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Volume 272, Number 51, Issue of December 19, 1997 pp. 32566-32572
(Received for publication, July 3, 1997, and in revised form, September 17, 1997)
,¶ and
From the Departments of § Medicine and
ABSTRACT A role for sphingolipids in the yeast heat stress
response has been suggested by the isolation of suppressors of mutants
lacking these lipids, which are unable to grow at elevated
temperatures. The current study examines the possible role of
sphingolipids in the heat adaptation of yeast cells as monitored by
growth and viability studies. The suppressor of long chain base
auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was
corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the
cause of the heat-sensitive phenotype of the SLC strain 7R4. The
ability of sphingolipids to rescue the heat-sensitive phenotype was
examined, and two endogenous yeast sphingoid backbones,
phytosphingosine and dihydrosphingosine, were found to be most potent
in this effect. Next, the effect of heat stress on the levels of the
three major classes of sphingolipids was determined. The inositol
phosphoceramides showed no change over a 1.5-h time course. However,
the four detected species of sphingoid bases increased after 15 min of
heat stress from 1.4- to 10.8-fold. The largest increases were seen in
two sphingoid bases, C20 phytosphingosine and
C20 dihydrosphingosine, which increased 6.4- and 10.8-fold
over baseline, respectively. At 60 min of heat stress two species of
yeast ceramide increased by 9.2- and 10.6-fold over baseline. The
increase seen in the ceramides was partially decreased by Fumonisin B1,
a ceramide synthase inhibitor. Therefore, heat stress induces
accumulation of sphingoid bases and of ceramides, probably through
de novo synthesis. Taken together, these results
demonstrate that sphingolipids are involved in the yeast heat stress
adaptation.
INTRODUCTION Saccharomyces cerevisiae has been shown to respond to a
transfer of 25-37 or 39 °C with the physiology defined as a heat
stress response (1, 2), which appears to involve two phases. The initial phase of the response is the gaining of thermotolerance, and an
increase in trehalose accumulation is proposed as a marker for this
event (3). This is accompanied by the induction of heat shock proteins
(4) and a G1 arrest in cell cycle that lasts for a period
of approximately 1 h (5). Once thermotolerance is gained, the
second phase of the response occurs when the yeast begin to grow at the
increased temperature. At this point, trehalose is degraded in an
HSP70-dependent process (6), and the cells begin to cycle
and resume growth. Therefore, the ability of yeast to grow under
increased temperature provides for an overall assessment of the heat
stress response. However, the mechanisms that mediate adaptation and
growth under the heat-stressed state are not fully defined.
The isolation of suppressors of mutants lacking sphingolipids in yeast
(Table I) has suggested a possible role
for sphingolipids in the heat stress response. The initial mutation is
a Ura disruption knockout of the serine palmitoyltransferase
(SPT) gene (LCB1) (7), which catalyzes the first
step of sphingolipid biosynthesis (Fig.
1). Consequently, the 1 Table I.
Yeast strains
[View Larger Version of this Image (17K GIF file)]
The rescue of the SLC lines via phytosphingosine indicates a possible
need for sphingolipids in the yeast heat stress response. The current
study examines the role of sphingolipids in the heat adaptation of
yeast cells. First, the SLC strain 7R4 was transformed with the serine
palmitoyltransferase gene to ensure that the observed heat-sensitive
phenotype was due to the lack of sphingolipids and not to the secondary
mutation. Next the specificity of sphingolipids that allowed for growth
during heat stress was examined. Furthermore, the levels of sphingoid
backbones, ceramides, and the inositol phosphoceramides (IPCs) were
measured in response to heat stress. Our results show that endogenous
sphingolipid levels change in response to a heat stress challenge and
that sphingolipids are necessary for the heat stress response of
yeast.
EXPERIMENTAL PROCEDURES Sphingosine, phytosphingosine,
dihydrosphingosine, and fumonisin B1 were from Sigma.
L-threo-Dihydrosphingosine, C2
phytoceramide, C2 and C6 ceramides, and
C2 and C6 dihydroceramides were synthesized as
described previously (11, 12).
Yeast strains used are all isogenic to the
wild-type strain SJ21R. Strain 1 The plasmid PLCB1-5 was a gift from
Dr. Robert Dickson. The LCB1 gene in the plasmid was cut at
its unique SmaI site at base 263 and EcoRI site
at base 936 to obtain a 573-base pair fragment with which to transform
the yeast strain 7R4. The transformation was done using the lithium
acetate protocol (13). To select for the desired recombination event,
the transformed yeast were plated on 5-fluoroorotic acid. Controls for
transformation efficiency were streaked on leucine-deficient plates.
Transformation results were confirmed by polymerase chain reaction.
Yeast strains were streaked and then
grown for 2 to 4 days at 37 °C. The plated strains were then
photographed and manually scored for growth. Plates containing
supplementary sphingolipids were made with or without detergent Nonidet
P-40 used as a dispersant at a concentration of 0.05%.
Yeast strains were seeded at 2 × 105 to 8 × 105 cells/ml and assayed
for growth under the given conditions and treatments. At the selected
time points, a 1-ml aliquot was taken out, and its optical density was
measured at 600 nm.
Yeast sphingolipids were extracted from
liquid cultures via the method of Bligh and Dyer (14). The resultant
lipids were resuspended and split into three thirds. One-third was for
organic phosphates, which were used to normalize samples to each other. The other two-thirds were used for the measurement of sphingoid bases
and ceramide measurements.
The resultant lipid extract was
analyzed after derivatization with ortho-phthaldialdehyde
via HPLC with a Shimadzu fluorescent detector (15). Samples were
compared by use of the unnatural sphingoid base
L-threo-dihydrosphingosine as an internal
standard added before the Bligh and Dyer extraction.
Lipid extracts were phosphorylated by
Escherichia coli diacylglycerol kinase (16). The
phosphorylated compounds were then separated on a thin layer
chromatography (TLC) plate and then exposed to film. For
quantification, the plates were subsequently exposed to a phosphoimager
screen. The screen was then scanned, and analysis of the resulting
image was performed via Image Quant.
Cells in liquid
culture were labeled with tritiated inositol overnight and then treated
as denoted. Yeast lipid extracts were prepared and run on TLC plates as
described (17). The resultant TLC plates were exposed to film to obtain
a permanent image. Quantification was done via Image Quant. An aliquot
for the measurement of organic phosphate was taken before the lipids
were run on TLC plates. The resultant phosphate value was used to
normalize the values obtained via Image Quant.
RESULTS The suppressor of auxotrophy for long chain bases,
7R4, displays a heat-sensitive phenotype (see Fig.
2). To ensure that this phenotype is not
a result of the compensatory mutation in the strain 7R4, we reinserted
the deleted LCB1 gene. A 573-base pair LCB1 gene
fragment was generated from the plasmid PLCB1-5 by separate digestion
with SmaI and EcoRI, and the fragment was used to
transform cells via lithium acetate. Colonies were selected for growth
on 5-fluoroorotic acid. This selection requires the yeast to have lost
the URA gene disruption of the endogenous LCB1 through homologous recombination to grow. Thus, the selected strain should contain this
SPT gene under its normal regulation. Eleven colonies from two transformations were identified. Seven of these were selected for
further study. The seven selected transformants did not grow upon
Ura
[View Larger Version of this Image (53K GIF file)]
The necessity of sphingolipids for SLC
growth at high temperature led to studies on the ability and
specificity of various mammalian and yeast sphingolipids to allow for
this growth. These studies were performed in solid and liquid
media.
The growth of the SLC strains was studied in liquid culture where a
quantitative assessment of the ability of sphingolipids to increase
growth was obtained. The heat-sensitive phenotype of the SLC strains
7R4 and 4R3 was overcome by both phytosphingosine and
dihydrosphingosine (Figs. 3 and 4). The growth of the SLC strain 7R4
was most potently increased by 1 µM dihydrosphingosine. However, 1 and 5 µM phytosphingosine also allowed for
heat-stressed growth of the 7R4 strain (Fig.
3). In the SLC strain 4R3, 1 and 5 µM phytosphingosine were more effective than 1 µM dihydrosphingosine in allowing for heat-stressed
growth of the SLC strain 4R3 (Fig. 4).
The heat-sensitive phenotype of the SLC strain 7R4 was not reversed by
the addition of 1 or 5 µM
D-erythro-sphingosine or C2
phytoceramide (data not shown). Furthermore,
D-e-sphingosine could not rescue the SLC strain
4R3. Therefore, the rescue of the SLC strains from their heat-sensitive
phenotype was accomplished only by the two endogenous yeast sphingoid
backbones phytosphingosine and dihydrosphingosine.
[View Larger Version of this Image (16K GIF file)]
[View Larger Version of this Image (16K GIF file)]
The growth studies on plates supported the findings of those found in
the liquid studies. The plate growth data at 37 °C showed that
phytosphingosine was the most potent long chain base in allowing for
growth with heat stress (data not shown). However, the other endogenous
sphingoid backbone, D-erythro-dihydrosphingosine
also allowed for SLC growth at 37 °C. In addition C2
phytoceramide, D-e-sphingosine, C2
and C6 ceramide along with C2 and
C6 dihydroceramide did not show an ability to overcome the
heat-sensitive phenotype of the 7R4 and 4R3 SLC strains (data not
shown). Overall, the plate and liquid studies showed that only yeast
endogenous sphingoid backbones allowed for growth at heat-stressed
temperatures.
The necessity of sphingolipids in the heat stress response
and the ability of the endogenous sphingoid backbones to allow for
growth at high temperature prompted us to examine the levels of
endogenous sphingolipids in the wild-type cells upon heat stress. Each
of the three major classes of sphingolipids was measured over a given
time course of heat stress.
The first class of sphingolipids measured in S. cerevisiae
were the inositol phosphoceramides. Liquid cultures were labeled overnight with tritiated inositol. Lipids from heat-stressed and nonheat-stressed cells were extracted by a previously described method
(17), and the resultant extracts were developed on TLC plates (Fig.
5A). Over a 1.5-h time course
of heat stress, these data showed no significant changes in IPC,
mannose IPC, and mannose (inositol phosphate)2 ceramide
bands (Fig. 5B). Furthermore, no changes were seen in
phosphatidylinositol and lysophosphatidylinositol (data not shown).
[View Larger Version of this Image (37K GIF file)]
The second class of sphingolipids measured were the sphingoid backbones
(long chain amino bases). The sphingoid backbones from liquid cultures
were first extracted under Bligh and Dyer conditions. The organic phase
was dried down and resuspended, and an aliquot was taken to measure the
organic phosphate level that was used to normalize measurements of
sphingolipids. The remaining extract was put through a mild base
hydrolysis and reextracted. The resultant lipids were derivatized with
ortho-phthaldialdehyde and separated via high performance
liquid chromatography using a C18 reverse phase column. The extraction
efficiency of each sample was evaluated by the use of a nonendogenous
sphingoid backbone, L-threo-dihydrosphingosine,
added prior to the initial Bligh and Dyer extraction. Four peaks on the
HPLC charts were identified as sphingoid bases. Four criteria were met
in order for these peaks to be identified as such. The first was the
resistance to mild base hydrolysis. Second, they contained a free amine
that was derivatized. The free amine is present in the sphingoid bases and not the other sphingolipids, because this is the attachment site of
the fatty acid. Third, these four peaks were not detected in the SLC
strain 7R4, which lacks sphingolipids (Fig.
6A). Finally, the two peaks
that comigrated with known standards of C18
phytosphingosine and C18
D-erythro-dihydrosphingosine were identified as
such, whereas the remaining two peaks were designated C20
phytosphingosine and C20 dihydrosphingosine due to their
longer retention times. Each time point measured was compared with a
time-matched nonheat-stressed control (Fig. 6C). Within 15 min of heat stress at 39 °C there was a large induction of the
sphingoid backbones (Fig. 6B). At the 15-min time point,
C18 phytosphingosine increased 1.4-fold, whereas
C18 D-erythro-dihydrosphingosine had
increased 2.2-fold over the time matched controls (Fig. 6D).
The largest increases were seen in the C20 phytosphingosine
and C20 dihydrosphingosine species. C20
phytosphingosine increased 6.4-fold over baseline (Fig. 6, C
and D). Also, at 15 min C20 dihydrosphingosine
increased 10.8-fold over its time-matched control (Fig. 6D).
These increases were transient in that the levels decreased to near
baseline by 1 h (Fig. 6D). The 7R4-LCB1 line showed
this phenomena, as did another wild-type yeast line W303 (data not
shown). Overall, the sphingoid backbones showed large and transient
increases with heat stress in several yeast lines.
[View Larger Version of this Image (32K GIF file)]
The third class of sphingolipids measured were the yeast ceramides. The
lipids from liquid cultures were extracted by the method of Bligh and
Dyer. After resuspension, an aliquot was taken for organic phosphate
measurement. The remaining lipids were phosphorylated by E. coli diacylglycerol kinase and ran out on TLC plates. We found two
bands of ceramide phosphates via TLC separation. These two bands were
identified as ceramides due to their absence in the SLC lines and
resistance to mild alkaline hydrolysis (data not shown). The ceramides
increased following 60 min (Fig. 7) of
heat stress and continued to be elevated until at least 2 h of
heat stress (Fig. 8A). As
shown, ceramide 1 increased by 9.2-fold (Fig. 7B), and the
ceramide 2 increased by 10.6-fold (Fig. 7C). A smaller
increase of 2.7-fold in diacylglycerol was also seen after an hour of
heat stress at 39 °C (data not shown). The increase seen in the
ceramides was partially inhibited by a 2-h preincubation period with
150 µM fumonisin B1 (Fig. 8, A and
B). Fumonisin B1 has been shown to inhibit the ceramide
synthase in yeast (see Fig. 1) (18). Therefore, these data indicate
that the increased ceramide derives at least partially from the
conversion of sphingoid backbones to ceramides.
[View Larger Version of this Image (28K GIF file)]
[View Larger Version of this Image (64K GIF file)]
The above data show that the sphingoid backbone species undergo a large
and transient increase upon heat stress. Also, ceramides show a large
increase with later kinetics and appear sustained for at least 1 h. This increase occurred after the return of the sphingoid backbones
to basal levels. Furthermore, the results with fumonisin B1 indicate a
conversion of the sphingoid backbones to ceramides in the heat stress
response.
DISCUSSION The question of the need for sphingolipids in the heat stress
response has arisen due to the isolation of suppressor mutations of the
knockout of serine palmitoyltransferase in yeast (7). These SLC mutants
are unable to grow under many states of cellular stress including
increased ambient temperature, unless the media they grow upon is
supplemented with phytosphingosine (10). Therefore, we set out to
determine whether the observed phenotype of heat sensitivity is due to
a lack of sphingolipids or due to the secondary suppressor mutations.
As shown, the 7R4-LCB1 strain with a reinserted serine
palmitoyltransferase gene (LCB1) is not heat sensitive. Thus
the heat sensitivity did not arise from the aforementioned suppressor
mutations. Therefore, we conclude that sphingolipids are necessary for
the yeast heat stress response.
The measurement of sphingolipids in heat-stressed wild-type yeast
showed dramatic increases in two classes of sphingolipids. Within 15 min of heat stress all four species of sphingoid backbones increased
with the C20 phytosphingosine and C20
dihydrosphingosine each showing over 6-fold increases. Furthermore, the
increases seen were transient, and levels returned to baseline by
1 h. Yeast ceramides also increased but not until 1 h of heat
stress and remained elevated for at least an additional hour. The
increases seen in the ceramides were partially inhibited by fumonisin
B1, indicating that at least part of the increase was from the
conversion of yeast sphingoid backbones to ceramide via ceramide
synthase. Furthermore, the increase in ceramides could be inhibited
nearly completely by Australifungin, a potent and specific inhibitor of
ceramide synthase.2 Moreover,
for up to 1.5 h of heat stress, the IPCs did not significantly change. Taken together these data suggest enhanced de novo
synthesis of these compounds, which could be the source of the
increased sphingoid bases and subsequently the increased ceramides.
One can speculate on the role of sphingolipids in the heat stress
response. There are two prominent possibilities. The first possibility
is that the role played by sphingolipids is of a structural nature.
This possibility is based upon the observation that membrane IPC's
comprise about 30% of the phospholipid content of plasma membrane in
yeast (19). Therefore, the reason that SLC strains may acquire
sensitivity to heat could be due to the lack of these sphingolipids in
the membranes. One piece of evidence that indicates that this might not
be the case is provided by the novel inositol glycerolipids. These
compounds are very similar to the IPCs in structure and are
hypothesized to fulfill their structural functions. This idea is
supported by the data that the SLC strains will grow at normal
temperatures, whereas the serine palmitoyltransferase knockout will
not. However, the novel inositol glycerolipids may not totally mimic
the function of wild-type sphingolipids. The second hypothesis suggests
that the sphingolipids play a more regulatory role that goes beyond
their structural role. This is supported by the observed changes in
sphingolipid levels following heat stress. These results raise the
possibility that yeast sphingolipids react to heat stress and may
participate in the adaptive responses, possibly as second messengers or
signal transducers. Such a role has been proposed in mammalian systems
where sphingolipids have been found to participate in senescence (20),
differentiation (21, 22), apoptosis (23), and cellular stress responses (24). In addition mammalian cells have been shown to display an
increase in ceramide in response to heat stress and that ceramide induces the expression of at least one heat shock protein, The results from the growth studies show that only endogenous yeast
sphingoid backbones are able to rescue the SLC cells from their
inability to grow in an increased ambient temperature. These data
indicate a possible importance for the sphingoid backbones in the yeast
heat stress response. In light of this idea one can speculate that the
yeast sphingoid backbones, which show major increases upon heat stress,
are important in a signaling role of this stress response. Second, the
increase in ceramides may also play a role. Indeed, these biphasic
changes in yeast sphingolipids may reflect the complex physiology of
the yeast heat stress response, which may be considered as a biphasic
physiology. First the yeast tries to survive the new temperature. Once
this is accomplished the yeast may then regain its ability to grow.
Questions such as what are the roles of sphingoid backbones and
ceramides in the yeast heat stress response should be addressed. Also,
one would like to delineate the necessity of either or both of the above sphingolipids in the gaining of thermotolerance and regrowth phases of heat stress. Finally, if there is indeed a signaling role for
sphingolipids in the heat stress response, the study of where and how
these molecules affect the heat stress response should be quite
interesting.
FOOTNOTES REFERENCES 
Cell Biology,
4 strain lacks
all sphingolipids. This yeast strain (14) is unable to grow at
normal temperatures without sphingoid backbone supplementation to the
media (7). Suppressors (7R4 and 4R3) of the aforementioned mutant have
been isolated, and were found to allow growth in the absence of
sphingolipid supplementation, possibly as a result of the formation of
novel inositol glycerolipids. The novel inositol glycerolipids contain: 1) the 26 carbon fatty acid that is ordinarily exclusively found in the
yeast sphingolipids; and 2) the same head groups that are found in the
inositol phosphoceramides, now attached to a glycerol backbone (8, 9).
Thus, these sphingolipid compensatory
strains are able to grow at normal
temperatures but with a slower doubling time than the wild-type strain
(SJ21R). However, the SLC1 strains still lack all
sphingolipids. The SLC strains were found not to grow under conditions
of cellular stress such as high osmolarity and increased ambient
temperature. However, the heat-sensitive phenotype could be reversed by
the addition of phytosphingosine to the media (10).
Strain
Genotype
Reference
SJ21R
MATa, ura3-52,
leu2-3, 112, ade1, MEL1
7
1
4MATa,
leu2-3, 112, ade1, MEL1, lcb1::URA3
7
4R3
MATa,
leu2-3, 112, ade1, MEL1, lcb1::URA3, SLC1-1
8
7R4
MATa, leu2-3, 112, ade1, MEL1, lcb1::URA3,
SLC2-1
8
7R4-LCB1
MATa, ura3-52, leu2-3, 112, ade1, MEL1,
SLC2-1
This study
Fig. 1.
Biosynthetic pathway of yeast
sphingolipids. The biosynthesis of sphingolipids is shown in the
above proposed pathway.
4 is the URA disruption of the
serine palmitoyltransferase gene (lcb1). Suppressors of the
long chain base knockout (SLC's) used in this study were the 4R3 and
7R4 strains, and they were obtained from Dr. Robert Dickson (University
of Kentucky) (7, 8). The strain 7R4-LCB1 was created for this study as
described below.
plates (data not shown), providing further evidence
that the gene had been properly inserted and had essentially deleted
the URA gene via recombination. Finally, polymerase chain
reaction was done with two sets of primers, and the selected mutant 7R4 2B (7R4-LCB1) was found to have the expected products (data not shown).
Strain 7R4-LCB1 was tested for its ability to grow at both 30 and
39 °C (Fig. 2). The 7R4 strain grew moderately at 30 °C and was
not able to grow at all at 39 °C (Fig. 2). On the other hand, the
7R4-LCB1 strain grew as well as the wild-type SJ21R at both
temperatures. At 30 °C, acquisition of sphingolipids by the strain
via the reinsertion of this SPT gene under its normal control allowed for wild-type growth as compared with the slower growth
of the 7R4 strain. These data indicate that indeed the slow growth of
the 7R4 strain is due to its lacking of sphingolipids. Furthermore, the
ability of 7R4-LCB1 to grow as well as the wild type at 39 °C
demonstrates that the secondary mutations did not impart the
heat-sensitive phenotype seen in the 7R4 strain. Therefore, these data
prove that the lack of sphingolipids causes the heat stress-deficient
phenotype seen in the SLC strain, and, thus, the presence of
sphingolipids is required for the heat stress response.
Fig. 2.
Growth of 7R4-LCB1 at both 30 and
39 °C. The strains SJ21R (wild type), 7R4 (suppressor of lcb1)
and 7R4-LCB1 (suppressor and LCB1) were streaked out on YEPD (yeast
extract, bacto-Peptone, dextrose) plates. One plate was grown at
30 °C and the other at 39 °C for 2 days.
Fig. 3.
Effects of phytosphingosine and
dihydrosphingosine on growth at 39 °C of the SLC strain 7R4.
Phytosphingosine (psph) (A) or dihydrosphingosine
(dhsph) (B) were added a half hour before the
zero time point of heat stress (39 °C) at the given final concentrations. Growth was assayed by absorbance at 600 nm of a 1-ml
aliquot for the indicated time points. Each point is the average of two
separate measurements shown with their standard deviation.
Fig. 4.
Effects of phytosphingosine and
dihydrosphingosine on growth at 39 °C of the SLC strain 4R3.
Phytosphingosine (psph) (A) or dihydrosphingosine
(dhsph) (B) were added a half hour before the
zero time point of heat stress (39 °C) at the given final concentrations. Growth was assayed by absorbance at 600 nm of a 1-ml
aliquot for the indicated time points. Each point is the average of two
separate measurements shown with their standard deviation.
Fig. 5.
Effects of heat stress on the levels of
inositol phosphoceramide. Extracted lipids (see Ref. 17) were
resuspended and run out on thin layer chromatography plates.
A, film of the TLC plate with lanes corresponding to the
following: lane 1, zero time; lane 2, 15 min heat
stress; lane 3, 30 min heat stress; lane 4, 60 min heat stress; lane 5, 90 min heat stress; lane
6, 15 min control; lane 7, 30 min control; lane
8, 60 min control; lane 9, 90 min control.
B, percent of time-matched controls of inositol
phosphoceramide (IPC), mannose inositol phosphoceramide (MIPC) and mannose (inositol phosphate)2
ceramide (M(IP)2C). Data are representative of three
experiments.
Fig. 6.
Effects of heat stress on levels of yeast
sphingoid backbones. Bligh and Dyer extracts were put through mild
base hydrolysis, derivatized, and run out on HPLC. Data are
representative of three experiments. A, HPLC charts showing
sphingoid peaks from the extracts of the SLC strain 7R4 and wild-type
strain SJ21R. Five peaks from the HPLC charts are identified as
follows: C18 phytosphingosine (peak 1),
C20 phytosphingosine (peak 2),
L-threo-dihydrosphingosine (peak 3),
C18 D-erythro-dihydrosphingosine
(peak 4) and C20 dihydrosphingosine (peak
5). B, comparison of sphingoid bases in control and
heat-stressed cells at 15 min. Peaks are identified as above.
C, C20 phytosphingosine (C20 psph)
levels in control versus heat-stressed cells. Each point is
the average of duplicate measurements given with their standard
deviation. D, fold increases of the sphingoid backbone species. Data are representative of three experiments.
Fig. 7.
Effects of heat stress on levels of
ceramides. Extracted lipids (see Ref. 14) were phosphorylated by
diacylglycerol kinase and run out on thin layer chromatography
plates. A, film of the TLC plate with lanes corresponding to
the following: lanes 1 and 2, zero time point;
lanes 3 and 4, 15 min control; lanes 5 and 6, 15 min heat stress; lanes 7 and
8, 30 min control; lanes 9 and 10, 30 min heat stress; lanes 11 and 12, 60 min control; and lanes 13 and 14, 60 min heat stress.
B, ceramide 1 control versus heat stress levels.
C, ceramide 2 control versus heat stress levels.
Each measurement is the average of duplicates displayed with their
standard deviation. Data are representative of more than three
experiments.
Fig. 8.
Effect of fumonisin B1 on heat stress-induced
increase of ceramides. Extracted lipids (see Ref. 14) were
phosphorylated by diacylglycerol kinase and run out on thin layer
chromatography plates. A, film of the TLC plate with lanes
corresponding to the following: lane 1, diacylglycerol
standard; lanes 2 and 3, 60 min controls;
lanes 4 and 5, 60 min heat stress; lanes
6 and 7, 60 min heat stress pretreated with 150 µM fumonisin B1 for 2 h; lanes 8 and
9, 120 min controls; lanes 10 and 11,
120 min heat stress; and lane 12 mammalian ceramide control.
B, comparison of ceramide 1 and ceramide 2 at 60 min with
and without heat stress and with heat stress preincubated with 150 µM fumonisin B1 for 2 h before heat stress. Each
measurement is the average of duplicates displayed with their standard
deviation.
B crystallin (25). In light of these roles in mammalian systems one can
easily theorize that yeast sphingolipids may play a similar role in the
yeast heat stress response.
To whom correspondence should be addressed: Box 3355, Duke
University Medical Center, Durham, NC 27710. Tel.: 919-684-2541; E-mail: hannu001{at}mc.duke.edu.
1
The abbreviations used are: SLC, sphingolipid
compensatory; IPC, inositol phosphoceramide; HPLC, high pressure liquid
chromatography; TLC, thin layer chromatography.
2
R. Dickson, personal communication.
Volume 272, Number 51,
Issue of December 19, 1997
pp. 32566-32572
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 A. Daquinag, M. Fadri, S. Y. Jung, J. Qin, and J. Kunz The Yeast PH Domain Proteins Slm1 and Slm2 Are Targets of Sphingolipid Signaling during the Response to Heat Stress Mol. Cell. Biol., January 15, 2007; 27(2): 633 - 650. [Abstract] [Full Text] [PDF]
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 J. L. Brace, R. L. Lester, R. C. Dickson, and C. M. Rudin SVF1 Regulates Cell Survival by Affecting Sphingolipid Metabolism in Saccharomyces cerevisiae Genetics, January 1, 2007; 175(1): 65 - 76. [Abstract] [Full Text] [PDF]
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G. Bultynck, V. L. Heath, A. P. Majeed, J.-M. Galan, R. Haguenauer-Tsapis, and M. S. Cyert Slm1 and Slm2 Are Novel Substrates of the Calcineurin Phosphatase Required for Heat Stress-Induced Endocytosis of the Yeast Uracil Permease Mol. Cell. Biol., June 15, 2006; 26(12): 4729 - 4745. [Abstract] [Full Text] [PDF]
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 L. J. Heung, C. Luberto, and M. Del Poeta Role of Sphingolipids in Microbial Pathogenesis Infect. Immun., January 1, 2006; 74(1): 28 - 39. [Full Text] [PDF]
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L. J. Heung, A. E. Kaiser, C. Luberto, and M. Del Poeta The Role and Mechanism of Diacylglycerol-Protein Kinase C1 Signaling in Melanogenesis by Cryptococcus neoformans J. Biol. Chem., August 5, 2005; 280(31): 28547 - 28555. [Abstract] [Full Text] [PDF]
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J. A. Rotolo, J. Zhang, M. Donepudi, H. Lee, Z. Fuks, and R. Kolesnick Caspase-dependent and -independent Activation of Acid Sphingomyelinase Signaling J. Biol. Chem., July 15, 2005; 280(28): 26425 - 26434. [Abstract] [Full Text] [PDF]
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K. Liu, X. Zhang, R. L. Lester, and R. C. Dickson The Sphingoid Long Chain Base Phytosphingosine Activates AGC-type Protein Kinases in Saccharomyces cerevisiae Including Ypk1, Ypk2, and Sch9 J. Biol. Chem., June 17, 2005; 280(24): 22679 - 22687. [Abstract] [Full Text] [PDF]
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B. Gaigg, B. Timischl, L. Corbino, and R. Schneiter Synthesis of Sphingolipids with Very Long Chain Fatty Acids but Not Ergosterol Is Required for Routing of Newly Synthesized Plasma Membrane ATPase to the Cell Surface of Yeast J. Biol. Chem., June 10, 2005; 280(23): 22515 - 22522. [Abstract] [Full Text] [PDF]
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 F. R. Pinto, L. A. Cowart, Y. A. Hannun, B. Rohrer, and J. S. Almeida Local correlation of expression profiles with gene annotations--proof of concept for a general conciliatory method Bioinformatics, April 1, 2005; 21(7): 1037 - 1045. [Abstract] [Full Text] [PDF]
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M. Germann, E. Swain, L. Bergman, and J. T. Nickels Jr. Characterizing the Sphingolipid Signaling Pathway That Remediates Defects Associated with Loss of the Yeast Amphiphysin-like Orthologs, Rvs161p and Rvs167p J. Biol. Chem., February 11, 2005; 280(6): 4270 - 4278. [Abstract] [Full Text] [PDF]
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 M. Kolaczkowski, A. Kolaczkowska, B. Gaigg, R. Schneiter, and W. S. Moye-Rowley Differential Regulation of Ceramide Synthase Components LAC1 and LAG1 in Saccharomyces cerevisiae Eukaryot. Cell, August 1, 2004; 3(4): 880 - 892. [Abstract] [Full Text] [PDF]
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X. Zhang, R. L. Lester, and R. C. Dickson Pil1p and Lsp1p Negatively Regulate the 3-Phosphoinositide-dependent Protein Kinase-like Kinase Pkh1p and Downstream Signaling Pathways Pkc1p and Ypk1p J. Biol. Chem., May 21, 2004; 279(21): 22030 - 22038. [Abstract] [Full Text] [PDF]
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L. J. Heung, C. Luberto, A. Plowden, Y. A. Hannun, and M. Del Poeta The Sphingolipid Pathway Regulates Pkc1 through the Formation of Diacylglycerol in Cryptococcus neoformans J. Biol. Chem., May 14, 2004; 279(20): 21144 - 21153. [Abstract] [Full Text] [PDF]
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 K. Malathi, K. Higaki, A. H. Tinkelenberg, D. A. Balderes, D. Almanzar-Paramio, L. J. Wilcox, N. Erdeniz, F. Redican, M. Padamsee, Y. Liu, et al. Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution J. Cell Biol., February 16, 2004; 164(4): 547 - 556. [Abstract] [Full Text] [PDF]
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H. Fyrst, D. R. Herr, G. L. Harris, and J. D. Saba Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster J. Lipid Res., January 1, 2004; 45(1): 54 - 62. [Abstract] [Full Text] [PDF]
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L. A. Cowart, Y. Okamoto, F. R. Pinto, J. L. Gandy, J. S. Almeida, and Y. A. Hannun Roles for Sphingolipid Biosynthesis in Mediation of Specific Programs of the Heat Stress Response Determined through Gene Expression Profiling J. Biol. Chem., August 8, 2003; 278(32): 30328 - 30338. [Abstract] [Full Text] [PDF]
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 A. D. Batheja, D. J. Uhlinger, J. M. Carton, G. Ho, and M. R. D'Andrea Characterization of Serine Palmitoyltransferase in Normal Human Tissues J. Histochem. Cytochem., May 1, 2003; 51(5): 687 - 696. [Abstract] [Full Text] [PDF]
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S. D. Kobayashi and M. M. Nagiec Ceramide/Long-Chain Base Phosphate Rheostat in Saccharomyces cerevisiae: Regulation of Ceramide Synthesis by Elo3p and Cka2p Eukaryot. Cell, April 1, 2003; 2(2): 284 - 294. [Abstract] [Full Text] [PDF]
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J. D. Hearn, R. L. Lester, and R. C. Dickson The Uracil Transporter Fur4p Associates with Lipid Rafts J. Biol. Chem., January 31, 2003; 278(6): 3679 - 3686. [Abstract] [Full Text] [PDF]
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J. Cheng, T.-S. Park, L.-C. Chio, A. S. Fischl, and X. S. Ye Induction of Apoptosis by Sphingoid Long-Chain Bases in Aspergillus nidulans Mol. Cell. Biol., January 1, 2003; 23(1): 163 - 177. [Abstract] [Full Text]
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G. M. Jenkins, L. A. Cowart, P. Signorelli, B. J. Pettus, C. E. Chalfant, and Y. A. Hannun Acute Activation of de Novo Sphingolipid Biosynthesis upon Heat Shock Causes an Accumulation of Ceramide and Subsequent Dephosphorylation of SR Proteins J. Biol. Chem., November 1, 2002; 277(45): 42572 - 42578. [Abstract] [Full Text] [PDF]
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H. Le Stunff, I. Galve-Roperh, C. Peterson, S. Milstien, and S. Spiegel Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis J. Cell Biol., September 16, 2002; 158(6): 1039 - 1049. [Abstract] [Full Text] [PDF]
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A. Kihara and Y. Igarashi Identification and Characterization of a Saccharomyces cerevisiae Gene, RSB1, Involved in Sphingoid Long-chain Base Release J. Biol. Chem., August 9, 2002; 277(33): 30048 - 30054. [Abstract] [Full Text] [PDF]
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E. Swain, K. Baudry, J. Stukey, V. McDonough, M. Germann, and J. T. Nickels Jr. Sterol-dependent Regulation of Sphingolipid Metabolism in Saccharomyces cerevisiae J. Biol. Chem., July 12, 2002; 277(29): 26177 - 26184. [Abstract]</ |
ABSTRACT
