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Journal of Bacteriology, February 1999, p. 1134-1140, Vol. 181, No. 4
Department of Biochemistry and Lucille P. Markey Cancer Center, University of Kentucky Medical Center,
Lexington, Kentucky 40536-0298
Received 18 May 1998/Accepted 30 November 1998
Sphingolipid long-chain bases and their phosphorylated derivatives,
for example, sphingosine-1-phosphate in mammals, have been implicated
as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we
present a simple and sensitive procedure for analyzing and quantifying
long-chain-base phosphates in S. cerevisiae cells. Our data
show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is
present at a low but detectable level in cells grown on a fermentable
carbon source at 25°C, while dihydrosphingosine-1-phosphate (DHS-1-P)
is only barely detectable. Shifting cells to 37°C causes transient
eight- and fivefold increases in levels of PHS-1-P and DHS-1-P,
respectively, which peak after about 10 min. The amounts of both
compounds return to the unstressed levels by 20 min after the
temperature shift. These data are consistent with PHS-1-P and DHS-1-P
being signaling molecules. Cells unable to break down long-chain-base
phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44°C heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3
single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as
signals for resistance to heat stress. Our procedure should expedite
experiments to determine how the synthesis and breakdown of these
compounds is regulated and how the compounds mediate resistance to
elevated temperature.
Sphingoid long-chain bases are
gaining appreciation for their role in cellular signaling processes.
Sphingosine, the primary long-chain base found in mammalian
sphingolipids, was first noted for its mitogenic activity (reviewed in
references 26 and 27), at least
some of which is mediated by sphingosine-1-phosphate (SPP)
(31), a phosphorylated derivative of sphingosine. SPP has
been shown also to inhibit cell motility and invasiveness of tumor
cells (1, 25, 28, 30). Recently SPP and
sphingosylphosphorylcholine have been found to bind G-protein-coupled
receptors that may play roles in regulating heart rate, the oxidative
burst, neurite retraction, and platelet activation (reviewed in
reference 32). Most of these observations have been
made for cultured cells, and it remains to be determined how
physiologically important SPP and related molecules are in whole
animals. To begin to assess the physiological role of long-chain-base
phosphates in regulating cellular processes, we have determined which
compounds are present before and during a heat shock in wild-type
Saccharomyces cerevisiae cells and in mutant cells defective
in breakdown of these compounds.
Studies of mutant S. cerevisiae strains lacking
sphingolipids indicated an essential role for these lipids in resisting
heat, osmotic, and low-pH stresses (22). Further analysis
during heat stress indicated that sphingolipids were not necessary for
induction of the major heat shock proteins but were necessary for
accumulation of trehalose (6), a disaccharide known to be
induced by heat stress and to be necessary for full thermoprotection of
log-phase (5) and stationary-phase (8) cells. In
addition, heat shock induced a 2- to 3-fold transient increase in the
concentration of C18-dihydrosphingosine (DHS) and
C18- phytotosphingosine (PHS), more that a 100-fold
transient increase in C20-DHS and C20-PHS, and
a stable 2-fold increase in ceramide containing C18-PHS and a 5-fold increase in ceramide containing C20-PHS (6,
12, 29). Finally, it was shown that treatment of cells with DHS or PHS induced transcription of a reporter gene containing either the
TPS2 promoter or seven copies of the global response
element, STRE. Transcription of TPS2, encoding a
subunit of trehalose synthase, has been shown to be induced by several
stresses including heat, and the promoter element responsible for these
responses has been shown to be STRE (9, 13, 18).
These results suggest that DHS, PHS, or a derivative thereof can act as
a signal to induce transcription.
To further characterize the function of long-chain-base phosphates, we
have developed, as described in this report, a quantitative method for
their analysis in S. cerevisiae cells. Using this method, we
show for the first time that S. cerevisiae cells have a low basal level of PHS-1-P and a barely detectable level of DHS-1-P. Heat
shock induces a rapid but transient increase in the concentration of
both compounds, suggesting that they have the potential to be signaling
molecules. Analysis of mutant cells unable to breakdown long-chain-base
phosphates reveals a large accumulation of both DHS-1-P and PHS-1-P in
log-phase cells. This accumulation of long-chain-base phosphates
correlates with increased survival at an elevated temperature, suggesting that long-chain-base phosphates may normally play a role in
heat stress resistance. Alternative explanations of our data are discussed.
Strains and media.
Strains used in this work are described
in Table 1. Strain MSS201 is a
Leu+ derivative of MSS200 made by transformation with the
LEU2 allele and selection for Leu+ cells. Strain
MSS207 is a derivative of MSS200 made by deleting DPL1 with
the
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Analysis of Phosphorylated Sphingolipid Long-Chain
Bases Reveals Potential Roles in Heat Stress and Growth Control
in Saccharomyces
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ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
1 allele (LEU2 replaces the region between
the NheI and BglII restriction sites [codons
10 to 536]), inserting TRP1 into LEU2 by a
swapping technique (3), and then deleting LCB3
with the
1 allele (LEU2 replaces 522 bp
between the BamHI and NsiI sites)
(23). The composition of PYED medium has been described
elsewhere (20).
TABLE 1.
Genotypes of strains used in this work
Analysis of long-chain-base phosphates. Lipids were uniformly labeled with 32Pi by growing yeast cells from 0.003 to 0.3 A600 units in PYED medium from which the phosphate had been omitted. The medium was supplemented with [32P]H3PO4 (0.1 mCi/ml; ICN, Costa Mesa, Calif.). Prior to lipid extraction, 5 ml of radiolabeled cells was mixed with 50 A600 units (4.5 ml) of nonradioactive carrier cells and treated with 5% trichloroacetic acid (0.5 ml of 100%) on ice for 30 min. The sample was centrifuged at 5,000 × g; the pellet was washed three times with 10 ml of ice-cold 5% trichloroacetic acid and then once with cold water. Residual liquid was carefully removed before extraction of total lipids by a 30-min incubation at 60°C with 0.5 ml of solvent A (95% ethanol, water, diethyl ether, pyridine, concentrated ammonium hydroxide [15:15:5:1:0.018, vol/vol] (10). The sample was centrifuged in a microcentrifuge at 13,000 × g before cooling. The supernatant fluid was dried under a stream of nitrogen.
Glycerophospholipids were deacylated by treatment of the sample with 0.5 ml of the monomethylamine reagent for 1 h at 50°C (2). The sample was dried under a stream of nitrogen. The remaining lipids were dissolved in 0.5 ml of solvent A lacking ammonia. To separate most sphingolipids and other unknown radiolabeled compounds from the long-chain-base phosphates, the lipid extract was applied to a 1-ml AG4-X4 (200/400 mesh; Bio-Rad, Hercules, Calif.) ion-exchange column which had been washed with 1 ml of water and 1 ml of methanol before equilibration with 3 ml of solvent A lacking ammonia. After loading the sample, the column was washed five times with 0.5 ml of solvent A lacking ammonia, followed by elution with 0.5-ml volumes of solvent A lacking ammonium hydroxide but acidified with glacial acetic acid (4 µl/ml). The 10 fractions were monitored, and those containing the small radioactive peak, usually fractions 6 and 7, were combined. This elution schedule was derived from pilot experiments in which [3H]DHS-1-P, mixed with a nonradioactive lipid extract and deacylated, was eluted with lipid extraction solvent containing increasing amounts of acid. Under the conditions described, more than 99% of the [3H]DHS-1-P eluted from the column. The combined fractions were dried under a stream of nitrogen, resuspended in 0.5 ml of solvent A, and chromatographed on thin-layer chromatography (TLC) plates (K5 Silica Gel 100A; Whatman, Clifton, N.J.) in solvent B (chloroform, methanol, water [60:35:8]) or solvent C (chloroform, methanol, 4.2 N ammonium hydroxide [9:7:2]) as indicated. Radioactivity was localized and quantified by using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).Synthesis of 32P-labeled long-chain-base
phosphates.
Soluble yeast proteins were prepared by vortexing (6 30-s bursts in a 15-ml Corex tube) 50 A600 units
of yeast cells in 1 ml of the extraction buffer (50 mM HEPES [pH
7.5], 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mg each of leupeptin, pepstatin, and aprotinin per ml) with 0.5 ml of
0.5-mm-diameter acid-washed glass beads. This and all other steps were
done at 4°C. The lysate was centrifuged for 5 min at 1,000 × g, and the resulting supernatant fluid was centrifuged at
100,000 × g for 15 min in a TLA 100.3 rotor (Beckman).
The final supernatant fluid, containing soluble yeast proteins, was
frozen and stored at
20°C and then thawed at 4°C for use in
long-chain-base kinase assays.
-32P]ATP (4,500 Ci/mmol; ICN).
The reaction was stopped by addition of 1.28 ml of chloroform-methanol
(1:1), and the mixture was used directly for TLC or the long-chain-base
phosphate was purified by using an AG4 column as described above.
Heat stress. Cells were grown overnight at 25°C in PYED medium to an A600 of 0.3 and then transferred to a 44°C water bath shaker. Viability was determined by diluting samples in distilled water and plating on PYED plates. Colonies were counted after 2 to 6 days incubation at 30°C.
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RESULTS |
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Synthesis of long-chain-base phosphates.
Both DHS and PHS are
intermediates in yeast sphingolipid biosynthesis, and it is possible
that both can be phosphorylated in S. cerevisiae. Since
radioactive standards for these long-chain-base phosphates are not
commercially available, we prepared them by incubating soluble yeast
proteins, a robust source of long-chain-base kinase activity, with a
nonradioactive long-chain base and [
-32P]ATP. The
radioactive products were partially purified on an AG4 column and
analyzed by TLC. DL-erythro-DHS and
D-erythro-sphingosine gave rise to a radioactive
product that chromatographed like the authentic phosphorylated
nonradioactive standards (Fig. 1).
Substituting PHS for DHS in the reaction produced radioactive PHS-1-P
(Fig. 1). The reaction is stereospecific because
L-threo-DHS was poorly phosphorylated (Fig. 1,
lane 3). The minor, rapidly moving product at the top of lane 4 is an
uncharacterized derivative produced from PHS during processing (see
below).
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Method for measurement of long-chain-base phosphates. Currently there is no simple and quantitative procedure for measuring long-chain-base phosphates in S. cerevisiae cells. Therefore, we sought to develop a simple procedure that used an inexpensive radiolabel to quantify and characterize all long-chain-base phosphates present in S. cerevisiae cells.
Cells were grown several generations in the presence of 32Pi to radiolabel all phosphorylated compounds to equilibrium. Lipids were extracted as described in Materials and Methods. Analysis of the deacylated lipid fraction by TLC did not clearly indicate the presence of long-chain-base phosphates because of a smear of radioactivity migrating where PHS-1-P and DHS-1-P were expected to migrate (Fig. 2A, before AG4).
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Types of long-chain-base phosphates present in S. cerevisiae cells. To determine the types of long-chain-base phosphates present in wild-type S. cerevisiae cells, we grew cells in the presence of 32P and extracted and processed the lipids as described in Materials and Methods. The two-dimensional TLC separation procedure revealed the presence of PHS-1-P (Fig. 3A, spots a and c) and almost no DHS-1-P (Fig. 3A, spot b) in log-phase cells grown at 25°C on complex medium having glucose as the carbon source.
Long-chain-base phosphates transiently increase during heat stress. If long-chain-base phosphates play a role in signal transduction during heat stress, their concentration should change after cells are shifted from a nonstressful to a stressful temperature. To determine if this was the case, we measured the quantity and type of long-chain-base phosphates in cells grown at 25°C and after a shift to 37°C.
At the earliest time point analyzed after the temperature shift, 5 min, we observed increases in PHS-1-P (Fig. 3B, spots a, c, and d) and DHS-1-P (Fig. 3B, spot b). Samples taken at all time points in this experiment were analyzed by two-dimensional TLC, and radioactivity was quantified by PhosphorImager analysis. At their peaks, which occurred around 10 min after the temperature shift, the concentrations of PHS-1-P and DHS-1-P were eight- and fivefold, respectively, above the baseline levels (Fig. 3C). The levels of both long-chain-base phosphates returned to the uninduced levels by about 20 min even though the cells were still growing at 37°C. We previously suggested that one process regulated by DHS, PHS, ceramide, or derivatives of them is transcriptional activation of the TPS2 gene (6). To determine if long-chain-base phosphates are components of a signaling pathway leading to induction of TPS2 transcription, we measured the kinetics of TPS2 transcription activation following a shift of cells from 25 to 37°C. Induction of TPS2 transcription was monitored by using a TPS2-lacZ reporter gene and measuring
-galactosidase activity.
-Galactosidase activity began to
increase at about 10 min after the temperature shift (Fig. 3C). These
kinetics are consistent with one or more long-chain-base phosphate
acting as a signal to activate transcription of TPS2.
Long-chain-base phosphates accumulate in mutant strains. To further understand the function of long-chain-base phosphates in S. cerevisiae, we deleted genes necessary for their catabolism. We anticipated that if there is a flux through long-chain-base phosphates, then deletions might disrupt the flux and cause accumulation of either or both intermediates. Analysis of phenotypes might suggest functions for long-chain-base phosphates. The genes deleted were DPL1 (see Fig. 6), necessary for long-chain-base phosphate lyase activity (24), and LCB3, necessary for the majority of phosphatase activity that dephosphorylates long-chain-base phosphates in yeast cells (16, 17).
The dpl1 deletion mutant (strain MSS204) had a 24-fold increase in the basal level of PHS-1-P but no increase in DHS-1-P; there was no detectable DHS-1-P (Fig. 4, time zero). The lcb3 deletion mutant (strain MSS205) had a 28-fold increase in the basal level of PHS-1-P and a 43-increase in the basal level of DHS-1-P (Fig. 4, time zero). The dpl1 lcb3 double mutant (strain MSS207) had more than a 500-fold increase in total long-chain-base phosphates, there being slightly more DHS-1-P than PHS-1-P (Fig. 4, time zero). These data suggest that there is a flux through DHS-1-P and PHS-1-P and that the Dpl1 lyase and Lcb3 phosphatase activities play roles in maintaining the basal rate of flux.
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Mutant strains are more heat resistant. The dpl1 (strain MSS204) and lcb3 (strain MSS205) single-deletion mutants grew normally in rich or synthetic medium at 25, 30, and 37°C. In contrast, the dpl1 lcb3 double mutant (strain MSS207) grew slowly under all of these conditions; for example, it had a generation time of about 5 h at 30°C in rich medium, compared to 2 h for the parental strain (MSS201) and the single mutant strains (data not shown). These data suggest that one or both long-chain-base phosphates that accumulate in the double mutant impair growth.
Survival at an elevated temperature was also examined since results from several laboratories implicate sphingolipids or their metabolites in resistance to heat stress (6, 12, 14, 16, 22). Log-phase cells of the dpl1 and lcb3 mutant strains were 2 to 4-fold more resistant to killing at 44°C than the parental strain, while the dpl1 lcb3 double-mutant strain was about 10-fold more resistant (Fig. 5). The correlation between increased long-chain-base phosphates and increased resistance to elevated temperature suggests that long-chain-base phosphates are components in cellular defenses against heat-induced damage.
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DISCUSSION |
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We have developed a simple procedure for quantifying the long-chain-base phosphates in S. cerevisiae cells. Using this procedure, we show for the first time that S. cerevisiae cells contain a barely detectable level of DHS-1-P and a higher level of PHS-1-P when grown on a fermentable carbon source at 25°C (Fig. 3A). We also show that switching cells from 25 to 37°C produces a transient eightfold increase in PHS-1-P and a fivefold increase in DHS-1-P, both peaking at about 10 min and then returning to near their starting levels (Fig. 3C). In addition, analysis of mutant strains disrupted for breakdown of DHS-1-P and PHS-1-P by the Dpl1 lyase or the Lcb3 phosphatase pathway (Fig. 6) showed a complex, strain-specific increase of one or both long-chain-base phosphates, some of which transiently increased with heat treatment (Fig. 4). Finally, we found a correlation between accumulation of long-chain-base phosphates in mutant strains and increased survival at an elevated temperature (Fig. 5).
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Sphingolipids have been implicated as signaling molecules in the heat stress response in S. cerevisiae (6, 12, 22). Two recent reports show that stationary-phase cells defective in DPL1 (14) or either of the long-chain-base phosphate phosphatase genes, LCB3 and LBP2 (16), are more resistant to killing at elevated temperature than the parental strains. These results must be interpreted with caution, however, because the concentration of long-chain-base phosphates in stationary-phase cells was not measured. More importantly, it remains to be determined if the elevation in long-chain-base phosphates is directly responsible for increased survival at elevated temperature. It is possible that the levels of long-chain-base phosphates are elevated in mutants strains grown to stationary phase but that increased heat resistance is a pathological response to elevated levels of long-chain-base phosphates. In wild-type cells, long-chain-base phosphates may not play a direct role in heat stress resistance.
Our experiments were performed on log-phase cells, but we also find that cells deleted for DPL1 or LCB3 survive somewhat (two- to fourfold) better than the parental strain at an elevated temperature in (Fig. 5). We also examined a strain deleted for both DPL1 and LCB3, the first time such a strain has been examined, and found that it survived 10-fold better than the parental strain (Fig. 4). Since our mutant strains had an elevated level of PHS-1-P (the dpl1 mutant) or both PHS-1-P and DHS-1-P (the lcb3 and the dpl1 lcb3 mutants [Fig. 4]) and increased survival at high temperature, it appears that long-chain-base phosphates play some role in mediating resistance to heat stress. However, it is unclear whether increased heat resistance is a physiological effect or a nonphysiological effect of greatly elevated levels of long-chain-base phosphates. Increased heat resistance of the dpl1 lcb3 double mutant may in fact be an indirect effect of a reduced growth rate, since slower-growing cells survive better at high temperature (7).
If long-chain-base phosphates act as signaling molecules during heat stress, their concentration should change transiently. Our data for PHS-1-P and DHS-1-P show such a transient increase following a heat shock (Fig. 3C), suggesting that one or both of these compounds are intracellular signals. The kinetics of the increase in long-chain-base phosphates are consistent with either of them acting as signals to induce transcription of TPS2 (Fig. 3C) and accumulation of trehalose (6), both of which occur during heat stress in S. cerevisiae (reference 5 and references therein).
We previously demonstrated that heat shock induces a 2 to 3-fold transient increase in the concentration of C18-DHS and C18-PHS and more than a 100-fold transient increase in C20-DHS and C20-PHS, with the maximum level occurring 10 to 15 min after the start of the heat shock (6, 12). These analyzes were done by using high-pressure liquid chromatography, which can separate C18 and C20 molecular species. The TLC procedure described here does not allow this distinction to be made, and we do not know which species of long-chain base is present in the measured PHS-1-P and DHS-1-P. The transient increase in C18-DHS, C20-DHS, C18-PHS, and C20-PHS may provide the substrates for producing the transient increase in DHS-1-P and PHS-1-P that we observe (Fig. 3). Previous data (6, 12) along with the data presented here show that all sphingolipid intermediates examined thus far increase during a heat stress. It remains to be determined if any of these compounds are signaling molecules, and if they are, which signaling pathway(s) they regulate. One or all of these changes in sphingolipid levels may represent a physiological adaptation to heat stress, and they may not be signaling molecules. Further work is needed to decide between these alternatives.
Strain MSS207 (
dpl1
lcb3) accumulated large amounts of
long-chain-base phosphates which amounted to about 2% of the total 32P found in a total lipid extract (Fig. 4). This level is
about 500-fold above the level seen in wild-type MSS200 cells and may account for the reduced growth rate (5 h versus 2 h) of MSS207 cells. The slow-growth phenotype could be due to activation of a signal
transduction pathway(s) that directly or indirectly regulates growth
rate. But we cannot exclude the possibility that slow growth results
from a nonphysiological effect of the high concentration of one of the
long-chain-base phosphates on a process necessary for a normal rate of
growth. In this case, the accumulation of long-chain-base phosphates
represents a pathological state as seen in the sphingolipid storage
diseases of mammals (19).
The accumulation of long-chain-base phosphates in cells deleted for DPL1, for LCB3, or for both genes (Fig. 4) strongly suggests that there is a flux through the metabolic pathway(s) that converts DHS and PHS to precursors for glycerolipid synthesis (Fig. 6). A direct demonstration of flux through the pathway by pulse-labeling cells with a radioactive precursor is not possible at this time for a variety of technical reasons, including the low level of PHS-1-P and DHS-1-P present in glucose-grown, log-phase cells. Pathways for breakdown of long-chain bases exist in all eucaryotes that have been examined including mammals, where sphingosine is converted to sphingosine-1-P. Our results suggest that there may be a constant flux through these pathways.
Since heat stress induces a transient accumulation of PHS-1-P and
DHS-1-P (Fig. 3) in S. cerevisiae and since one or both of
these compounds accumulate to a high level in mutant cells (Fig. 4),
there must be a mechanism(s) for regulating their level in wild-type
cells. Regulation is indicated also by the presence of only PHS-1-P and
not DHS-1-P in dpl1 mutant cells (Fig. 4). The lack of
DHS-1-P may be due to the absence of Dpl1 lyase or the long-chain
aldehyde produced by its action on DHS-1-P. Either the lyase or the
aldehyde could regulate the Lcb4 and Lcb5 kinases, the Lcb3 or Lpb2
phosphatase, or both (Fig. 6). It should be possible to determine the
mechanism(s) for regulating the basal level of long-chain-base
phosphates and transient changes in their level, since all of the genes
necessary for metabolism of long-chain-base phosphates
the lyase
(DPL1 [24]), the phosphatase
(LCB3 and LBP2 [16, 17, 23]),
and the kinase (LCB4 and LCB5
[21]) genes
have been identified.
Our procedure for quantifying long-chain-base phosphates in S. cerevisiae cells is simple, sensitive, and inexpensive and can be performed with a small quantity of cells. It should allow the level of long-chain-base phosphates to be measured under a variety of growth conditions including stressful insults in log-phase, stationary-phase, and starved cells, conditions for which there is precedent for believing that sphingolipids are essential for survival of S. cerevisiae cells (reference 22 and unpublished observations). The method should also be applicable to other fungi and single-cell organisms whose long-chain-base phosphates have not been analyzed.
Sphingosine and related second messengers are generally thought to be derived from breakdown of complex sphingolipids, especially sphingomyelin. For example, Swiss 3T3 fibroblasts stimulated to divide by binding of platelet-derived growth factor to its plasma membrane receptor causes hydrolysis of sphingomyelin to yield ceramide. The ceramide is cleaved by ceramidase to give sphingosine and a fatty acid, and the sphingosine is phosphorylated by sphingosine kinase to yield SPP (reviewed in references 26 and 27). The increases in PHS-1-P and DHS-1-P seen during heat shock of S. cerevisiae cells are not likely to be derived by breakdown of complex sphingolipids because there is no measurable breakdown (29). Also, complex sphingolipids in S. cerevisiae contain only PHS, not DHS, so the increase in DHS-1-P that we observe cannot be due to breakdown, unless there is an unknown pathway for converting PHS back to DHS. Current data indicate that DHS is a precursor to PHS (6, 15). Thus, S. cerevisiae cells may be a new paradigm for studying the generation of long-chain-base phosphates during stress responses and perhaps during other cellular processes.
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ACKNOWLEDGMENT |
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This work was supported by Public Health Service grant GM41302 to R.L.L. and R.C.D.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536-0298. Phone: (606) 323-6052. Fax: (606) 257-8940. E-mail: bobd{at}pop.uky.edu.
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