From the Department of Biochemistry and the Lucille P. Markey
Cancer Center, University of Kentucky,
Lexington, Kentucky 40536-0084
Sphingolipid-related metabolites have been
implicated as potential signaling molecules in many studies with
mammalian cells as well as in some studies with yeast. Our previous
work showed that sphingolipid-deficient strains of Saccharomyces
cerevisiae are unable to resist a heat shock, indicating that
sphingolipids are necessary for surviving heat stress. Recent evidence
suggests that one role for the sphingolipid intermediate ceramide may
be to act as a second messenger to signal accumulation of the
thermoprotectant trehalose. We examine here the mechanism for
generating the severalfold increase in ceramide observed during heat
shock. As judged by compositional analysis and mass spectrometry, the
major ceramides produced during heat shock are similar to those found
in complex sphingolipids, a mixture of
N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous
enzyme. Using [3H]phytosphingosine and
[3H]inositol-labeled yeast sphingolipids, a novel
membrane-associated phospholipase C-type activity that generated
ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and
mannose(inositol-P)2-ceramide was demonstrated. The
sphingolipid head groups were concomitantly liberated with the expected
stoichiometry. However, other data demonstrate that the ceramide
generated during heat shock is not likely to be derived by breakdown of
complex sphingolipids. For example, the water-soluble fraction of
heat-shocked cells showed no increase in any of the sphingolipid head
groups, which is inconsistent with complex sphingolipid hydrolysis.
Rather, we find that de novo ceramide synthesis involving
ceramide synthase appears to be responsible for heat-induced ceramide
elevation. In support of this hypothesis, we find that the potent
ceramide synthase inhibitor, australifungin, completely inhibits both
the heat-induced increase in incorporation of
[3H]sphinganine into ceramide as well as the heat-induced
increase in ceramide as measured by mass. Thus, heat-induced ceramide
most likely arises by temperature activation of the enzymes that
generate ceramide precursors, activation of ceramide synthase itself,
or both.
 |
INTRODUCTION |
Widespread research, primarily in mammalian cells, has
focused on sphingolipids as possible mediators of stress responses. Sphingolipid metabolites such as sphingosine, sphingosine-phosphate, and ceramide have been proposed as signaling molecules in a host of
cellular processes (for recent reviews, see Refs. 1-4); however, in
most cases the precise molecular interactions in a
sphingolipid-mediated signaling cascade await definition.
Ligand-activated sphingomyelinase activity acting on plasma membrane
sphingomyelin is the most studied mechanism for generating the second
messenger ceramide (5). Some reports suggest an alternative mechanism;
altered ceramide synthase activity produces the ceramide increase seen
in apoptosis (6) and arachidonic acid signaling in macrophages (7).
Saccharomyces cerevisiae is an exemplary organism in which
to sort out the sphingolipid-related genes and proteins necessary for
mounting a response to stress. Not only has the genome sequence been
determined, but the sphingolipid composition is relatively simple as
compared with mammals. The principal ceramide of S. cerevisiae, phytosphingosine (4-OH sphinganine)
N-acylated with an
-OH C26 fatty acid, is
phosphodiester-linked to inositol
(IPC),1 mannosylinositol
(MIPC), or inositolphosphorylmannosylinositol (M(IP)2C)
(8). The synthesis of the Saccharomyces
inositolphosphoceramides is schematized in Fig.
1, indicating inhibition of ceramide
synthase by the potent antifungal agent australifungin (9).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Sphingolipid synthesis in S. cerevisiae. The pathway reflects the available data;
however, it is unclear whether long chain base 4-hydroxylation occurs
at the level of the free long chain base(s) or at the level of
ceramide. Also unclear is the nature of the substrate(s) for fatty acid
-hydroxylation.
|
|
Sphingolipids were first implicated in stress responses in S. cerevisiae when it was observed that strains unable to make sphingolipids failed to grow under conditions such as high temperature, high osmotic pressure, and low pH, whereas such strains could withstand
these stresses when cultured so as to contain sphingolipids (10).
Subsequent reports implicated sphingolipids in signaling roles in
S. cerevisiae. N-Acetyl sphingosine (C2-ceramide)
was shown by some (11, 12) but not all (13) laboratories to inhibit
growth (12) via the proposed activation of a protein phosphatase (11,
12).
Shifting the temperature from 24 to 39 °C is known to induce a
variety of responses such as heat shock protein synthesis (14) and
trehalose accumulation (see Ref. 15 and references cited therein). We
recently demonstrated that such a temperature shift caused a
severalfold elevation of ceramide (16) as well as a transient increases
in sphinganine and phytosphingosine (17). Recent work also indicates
that sphingolipids could be involved in the trehalose accumulation
response, including the failure to accumulate trehalose in
sphingolipid-deficient cells, exogenous sphinganine induction of
trehalose accumulation in wild type cells, and exogenous sphinganine
induction of the transcription of the TPS2 gene required for
trehalose synthesis (17).
In this paper, we examine the biochemical basis for the
heat-induced ceramide elevation. A recent study has raised questions concerning the validity of many reports of ceramide elevation based on
using diacylglycerol kinase to measure ceramide (18). We have used
direct chemical methods to establish the nature of the heat-induced
ceramides in S. cerevisiae. We show that, although there
exists a membrane-associated phosphodiesterase C-type enzyme activity
that generates ceramide from yeast sphingolipids, it appears that the
heat-induced ceramide increase results from de novo
synthesis rather than from phosphodiesterase-mediated sphingolipid catabolism. This finding is different from the most studied explanation of ceramide elevation in mammalian cells, namely stimulated
sphingomyelinase activity as part of a "sphingomyelin cycle"
(5).
 |
EXPERIMENTAL PROCEDURES |
Materials, Culture Media, Yeast Strain
Ceramides were prepared from autolysed commercial bakers' yeast
(Fleischmann) by modifications of the procedure of Oda and Kamiya (19).
C18-Celite was prepared as follows. Acid-washed Celite 545 (Fisher)
freed of phosphate impurities was treated with 10%
octadecyltrichlorosilane (PCR Research Chemicals, Inc.) in toluene
overnight at room temperature, washed with toluene and methanol, and
dried under vacuum. Silica gel-impregnated paper (Whatman SG-81) was
treated with EDTA as described (20). Complex media consisted of 4%
glucose, 1% peptone, 1% yeast extract, 1% KH2PO4, 0.05 M sodium succinate, pH
5.5, 0.05% Tergitol, 10 mg of myo-inositol/liter. S. cerevisiae strain YPH252 (MAT
ura3-52 lys2-801amber
ade2-201ochre trp1-
1 his3-
200
leu2
1) (21) was used throughout unless otherwise noted. Where
indicated, strains RCD113 (MATa ura3-52
lys2-801amber
ade2-101ochre trp1-
1 his3-
200 leu2-
1
ipt1-
1) (22) and YPH250 (21) were employed. Australifungin was
a generous gift of Dr. S. Mandala (Merck).
[4,5-3H]Sphinganine
N-[4,5-3H]Acetylsphinganine (1.5 × 107 dpm/nmol), prepared as described (23), was
hydrolyzed with 1.0 N HCl in methanol-H2O (82:18) for 18 h at 80 °C. After removing unhydrolyzed
acetylsphinganine by twice extracting with 2 volumes of hexane, the
hydrolysate was dried and stored in 95% ethanol. Aliquots were dried
and dispersed in 0.5% Tergitol in a sonic bath prior to the addition
to culture medium.
Ceramide Analysis in Unlabeled Cells
Free ceramides were extracted from cells, perbenzoylated, and
quantitated after HPLC as described previously (23). Underivatized ceramide was sometimes directly analyzed. The initial
chloroform/methanol (1:1) extract was filtered (Gelman 0.45 µ ACRO
LC3S filters), dried, and dissolved in 200 µl of chloroform. A
50-µl aliquot was subjected to HPLC on a 0.45 × 30-cm 5 µ Lichrosorb Si 60 column equilibrated with chloroform and eluted (1 ml/min) with 1 ml of chloroform followed by a 19-min linear gradient
between chloroform and chloroform/methanol (3:1). Ceramide-III eluted
at about 15 min and was detected with a Varex evaporative light
scattering detector (drift tube temperature, 70 °C; nitrogen flow
rate, 1.1 liters/min).
Preparative Isolation and Analysis of Ceramides from
Heat-shocked Cells
An overnight culture grown at 24 °C (2 liters;
A650 = 1.0) was warmed to 39 °C and incubated
with shaking for 40 min, followed by termination with trichloroacetic
acid to 5%. The water-washed cell pellet was extracted with 60 ml of
chloroform/methanol (1:1) at 50 °C for 30 min and centrifuged while
warm, and the supernatant was dried and suspended in 2 ml of
chloroform. The extract was applied to a 6-ml silica gel column
(Adsorbosil, 100-200 mesh, Applied Science Labs) equilibrated with
chloroform, and eluted with 9 ml of chloroform, 9 ml of
chloroform/methanol (9:1), and 9 ml of chloroform/methanol (1:1).
Ceramide-III assay by HPLC of the three fractions showed 0, 471, and 34 nmol, respectively. Fraction 2 was further purified by HPLC on a
0.45 × 30-cm column of 5-µm Lichrosorb Si60 (Merck)
equilibrated with chloroform. The elution schedule (flow rate of 1 ml/min) was 1 min chloroform, 19 min linear gradient between chloroform
and chloroform/methanol (75:25). The eluate was monitored with a Varex
evaporative light scattering detector, and the fractions expected for
ceramide-III were pooled, dried, and dissolved in chloroform. Final
purification was on the same Lichrosorb column with isocratic elution
with chloroform/methanol (9:1). The final ceramide fraction was
analyzed for long chain base and fatty acid (24) as well as assayed by fast atom bombardment mass spectrometry. Part of the sample was benzoylated and separated by HPLC by a scaled up version of the protocol described above for quantitative ceramide analysis. Samples from each of the twin peaks were subjected to electron impact mass
spectrometry.
Mass Spectrometry
Positive ion fast atom bombardment spectra of underivatized
ceramides were measured with a Concept IH (Kratos) two-sector mass
spectrometer equipped with an Ion Tech Ltd. saddle field gun operating
with a xenon gas and set at a resolution of about 1500 at the
acceleration voltage of 5.3 kV. Samples (~2 nmol/2 µl) in methylene
chloride were added to 3-nitrobenzyl alcohol matrix (2-3 µl) on a
7-mm diameter stainless steel probe tip. Spectra were acquired in a raw
data mode, in a 100-2100 atomic mass unit range, 3 s per decade,
using external CsI mass calibration. Spectra representing the best
response were averaged and digitally smoothed. Electron impact mass
spectra of perbenzoylceramides were measured at 70 eV using a CONCEPT
IH (Kratos) two-sector instrument. Samples on the platinum wire were
directly introduced to the ion source at 250 °C. Spectra were
acquired from 40 to 1400 atomic mass units, 3 s per decade, and
the instrument was set to about 2000 atomic mass units resolving
power.
Preparation of [3H]Phosphosphingolipids
Sphingolipids labeled with either [3H]inositol or
[3H]phytosphingosine were prepared by metabolic labeling.
Ethanol solutions of myo-[2-3H]inositol (1 mCi, NEN Life Science Products) or [4,5-3H]sphinganine
(775 × 106 dpm; prepared as described above) were
dried in a sterile culture flask, sonic treated with 10 ml of culture
medium, inoculated with a starter culture of strain YPH252 to a
starting A650 of 0.2, and shaken for 22 h
at 30 °C, the A650 reaching 11. The cells were treated with trichloroacetic acid (5% final concentration), centrifuged, and washed twice with water. Lipids were extracted by
treating each pellet with 2 ml of solvent B (diethylether, 95%
ethanol, water, pyridine; 5:15:15:1 (v/v/v/v) containing 0.5 ml/liter
concentrated ammonia) for 30 min at 60 °C. Further purification proceeded by slightly different routes.
The solvent B extract from the
[4,5-3H]sphinganine-labeled cells was added to a 1-ml
column of BioRex 70 resin (H+ form, 200-400 mesh, Bio-Rad)
in a Pasteur pipette (packed in water and equilibrated with methanol)
and washed with 3 ml of solvent B followed by 2 ml of methanol. The
eluates, now free of long chain bases, were combined, dried, and
dissolved in 1 ml of solvent B. Acyl ester lipids were deacylated by
adding 1 ml of 0.2 N KOH in methanol and incubating for 30 min at room temperature. Further work up by adsorbtion to and elution
from a Chelex resin C18 Celite mixture was as described previously (25). The sphingolipid fraction was dried and suspended in 1 ml of
chloroform/methanol (1:1) and applied to a 3-ml column of silica gel
(Adsorbosil, 100/200 mesh, Applied Sciences, Inc.) equilibrated with
chloroform/methanol (1:1), and neutral catabolites of
[4,5-3H]sphinganine (~11% of the radioactivity) were
eluted with 6 ml of chloroform/methanol (1:1). The
[3H]sphingolipids were eluted with 15 ml of solvent A
containing three drops of concentrated ammonia/5 ml. Only radioactive
sphingolipids were evident by thin layer chromatography (yield,
170 × 106 dpm).
The solvent B extract from the [3H]inositol-labeled cells
was deacylated directly and purified over a C18-Celite
column as above; this procedure was repeated once. Thin layer
chromatography of the final base-stable Celite eluates showed all the
radioactivity to be in various species of sphingolipids (yield, 99 × 106 dpm).
Separation into molecular species was carried out by thin layer
chromatography (Whatman HP-K 200 µm; 10 × 20 cm, solvent C). Each radioactive zone was eluted with solvent A, dried, dissolved in 1 ml of solvent A, and filtered (Acrodisc 4 CR 4 mm; 0.45-µm filters,
Gelman Sciences, Inc.). Purity and identity was verified by subjecting
each molecular species to high performance thin layer chromatography as
above. Unlabeled standards were incorporated in each lane and were
detected by charring (25) after the radioactivity was located (BioScan
apparatus).
Product Analysis of Putative Yeast Membrane Phospholipase C
Action on [3H]Phosphosphingolipids
The reaction mixtures (0.3 ml) consisted of 22 mM
potassium phosphate, pH 7.0, 2 mM dithiothreitol, 5 mM MgCl2, 0.6%
n-octyl-
-glucopyranoside, 0.22 µg of membrane (27)
protein. Also added were 10 nmol of unlabeled sphingolipid (IPC-III,
MIPC-III, or M(IP)2C-III) and equal amounts of
radioactivity of the corresponding [3H]inositol- and
[3H]sphinganine-labeled sphingolipids: IPC-III,
101,000 cpm; MIPC-III, 39,000 cpm; M(IP)2C-III, 200,000 cpm. The
sphingolipids were dried and suspended by sonic treatment in the assay
mixture before the addition of enzyme. After 60 min at 24 °C, the
reaction was terminated by 2-min heating (100 °C) followed by adding
30 µl of 0.5 M Na-EDTA, pH 7.1. Aliquots were
chromatographed on silica gel paper (solvent C). One-cm zones were
subjected to scintillation counting. Water-soluble products were at the
origin, ceramides migrated near the solvent front, and sphingolipids
migrated at these RFs: IPC-III, 0.65; MIPC-III,
0.54; M(IP)2C-III, 0.30. Separate 20-µl aliquots of the
reaction mixtures were chromatographed on silica gel paper with the
solvent chloroform/methanol (19:1.5, v/v) along with ceramide-III
standard. Radioactivity was determined for each lane as above.
Water-soluble products as well as the phosphosphingolipids remain at
the origin with this solvent, while ceramide-III migrates to an
RF factor of 0.66.
To identify the water-soluble fragments of sphingolipids resulting from
a putative phospholipase C reaction, 106 cpm each of
[3H]inositol-labeled IPC-III and M(IP)2C-III
were incubated as above except that no unlabeled sphingolipid was
added, and the reaction was carried out for 4 h, adding an equal
amount of membranes after 2 h. The reaction was terminated as
above. To each sample was added 30 µl of 0.5 M Na-EDTA,
pH 7.1. Aliquots were chromatographed on silica gel paper (solvent C).
Radioactivity in each lane was quantitated as described above. The
polar product(s) at the origin derived from IPC-III and
M(IP)2C-III were 44 and 16% of the total counts,
respectively. Water-soluble products were separated by ion exchange
chromatography by diluting the reaction mixtures with 5 ml of water
followed by centrifugation and application of the supernatant to a
0.6 × 81-cm column of AG1-X2 (200-400 mesh bicarbonate form,
Bio-Rad) equilibrated with 50 ml of 0.1 M ammonium
bicarbonate. Elution was with 0.3 M ammonium bicarbonate, pH 7.9 (flow rate 1.22 ml/min), collecting 8.8-ml fractions.
Radioactive peaks were located and quantitated, pooled, and dried
in vacuo at 70 °C to remove ammonium bicarbonate. Some of
each pooled peak was dissolved in 0.1 ml of 0.2 M ammonium
acetate, pH 8.6, treated with 30 µl of Escherichia coli
alkaline phosphatase (type III, Sigma, 0.33 units/µl) for 3 h at
room temperature, diluted with 2 ml of water, and
chromatographed/quantitated on an AG1-X2 column exactly as described
above. Another portion of the pooled peaks was dried and treated with 1 ml of 10 N NH4OH for 18 h at 150 °C. The labeled ammonolysis products, mannosylinositol and inositol, were
resolved by thin layer chromatography (Whatman HP-K) and developed with
acetonitrile/water (3:1), followed by detection/quantitation with a
BioScan apparatus.
Phospholipase C Assay with Yeast Membranes and
Phosphosphingolipids
The reaction mixture was 25 mM potassium phosphate,
pH 7.0, 2.5 mM dithiothreitol, 5 mM
MgCl2, 0.6% n-octyl-
-glucopyranoside, 33.3 µM [3H]sphingolipid (~105
cpm), 100-400 µg of membrane protein in a 0.3-ml volume. The [3H]sphingolipids were added to the reaction tube, dried,
and suspended by sonic treatment with the assay mixture before the
addition of enzyme. After incubation for 60 min at 24 °C, the
reaction was terminated and processed by two different methods.
Method A--
Sodium dodecylsulfate (0.9 ml, 2.27%) was added
to the reaction mixture. Ceramide was extracted from the mixture with
two 2-ml portions of methyl tert-butylether. The pooled
extracts were washed with 1 ml of water and dried, and radioactivity
was measured. A small no enzyme blank reaction, equivalent to <1%
apparent substrate breakdown, was subtracted from the
membrane-containing samples to calculate the specific activity.
Method B--
The reaction was stopped by the addition of 3 ml
of chloroform/methanol (1:1) and centrifuged. The supernatant was added
to a 1-ml column of AG4-X4 (acetate form, 100-200 mesh, Bio-Rad; packed in water and equilibrated with methanol) and eluted with 3 ml of
chloroform/methanol (1:1). The substrates bound to the resin. The
eluted ceramide was dried and assayed for radioactivity. A no enzyme
blank value was subtracted.
Turnover of Inositol-labeled Lipids
An overnight log phase culture was transferred to 15 ml of fresh
medium containing 1.5 mCi of
myo-[2-3H]inositol (American Radiochemicals,
Inc.) to give a starting A650 = 0.2 and cultured
for 6 h at 24 °C. The cells were rapidly resuspended in 15 ml
of fresh nonradioactive medium and divided in three parts. One part
(zero time) was centrifuged, and the pellet was quenched with 1 ml of 1 M HClO4. The other two parts were incubated at
24 and 39 °C for 20 min followed by rapid centrifugation and
quenching of the cell pellets with HClO4 as above, yielding HClO4 extracts and culture medium fractions, which were
filtered (0.2-µ Teflon Acrodiscs CR, Gelman Sciences). After standing
for 15 min at 0 °C, the HClO4-treated cell pellets were
frozen and thawed twice in dry ice/ethanol and centrifuged at 0 °C.
The supernatants were slowly neutralized (chlorophenol red pH
indicator) at 0 °C with 2.6 M KOH and centrifuged, and
the final supernatants were reserved for ion exchange chromatographic
analysis. The cell pellets were washed twice with water and extracted
for sphingolipids with 1 ml of solvent B for 30 min at 60 °C,
followed by centrifugation while warm. The extract was dried and
deacylated by treatment with 0.5 ml of monomethylamine reagent (28) for
30 min at 50 °C. After evaporation of the reagent, the sample was
dissolved in 1 ml of solvent A, and aliquots were chromatographed on
silica gel paper (solvent C). One-cm zones from each lane were analyzed by scintillation counting to give three groups: the counts near the
origin, representing deacylated phosphatidylinositol, the M(IP)2C zone, and the zones representing incompletely
resolved MIPC plus IPC. Aliquots (0.5 ml) of the three neutralized
HClO4 extracts as well as the filtered culture media were
diluted 10- and 20-fold, respectively, with water and applied to
0.6 × 81-cm columns of AG-1-X2 200-400 mesh resin equilibrated
with 0.1 M ammonium bicarbonate. Elution was carried out at
1.2 ml/min with 0.3 M ammonium bicarbonate, collecting 20 8.6-ml fractions, which were analyzed for radioactivity. Peak fraction
numbers for labeled products were as follows: inositol, 2;
glycerophosphorylinositol, 4; inositol-P and mannosylinositol-P, 6;
mannose-(inositol-P)2, 11.
Incorporation of [3H]Sphinganine into Ceramide and
Phosphosphingolipids
About 7.5 A650 units of mid-log phase
cells were suspended in 5 ml of fresh medium containing 47 × 106 cpm [3H]sphinganine and incubated at 24 or 39 °C. Aliquots (1.4 ml) were removed and terminated with 0.07 ml
of 100% trichloroacetic acid and incubated at least 15 min on ice. The
cells were centrifuged and washed twice with 1 ml of cold water, and
the final pellet was extracted with 0.4 ml of solvent B for 30 min at
60 °C. After centrifuging while still warm, the soluble extract was
processed for further analysis.
For labeled ceramide analysis, [3H]sphinganine was first
removed by applying the solvent B extract to a 0.5-ml column of BioRex 70 (H+) resin (200-400 mesh) packed in water and equilibrated with
methanol. Elution was carried out with 1.5 ml of solvent A and then
with 1 ml of methanol. The combined eluates were dried and dissolved in
0.2 ml of solvent A and subjected to thin layer chromatography (Whatman
K5 plates, solvent CHCl3/methanol, 19:1.5). To each lane,
3.5 nmol of ceramide-III was added. Following radioactivity determination (BioScan apparatus), the plates were sprayed with 10%
(w/v) CuSO4·5H2O in 8%
H3PO4 and charred at 160 °C (26) to locate
the added ceramide standard. The total radioactivity in the ceramide
zone was calculated from the BioRex 70 eluates and from the percentage
distribution of radioactivity from the BioScan analysis of the thin
layer plates.
For phosphosphingolipid analysis, the solvent B extract was dried and
deacylated with 0.5 ml of monomethylamine reagent as above followed by
thin layer chromatography (200-µ Whatman HP-K plates, solvent C).
Each lane contained a mixture of sphingolipid standards, 1-2 µg each
of IPC-III, IPC-III, MIPC-III, and M(IP)2C-III. Following
radioactivity determination (BioScan apparatus), the added sphingolipid
standards were located by charring (26). The total radioactivity in the
phosphosphingolipid zones was calculated from the deacylated solvent B
extracts and from the percentage distribution of radioactivity (Bioscan
apparatus) on the thin layer plates.
Effects of Australifungin and Cycloheximide on Conversion of
[3H]Sphinganine to Ceramide and Phosphosphingolipids
A log phase culture grown at 24 °C was transferred to fresh
medium without Tergitol (A650 = 1.7), and 4.5-ml
samples were incubated for 10 min at 24 °C after the addition of 3 µl of australifungin (500 µg/ml ethanol) or 3 µl of ethanol to
the controls. After the addition of 0.5 ml of 0.5% Tergitol containing
2.5 × 108 dpm [3H]sphinganine,
incubation was continued at 24 and 39 °C. At 20, 40, and 60 min,
1.4-ml aliquots were quenched with 0.07 ml of 100% trichloroacetic
acid. Another experiment was carried out as above, except
cycloheximide, 1 mM final concentration, was added at zero
time. Labeled ceramides and phosphosphingolipids were extracted,
processed, and analyzed by thin layer chromatography as described
above.
 |
RESULTS |
Evidence for Heat Induction of Two Molecular Species of
Ceramide--
S. cerevisiae cells were cultured at 24 °C
and switched to 39 °C, and their extracted lipids were derivatized
and separated by HPLC. It can be seen (Fig.
2) that a significant increase occurs at
39 but not at 24 °C in a double peak that migrates similarly to
ceramide prepared from autolysed commercial bakers' yeast. To
positively identify these two peaks as ceramides, we isolated the
putative ceramide fraction from heat-shocked cells and subjected the
ceramide fraction and the two peaks formed after benzoylation and HPLC
separation to compositional analysis and mass spectrometry. Hydrolysis
of the putative ceramides yielded equimolar amounts of
hydroxyhexacosanoic acid and C18 and C20
phytosphingosines. Fast atom bombardment mass spectrometry of the
ceramide fraction showed two molecular ions with masses 712.7 and
740.7, consistent with N-hydroxyhexacosanoyl-C18
phytosphingosine and N-hydroxyhexacosanoyl-C20 phytosphingosine, respectively (Table I).
After benzoylation and separation by HPLC, peaks I and II exhibited
molecular ions with masses of 1155.8 and 1127.8, respectively, as
expected for tetrabenzoyl-N-hydroxyhexacosanoyl-C20
phytosphingosine and
tetrabenzoyl-N-hydroxyhexacosanoyl-C18 phytosphingosine, respectively (Table I). These molecular species of
ceramide are consistent with the ceramide found in complex yeast
phosphosphingolipids (8).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Assay of ceramide (Cer-III Std)
in heat-shocked cells. Aliquots (50 ml) of a culture grown at
24 °C to an A650 of 0.57 were removed at zero
time and after 30 min of incubation at 24 or 39 °C. Lipid extracts
were benzoylated and subjected to HPLC as described under
"Experimental Procedures."
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Identification of ceramides isolated from heat-shocked cells
A putative ceramide-III fraction was isolated from heat-shocked cells.
Its fatty acid and long chain base composition was analyzed. Masses of
the most abundant molecular ions were determined by fast atom
bombardment (FAB) mass spectrometry. After perbenzoylation, peaks I and
II (Fig. 2) were separated by HPLC, and these molecular species were
subjected to electron impact (EI) mass spectrometry to determine their
molecular ions.
|
|
Accumulation of Ceramide Is Rapid and Specific to Heat
Stress--
We hypothesized that mutant strains lacking sphingolipids
cannot grow at low pH, in high salt, or at high temperature (10) because they are unable to generate sphingolipid second messengers such
as ceramide. To test this hypothesis, we examined wild type cells for
changes in ceramide following stress. A rise in ceramide concentration
can be detected after 10 min of heat treatment at 39 °C. The rise
peaks after 30-40 min and is sustained for at least 2 h (Fig.
3). The 4-5-fold heat-induced ceramide
elevation appears to be specific to heat stress, since neither high
osmotic pressure (Fig. 3A) nor low pH (Fig. 3B)
at 24 °C resulted in marked elevation of ceramide.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Kinetics of ceramide response to stress.
Separate experiments (A, B) were carried out,
starting with cells grown overnight at 24 °C to an
A650 of 0.57 (A) or 0.68 (B). At zero time, solid KCl was added to one set of
cultures to a concentration of 0.75 M (A).
B, one set of cultures was received per 300 ml of culture (2.5 ml concentrated HCl plus 30 ml of 0.5 M glycylglycine,
pH 1.4, to achieve a final pH of 3.0). Aliquots (50 ml) were withdrawn at the indicated times and analyzed for ceramides either as benzoyl derivatives (A) or as the neat lipid extract (B)
as described under "Experimental Procedures." Zero time ceramide
concentrations were 30 (A) and 48 (B)
pmol/A650 unit.
|
|
Enzymatic Hydrolysis of Yeast Phosphosphingolipids Can Generate
Ceramide--
Activation of a sphingomyelinase activity seems to be
responsible for the generation of ceramide in mammalian cells following various stimuli (1, 2). Therefore, we sought evidence for the existence
of a comparable phosphodiesterase, with phospholipase C specificity,
that would hydrolyze yeast phosphoinositol sphingolipids to generate
ceramide. Ceramide
(N-hydroxyhexacosanoylphytosphingosine)-labeled sphingolipids were isolated from cells metabolically labeled with [3H]sphinganine and used as substrates. In one enzyme
assay (method A), the radiolabeled ceramide product was extracted with
methyl tert-butylether. In an alternate assay (method B),
the solvent-treated assay mixture was chromatographed on a small anion
exchange column, which retained the acidic substrates but not the free
ceramides.
[3H]Ceramide was released from [3H]IPC,
[3H]MIPC, and [3H]M(IP)2C by
incubation with crude membrane preparations in a reaction that
absolutely required octyl glucoside and MgCl2 (not shown). Ceramide release was reasonably linear with time, and protein concentration and optimum hydrolysis activity was obtained with 2-3
mol % of M(IP)2C in the mixed micelles, bulk concentration 33 µM (Fig. 4). The pH
optimum for the reaction was about 6-6.5 (Fig. 4).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Ceramide formation by reaction of
[3H]M(IP)2C and [3H]IPC with
putative membrane phospholipase C. [3H]M(IP)2C was reacted with yeast membranes
and processed as per "Experimental Procedures." A and
B, method B; C, method A; D, [3H]IPC-III was the substrate and was processed as
described in the legend to Table II.
|
|
Product Analysis of Yeast Membrane Phosphodiesterase Action on
[3H]Phosphosphingolipids--
Since phosphodiesterase
activity toward yeast phosphosphingolipids has not been previously
reported, it seemed essential to define the stoichiometry and nature of
the products in order to establish whether hydrolysis is between the
phosphorus and ceramide (phospholipase C) or between the phosphorus and
the inositol (phospholipase D), the latter generating phosphoceramide
that would have to undergo further hydrolysis by a phosphatase to yield
ceramide. All of the evidence described below is consistent with
membranes containing an enzyme(s) with phospholipase C-type
phosphodiesterase activities catalyzing the following reactions.
We first reacted sphingolipid substrates containing equal
radioactivity in their ceramide and inositol portion and
chromatographed the entire reaction mixture on silica gel-impregnated
paper. In this system, the water soluble product(s) remain at the
origin, while ceramide and other sphingolipids migrate at the
RFs indicated under "Experimental Procedures."
In the case of IPC-III, equal amounts of radioactivity were found in
the ceramide and origin regions, consistent with phosphodiesterase
activity (Table II). In the case of
M(IP)2C, in addition to ceramide, some MIPC was formed,
requiring that some inositol-P be one of the polar products. The ratio
of ceramide to total polar product radioactivity was as expected for
the action of a phosphodiesterase(s) cleaving M(IP)2C to
yield free ceramide as well as yielding equimolar amounts of
inositol-P and MIPC (Table II). The observed stoichiometry is
consistent with a phosphodiesterase acting on the
phosphosphingolipids.
View this table:
[in this window]
[in a new window]
|
Table II
Analysis of products produced by action of yeast membranes on
phosphosphingolipids
Phosphosphingolipids labeled equally with [3H]inositol and
[3H]sphinganine were reacted with yeast membranes for 60 min,
the reaction mixture was subjected to chromatography on silica
gel-impregnated paper, and the distribution of radioactivity in each
lane was measured as described under "Experimental Procedures."
|
|
Evidence for a phospholipase C-type mechanism for sphingolipid
hydrolysis was obtained by showing that the polar products had alkaline
phosphatase-susceptible phosphomonoester groups. The polar products
generated from [3H]inositol-labeled sphingolipids treated
with membranes were isolated by anion exchange chromatography and then
treated with alkaline phosphatase. The water-soluble products generated
from [3H-inositol]IPC gave a major peak at the retention
time expected for inositol monophosphate (52 ml) as well as a smaller
peak where free inositol would emerge (Table
III). Anion exchange chromatographic analysis after phosphatase treatment showed complete conversion of the
putative inositol phosphate radioactivity to free inositol (Table
III).
View this table:
[in this window]
[in a new window]
|
Table III
Analysis of the water-soluble products formed by the action of yeast
membranes on IPC and M(IP)2C
[3H]IPC-III and M(IP)2C-III were incubated with
membranes, and the water-soluble products were resolved by anion
exchange column chromatography as described under "Experimental
Procedures." The resulting peaks were pooled, treated with alkaline
phosphatase, and subjected to the same anion exchange chromatographic
system.
|
|
Analysis of the water-soluble products derived from
[3H-inositol]M(IP)2C disclosed a small amount
of free inositol and two major products, the first with a net charge of
about
2, consistent with inositol-P, mannosylinositol-P, or both, and
the second with a net charge of about
3, consistent with
inositol-P-mannosylinositol-P (Table III). The charge
2 product, when
treated with phosphatase, completely migrated in the zero net charge
region (Table III), consistent with the reaction(s) inositol-P
inositol + Pi and/or mannosylinositol-P
mannosylinositol + Pi. The second product in the net charge
3 region was completely converted by phosphatase to a product that
migrated to the net charge
1 region (Table III), consistent with the
reaction inositol-P-mannose-inositol-P
inositol-P-mannose-inositol + Pi. The identities of the two radioactive major
M(IP)2C products (Table III) were further established by
subjecting each to ammonolysis conditions that hydrolyze all phosphate
bonds, leaving the mannose-inositol glycosidic bond intact (29). The
radioactive ammonolysis products were resolved by TLC with the
radioactivity being accounted for as inositol and mannosylinositol.
Table IV shows that each major peak gave the same ratio of mannosylinositol to inositol counts as the original M(IP)2C. These data are consistent with the interpretation
that the charge
3 peak (Table III) composition was
inositol-P-mannose-inositol-P and that the charge
2 peak consisted of
about equal amounts of inositol-P and mannose-inositol-P. The digestion
of M(IP)2C (Table III) was carried out for much longer than
the experiment described in Table IV (see "Experimental
Procedures"), probably accounting for a higher proportion of the
reaction M(IP)2C
MIPC + inositol-P.
View this table:
[in this window]
[in a new window]
|
Table IV
Ammonolysis of the water-soluble products produced by the action of
putative phospholipase C on [3H]M(IP)2C
[3H]M(IP)2C was incubated with membranes, and the
water-soluble products were resolved by anion exchange column
chromatography. The resulting peaks were pooled and subjected to
ammonolysis, and the ammonolysis products were resolved by thin layer
chromatography as described under "Experimental Procedures." The
data are the average of two experiments.
|
|
Does Sphingolipid Breakdown by a Phospholipase C Type Enzyme
Account for Heat-induced Ceramide Elevation?--
Since we established
that S. cerevisiae has phospholipase(s) C that can generate
ceramide from sphingolipids, we looked for evidence that catabolism of
phosphosphingolipids might be responsible for heat-induced increases in
ceramide. Because ceramide increases represent only a few percent of
the total potential ceramide in sphingolipids, it was not practical to
look for a heat-induced decrease in sphingolipid levels. We therefore
looked for the increases in the free, water-soluble inositol-containing
sphingolipid head groups that would be generated concomitant with
ceramide formation. We cultured cells at 24 °C in the presence of
[3H]inositol to label the sphingolipids, followed by
incubation in fresh medium for 20 min at 24 and 39 °C. Water-soluble
substances were extracted from the cells with perchloric acid followed
by lipid extraction. The distribution of radioactive lipids was
obtained after paper chromatography. The aqueous cellular fractions as well as the culture medium were subjected to anion exchange
chromatography to look for peaks of radioactivity that increase in the
39 versus 24 °C samples at elution volumes expected for
the sphingolipid head groups (Table III). Based on the radioactivity in
the total sphingolipids (Table V) at zero
time (4,077,000 cpm) and the increase in ceramide expected to result
from heat treatment (Fig. 3) equivalent to breakdown of about 6% of
the sphingolipid (see legend to Table V), we can calculate that the
acidic sphingolipid catabolites should increase by about 258,000 cpm in
the 39 °C sample. One-tenth of that value could be readily detected,
but as can be seen from the data (Table V), there is no increase in
radioactivity at 39 °C compared with 24 °C, in either the
perchloric acid cell extract or the culture medium. It thus appears
unlikely that the ceramide increase following heat shock is due to
breakdown of sphingolipids. We cannot rule out the possibility of an
extremely rapid catabolism of sphingolipid head groups, which would
prevent their detection.
View this table:
[in this window]
[in a new window]
|
Table V
Does heat-induced ceramide elevation arise from catabolism of
inositol sphingolipids?
Cells were cultured with [3H]inositol for 6 h at
24 °C, and then with fresh unlabeled medium for 20 min at either 24 or 39 °C. The cellular HClO4-soluble fraction and the
culture medium were analyzed by anion exchange chromatography to
separate and quantify potential water-soluble sphingolipid head groups
arising from sphingolipid breakdown. Sphingolipids, extracted from the
perchloric acid-treated cells, were separated and measured after paper
chromatography.
|
|
Further support for this conclusion was obtained with a mutant (RCD113)
defective in M(IP)2C synthesis (22), which makes no
detectable M(IP)2C due to the deletion of the
IPT1 gene but accumulates increased levels of MIPC. Strain
RCD113 gave a heat-induced ceramide response equivalent to its cognate
wild type strain (Table VI). Thus,
M(IP)2C breakdown cannot be the source of the elevated ceramide level observed during heat shock.
View this table:
[in this window]
[in a new window]
|
Table VI
Heat-induced ceramide elevation in a strain devoid of M(IP)2C
Cells cultured at 24 °C at a starting A650 = 0.6 were incubated for 20 min at 24 and 39 °C. Ceramide-III was measured
by HPLC after perbenzoylation as described under "Experimental
Procedures."
|
|
Is de Novo Synthesis of Ceramide Responsible for the Heat-induced
Increase?--
In view of all of the results, we considered whether
increased de novo synthesis accounts for heat-induced
increases in ceramide. We showed previously (17) that a change from 24 to 39 °C results in a temporary elevation of the concentration of
sphinganine and phytosphingosine. We therefore determined if exogenous
long chain base alone could increase ceramide levels. When
phytosphingosine was added to cultures at 24 °C, ceramide did not
increase to the level achieved by raising the temperature to 39 °C,
although phytosphingosine, when added at 39 °C, increased ceramide
levels somewhat (Fig. 5). A similar
experiment carried out with 50 µM
DL-sphinganine at 24 °C induced little increase in
ceramide above the untreated control (data not shown). We conclude that
increased long chain base synthesis alone is insufficient to account
for the ceramide increase observed at 39 °C.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of exogenous phytosphingosine
(PHS) on ceramide levels. Cultures (100 ml) grown at
24 °C to an A650 of 0.8 were mixed with 10 ml
of fresh medium with or without phytosphingosine to yield 0, 20, and 100 µM. Incubation was continued at 24 °C (open circles) or 39 °C (closed circles)
with samples processed at 0, 15, and 30 min for ceramide-III analysis
(perbenzoyl derivatives, "Experimental Procedures").
|
|
Further evidence that de novo ceramide synthesis is
responsible for heat-induced ceramide accumulation was sought by
studying the incorporation of [3H]sphinganine into
ceramide at 24 and 39 °C and in the presence of a ceramide synthase
inhibitor. The rate and extent of ceramide-III labeling was higher at
39 than 24 °C (Fig. 6A).
Australifungin, a potent antifungal agent and inhibitor of ceramide
synthase (9), abolished labeling of ceramide at either temperature
(Fig. 6A), indicating that the ceramide synthase responsible
for ceramide labeling in this experiment was sensitive to this
antibiotic. To further implicate ceramide synthase in the
temperature-induced accumulation of ceramide, we determined if
australifungin would prevent the temperature-induced increase in
ceramide as measured by mass. Australifungin at 0.3 µg/ml totally
prevented the temperature-induced accumulation of ceramide (Table
VII). In fact, the ceramide level fell
below the control probably because it was further metabolized to
complex sphingolipids. It thus seems likely that ceramide synthase plays a key role in the temperature-induced accumulation of ceramide.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of australifungin and cycloheximide
on conversion of [3H]sphinganine to ceramide and
phosphosphingolipids. Cells were incubated at 24 °C
(squares) or 39 °C (circles) in the presence of 3.3 µM [3H]sphinganine as described
under "Experimental Procedures." A and C,
ceramide-III and total phosphosphingolipid labeling, respectively, with
0.3 µg/ml australifungin added at zero time (dotted lines) and without australifungin (solid lines). B,
accumulation of radiolabeled ceramide-III in the presence of 1 mM cycloheximide (dotted lines) or in the
absence of cycloheximide (solid lines).
|
|
View this table:
[in this window]
[in a new window]
|
Table VII
Australifungin inhibits heat induction of ceramide-III as
determined by mass measurement
Cells were grown at 24 °C to an A650 of 0.4 and
then incubated for 30 min at 24 or 39 °C in the presence or absence
of 0.3 µg/ml australifungin. Ceramide-III was measured by HPLC after perbenzoylation as described under "Experimental Procedures." The
data represent the mean of duplicate samples.
|
|
If the 39 °C induced increase in ceramide were due to increased
synthesis of the enzyme ceramide synthase, then the enhanced conversion
of [3H]sphinganine to ceramide should be blocked by
cycloheximide. Cycloheximide (1 mM) did not inhibit
ceramide synthesis at either temperature (Fig. 6B) but
actually increased the extent of labeling; thus, it is unlikely that
increased synthesis of ceramide synthase or any other protein mediates
the temperature-induced synthesis of ceramide.
Another explanation for enhanced ceramide accumulation is a reduction
in the rate of conversion of ceramide to complex sphingolipids following a temperature shift, for example, by inhibition of IPC synthase (Fig. 1). However, the temperature shift from 24 to 39 °C does not inhibit but rather increases total radiolabeling of complex sphingolipids by [3H]sphinganine, although not to the
extent that it enhances ceramide labeling; as expected, sphingolipid
labeling is abolished by australifungin (Fig. 6C).
Finally, although australifungin is a potent ceramide synthase
inhibitor, it has not been studied enough to know if other reactions
are affected. Specifically, we sought to rule out the possibility that
it might inhibit enzymatic hydrolysis of complex sphingolipids yielding
ceramide. Australifungin, at concentrations up to 10-fold higher than
those that abolished in vivo ceramide synthesis (Fig. 5,
Table VI), was without effect on the membrane-catalyzed hydrolysis of
IPC and M(IP)2C (not shown). We conclude from this experiment and all of the other data presented in this section that
increased de novo ceramide synthesis accounts for the
heat-induced ceramide elevation.
 |
DISCUSSION |
De Novo Synthesis Is Responsible for Heat-induced Elevation of
Ceramide--
Our experiments demonstrate that de novo
synthesis, not breakdown of sphingolipids, is the primary mechanism for
generating an increased level of ceramide following a shift from 24 to
39 °C. To show that de novo synthesis of ceramide via
ceramide synthase (Fig. 1) was essential for a increased ceramide, we
employed australifungin, a potent antifungal drug shown to be an
inhibitor of ceramide synthase (9). The heat-induced increase in
ceramide as measured directly by mass as well as by incorporation of
[3H]sphinganine into ceramide, were completely inhibited
by australifungin (Table VII, Fig. 6), thus identifying the
essentiality of ceramide synthase for the heat-induced ceramide
accumulation.
The question of what reaction(s) leading to increased ceramide are
affected by temperature elevation is complex and largely unanswered.
One possible explanation could be differential temperature effects on
the rate constants of the enzymatic reactions leading to ceramide
synthesis and to its further metabolism. An additional explanation is
that elevated temperature could lead to alterations of the amount
and/or structure of one or more enzymes of ceramide metabolism.
Increased enzyme synthesis does not play a role as judged by the lack
of effect of cycloheximide on ceramide synthesis (Fig. 6B).
A heat-induced decreased rate of ceramide conversion to IPC and more
complex sphingolipids, which could explain ceramide accumulation, is
also not evident from the experiment measuring the incorporation of
[3H]sphinganine into sphingolipids (Fig. 6C).
Thus, the enzymes that are likely candidates for temperature regulation
are those involved in generating ceramide precursors as well as
ceramide synthase itself. Earlier work showed (17) that heat shock
causes a rapid and temporary rise in the concentration of sphinganine and phytosphingosine, precursors of ceramide. However, increased exogenous long chain base by itself appears to be inadequate to account
for increased ceramide, since exogenous long chain bases added in
excess at 24 °C did not stimulate ceramide concentrations to the
level achieved at 39 °C (Fig. 5). The mechanism of heat-induced ceramide accumulation merits further analysis, especially since exogenous sphinganine activates trehalose accumulation at 24 °C via
gene activation (17), thus implicating yeast sphingolipids in a well
known stress response.
Ligand activation of a sphingomyelinase, the "sphingomyelin cycle"
(5), has been the most studied reaction to account for stress-induced
ceramide generation in animal cells. However, two studies with animal
cells implicated ceramide synthase in ceramide generation.
Daunorubicin-induced apoptosis and ceramide elevation were prevented by
the ceramide synthase inhibitor, fumonisin B1 (6); however, other
workers claim sphingomyelin hydrolysis is associated with
daunorubicin-induced apoptosis (30). Fumonisin has been reported to
inhibit ceramide elevation associated with macrophage activation (7).
Two studies in animals cells have observed heat shock-induced elevation
in ceramide by as yet undefined mechanisms (31, 32). Future work needs
to be directed at the unknown mechanism(s) of temperature regulation of
ceramide synthase activity in both animal cells and yeast.
Phospholipase C Type Activity in Yeast Utilizing
Phosphoinositol-containing Sphingolipids--
Generation of
ceramides by activation of a sphingomyelinase,
is the predominant paradigm in mammalian cells for the formation
of mediators in various signaling pathways with diverse outcomes
(1-4). We therefore looked for a comparable phosphodiesterase activity
in yeast to explain the heat-induced increase in ceramide. Our data
suggest the existence of one or more phosphodiesterases in S. cerevisiae membranes capable of catalyzing the hydrolysis of yeast
sphingolipids to yield ceramide,
where R represents hydrogen, mannose, or inositol-P-(mannose).
With sphingolipids containing [3H]ceramide as well as
[3H]inositol in the head groups, the above stoichiometry
was demonstrated (Tables II-IV). Furthermore, the ceramides from
heat-shocked cells were isolated, and their chemical composition was
confirmed by chemical analysis and by mass spectrometry (Table I).
These analyses as well as ceramide analysis in the various heat shock
experiments were all performed by chemical methods, a noteworthy
observation in light of the recent challenge of the validity of
ceramide measurements made by many investigators employing the enzyme
diacylglycerol kinase for ceramide analysis (18).
Another phosphodiesterase with activity toward substrates containing
ceramide has been described in S. cerevisiae. Ella et al. (13) characterized a sphingomyelinase activity, partially purified from S. cerevisiae membranes, capable of generating
ceramide from sphingomyelin. However, this enzyme preparation had no
activity toward yeast phosphoinositol sphingolipids. This enzyme was
dependent on a divalent cation, as was the sphingolipase activity we
describe, but was inhibited by octyl glucoside and other detergents
unlike our enzyme activity. It should be noted that sphingomyelin has not yet been reported to occur in S. cerevisiae. The
sphingomyelinase of Ella et al. (13) is not likely to be a
phosphoinositol sphingolipid hydrolase.
Several groups (33-36) have reported on a putative phospholipase C
gene (PLC1) in S. cerevisiae, which upon
deletion, results reportedly in differing phenotypes such as lethality
or very slow, temperature-sensitive growth, etc. Plc1p was purified as
a soluble enzyme after overexpression and was shown to catalyze the
following reactions (36).
We tested a sample of this enzyme (generously supplied by Dr.
Jeremy Thorner) and found it to be without effect on yeast phosphoinositol sphingolipids when assayed (data not shown) as described (36). Furthermore, we assayed hydrolysis of
[3H]IPC and [3H]M(IP)2C with
membranes prepared from strain YJF132 (36) carrying a plc1
deletion as well as from the cognate wild type strain YJF131 (36). The
plc1-deleted strain had about 50% of the sphingolipid hydrolase wild type activities. Whether the lowered activity is related
to the very slow growth of the mutant strain and/or some indirect
effect of the lack of plc1p on the regulation of sphingolipid phospholipase activity is unclear. Nonetheless, it is clear that substantial sphingolipid hydrolase activity remains in the
plc1 deletion strain, and thus plc1 is unlikely
to code for sphingolipid hydrolase activity.
We previously examined the turnover of [3H]inositol
labeled lipids with uniformly labeled S. cerevisiae cells
transferred to nonradioactive growth medium. Although a large decrease
in the phosphatidylinositol pool could be readily observed, consonant with its conversion to sphingolipids and extracellular
glycerophosphoinositol, no decrease in the total sphingolipid pool
consistent with sphingolipid turnover was observed after many hours
except for that interpreted as the conversion of IPC plus MIPC to
M(IP)2C (37). If heat-induced ceramide elevation were due
to sphingolipid breakdown, then only quite small sphingolipid changes
could be expected. To obtain in vivo evidence of a small
sphingolipid turnover (about 6% of the total sphingolipid) that might
be associated with the heat-induced ceramide increase, the approach
taken was to look for the appearance of
[3H]inositol-labeled, water-soluble sphingolipid head
groups that could result from the action of a phospholipase C on the
various yeast sphingolipids. No more than 3% of the expected products could be found (Table V), making it unlikely that sphingolipid hydrolysis was generating ceramide. One cannot exclude an extremely rapid catabolism of sphingolipid head groups masking any accumulation. In conclusion, the absence of sphingolipid head group accumulation and
the observed australifungin-sensitive nature of ceramide elevation make
de novo ceramide synthesis the most likely mechanism for heat-induced ceramide elevation.