal order of PHS generation and/or cellular context (26).

Considering the structural similarities between PHS and other metabolically related

molecules including PHC, DHS, and KDS, the specificity of growth inhibition by PHS is

remarkable. PHS differs from PHC in that the amino group at the C2 position is acylated in

PHC, and it differs from DHS in that the hydroxyl group at the C4 position is absent in DHS

(Fig. 2). Computer-simulated three-dimensional modeling of PHS showed that the amino group

at the C2 position and the hydroxyl group at the C4 position are clustered in a close proximity at

one end of the hydrocarbon chain (data not shown). It is likely that PHS is embedded in

membrane bilayers with these functional groups protruding out of the membrane. The combined

amino and hydroxyl groups could provide an interface to other macromolecules that relay

growth-inhibitory signals, and the abolishment of these features could result in failure to recruit

signaling macromolecules.

We used genetics methodology to demonstrate the specificity of PHS. We believe this kind

of approach should be more extensively utilized in many other studies requiring the specificity of

molecular actions. Because of the dynamics of many signaling molecules in context of

metabolism, it is not guaranteed whether the biological effects of a particular molecule are really

originated from itself. The combination of pharmacological and genetic tools could eliminate

this kind of doubts.


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Acknowledgements––We thank Jon Y. Takemoto, Charles E. Martin, Alicja Bielawska, Per

Ljungdahl, and Anja Schmidt for reagents and discussions. Supported by the following grants:

AG16583 (L.M.O.), GM43825 (Y.A.H.), HL43707 (Y.A.H.), and AI41937 (J.H.). N.C. was

supported in part by a predoctoral fellowship from the Korea Foundation for Advanced Studies.

J. H. is an Associate Investigator of the Howard Hughes Medical Institute and a Burroughs

Wellcome Scholar in Molecular Pathogenic Mycology.


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FOOTNOTES

1The abbreviations used are: PHS, phytosphingosine; DHS, dihydrosphingosine or sphinganine;

KDS, 3-keto-dihydrosphingosine; PHC, phytoceramide; DHC, dihydroceramide; STA,

stearylamine; IPC, inositol phosphoceramide; MIPC, mannose inositol phosphoceramide;

M(IP)2C, mannose diinositolphosphoceramide; PE, phosphatidylethanol; PC,

phosphatidylcholine; PI, phosphatidylinositol; CAPP, ceramide-activated protein phosphatase;

TLC, thin-layer chromatography. VLCFA very long chain fatty acids.


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FIGURE LEGENDS

Fig. 1. Biosynthesis and degradation of sphingolipids.

Fig. 2. Specificity of growth inhibition by PHS. A, The yeast strain JK9-3d was grown to

mid-log phase, and approximately 200 cells were plated onto YPD medium containing 20 µM

PHS or 0.1% ethanol (Control). Plates were incubated at 30 ˚C for 2 days (left). The same cells

grown in YPD liquid culture. Growth was monitored every 2 hours by measuring absorbance at

600 nm (A600; right). B, The JK9-3d strain was streaked onto YPD containing 20 µM of

indicated lipids or 0.1% ethanol (Control) and grown at 30 ˚C for 2 days. In the sphingolipid

biosynthetic pathway, KDS is converted to DHS, DHS to PHS, and PHS to PHC. C2-PHC is a

cell-permeable synthetic PHC, and STA is a long chain amine that is structurally similar to

sphingoid bases. Structures of sphingoid bases are shown.

Fig. 3. Specificity of nutrient import inhibition by PHS. A, The specificity of PHS in

inhibiting tryptophan import activity (left panel) and decrease in the levels of the general amino

acid permease (right panel) was demonstrated by comparison with other metabolically and/or

structurally related lipid molecules. 20 µM of indicated lipids were used: 0.1% ethanol as a

control (open circle), PHS (closed triangle), DHS (closed rectangle), KDS (closed diamond), C2-

PHC (open rectangle), and STA (open diamond). B, Correlation between PHS sensitivity and

auxotrophic status of yeast strains. The JK9-3d strain (trp1 his4 leu2) was mated to an isogenic

strain with prototrophic markers (TRP1 HIS4 LEU2) and an opposite mating type. The resulting


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diploid was sporulated and tetrads were dissected to obtain strains with genotypes as shown in

the figure. PHS sensitivity was tested by streaking the strains onto YPD medium containing 20

µM or 60 µM PHS. Photographs were taken after incubation at 30 ˚C for 2 days. C, The JK9-3d

strain was grown to mid-log phase, and nutrient import activities for tryptophan, leucine,

histidine, and uracil were measured in the presence (open circles) or absence (closed circles) of

20 µM PHS

Fig. 4. DHS needs to be converted to PHS to inhibit growth. A, The syr2 mutant is

defective in the conversion from DHS to PHS. An equal number of cells were labeled with [3H]-

DHS with or without pretreatment with 150 µM fumonisin B1, and sphingolipids were extracted

and resolved by TLC. Without fumonisin B1 treatment, PHS is not accumulated to visible levels

and it is probably quickly metabolized away as soon as it is produced. B, DHS needs to be

converted to PHS to inhibit growth. The syr2 /SYR2 heterozygous strain was sporulated and

tetrads were dissected to obtain the wild-type (SYR2) and the syr2 sibling mutant strains. These

sibling strains were streaked onto YPD medium containing either 40 µM DHS or PHS and

grown at 30 ˚C for 2 days. The picture shown here is a representative of ten tetrad analyses.

Fig. 5. PHS does not need to be converted to PHC to inhibit growth. A, PHS accumulates in

the elo2 mutant strain. The JK9-3d strain (ELO2) and isogenic elo2 and elo2 SEL1-1 strains

were grown to an early log phase in SC (synthetic complete) medium, changed to SC-Ser

medium supplemented with [3H] serine (20 µCi/ml), incubated for 6 hours, and sphingolipids

were extracted and resolved by TLC. B, PHS does not need to be converted to PHC and is

sufficient to inhibit growth. The JK9-3d strain and isogenic elo2 and elo2 SEL1-1 strains


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were streaked onto YPD medium containing 5 µM PHS with or without 100 µg/ml tryptophan,

and incubated at 30 ˚C for 2 days

Fig. 6. The elo2 TRP1 strain accumulates PHS but is resistant to PHS. The TRP1 and

elo2 TRP1 strains isogenic to the JK9-3d strain were analyzed for sphingolipid profile (A) as in

Figure. 5, and for resistance to PHS (B) as in Figure 3.


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TABLE

Table I. Summary of PHS phenotypes of sphingolipid metabolic mutant strains.

Indicated mutant strains were tested and showed normal sensitivity (S) to PHS, except the elo2

strain which were hypersensitive (HS) to PHS. The syr2 strain is resistant to 40 µM DHS (see

text).

Defects Genotype PHS Phenotype

WT S

DHS à PHS syr2 S

PHS à PHC elo2 HS

PHS à PHS 1-P lcb4 S

PHS à PHS 1-P lcb5 S

PHS à PHS 1-P lcb4 lcb5 S

PHS 1-P à PHS ysr2 S

PHS 1-P à PHS ysr3 S

PHS 1-P à PHS ysr2 ysr3 S

PHS 1-P à degradation dpl1 S


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Ser + Palmitoyl-CoA

KDS

DHS

PHS

PHC

DHS 1-Pi

Palmitaldehyde+

Ethanolamine Pi

Lcb4, Lcb5

Ysr2, Ysr3

Dpl1

Lcb1+Lcb2

Syr2

Tsc10

PHS 1-PiLcb4, Lcb5

Ysr2, Ysr3

C14

C24

C26

C16

Elo1

Elo2

Elo3

C22


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PHS

DHS

STA

PHC2

Control

KDS

PHS

Control

0

1

2

0 5 10 15 20hrs

A60

0

A B

PHS

Control

HOCH2

O

NH3+

HOCH2

OH

NH3+

NH3+

HOCH2

OH

OH

HN

HOCH2

OH

OH

O

H3+N

KDS

DHS

PHS

C2-PHC

STA


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20 µM

60 µM

A

HIS4

his4leu2trp1ura3

LEU2

TRP1

HIS4LEU2

HIS4TRP1

LEU2TRP1

HIS4LEU2TRP1

HIS4LEU2TRP1URA3

0

100

200

300

400

0 25 50 75 100

Tryptophan

0

100

200

300

400

0 25 50 75 100

Leucine

0

100

200

300

400

0 25 50 75 100

Histidine

0

2

4

6

8

0 25 50 75 100

Uracil

0

100

200

300

400

0 25 50 75 100

Tryptophan

B

Tot

al U

ptak

e (c

pm/A

600,

x1,

000)

Time (min)

Time (min)

Tot

al U

ptak

e (

cpm

/A60

0, x

1,00

0)

C

PHS

Con

trol

PH

S

DH

S

ST

A

Gap1

Tat2

Fur4


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DHS

[ PHS ]

A B KDS

Growth

syr2

40 µM DHS 40 µM PHS Control

syr2

syr2 SYR2

SYR2

PHSPE

Sphingo-lipids

PCPI

syr2SYR2

- + +-

DHS

Fum B1

1 2 3 4


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DHS

[ PHS ]

PHC

A B

Growthelo2

5 µM PHS 5 µM PHS + Trp

ELO2elo2

SEL1-1

elo2

ELO

2

elo

2elo

2S

EL1-1

KDSDHS

PHS

Sphingo-lipids

1 2 3


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5 M PHSELO2 trp1

elo2 trp1

ELO2 TRP1

elo2 TRP1

B

76

ELO

2

elo

2

KDS

DHS

PHS

Sphingo-lipids

SolventFront

Origin

TRP1

A


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Namjin Chung Chung, Cungui Mao, Joseph Heitman, Yusuf A. Hannun and Lina M. ObeidSaccharomyces cerevisiae

Phytosphingosine as a specific inhibitor of growth and nutrient import in

published online July 23, 2001J. Biol. Chem.

10.1074/jbc.M105653200Access the most updated version of this article at doi:

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