From the Department of Biochemistry and Molecular
Biology, Medical University of South Carolina, Charleston, South
Carolina 29482 and the § Department of Cell Biology, Duke
University, Durham, North Carolina 27710
Received for publication, August 15, 2000, and in revised form, September 27, 2000
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ABSTRACT |
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The recent findings of sphingolipids
as potential mediators of yeast heat stress responses led us to
investigate their possible role in the heat-induced cell cycle arrest
and subsequent recovery. The sphingolipid-deficient yeast strain 7R4
was found to lack the cell cycle arrest seen in the isogenic wild type.
Furthermore, strain lcb1-100, which harbors a temperature-sensitive
serine palmitoyltransferase, lacked increased de novo
generated sphingoid bases upon heat stress. Importantly, this strain
was found to lack the transient heat-induced
G0/G1 arrest. These results indicate a
role for sphingolipids and specifically those generated in the de
novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an
analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base
kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the
knockout of a fatty acyl elongation enzyme, which severely attenuates
ceramide production, displayed the arrest. These experiments suggested
that the active species for cell cycle arrest were the sphingoid bases.
In further support of these findings, exogenous phytosphingosine (10 µM) was found to induce transient arrest. Stearylamine
did not induce an arrest, demonstrating chemical specificity, and
L-erythro- was not as potent as
D-erythro-dihydrosphingosine showing
stereospecificity. To investigate a possible arrest mechanism, we
studied the hyperstable Cln3 (Cln3-1) strain LDW6A that
has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found
to be dependent upon the sphingoid base kinases (LCB4,
LCB5). Overall, this combination of genetic and
pharmacologic results demonstrates a role for de novo
sphingoid base biosynthesis by serine palmitoyltransferase in the
transient G0/G1 arrest mediated through Cln3
via a novel mechanism.
Saccharomyces cerevisiae has been shown to respond to
an increase in temperature from 30 to 39 °C with a physiology known as the heat stress response (1, 2), which involves two phases. The
initial phase is the gaining of thermotolerance through an accumulation
of trehalose (3), an induction of the heat shock proteins (4), and a
transient arrest of the cells in the G0/G1 phase of the cell cycle (5). The transient cell cycle arrest is
characterized as a decrease in budding after 1 h of heat stress (6). The decrease in budding has been shown to be blocked by expression
of a hyperstable Cln3, indicating a role for G1 cyclins in
this response (7). Furthermore, G1 cyclins are regulators of entry into S phase at the point known as START, and, upon
heat stress, transcript levels of Cln1 and Cln2 are decreased (7). A
decrease in available G1 cyclins induces a
G0/G1 arrest. After thermotolerance has been
achieved, the second phase of the heat stress response is characterized
by a resumption of normal growth at the elevated temperature. This
phase is marked by a HSP-70-dependent process of trehalose
degradation (8) and resumption of a normal cell cycle as seen by a
recovery of budding by 2 h of heat stress (6). The mechanisms
involved in regulating the cell cycle response to heat are not yet defined.
Examination of suppressor mutants of the lethal deletion of serine
palmitoyltransferase subunit (LCB1) has begun to shed light on the roles of sphingolipids in the yeast heat stress response (9-11). The suppressor makes novel inositol glycerolipids that mimic
the inositol phosphoceramides, thus allowing for growth under normal
conditions (12, 13). However, under heat stress and other stress
conditions, the suppressor mutants were found to be defective in growth
as compared with the isogenic wild type yeast strain (14). Thus, the
study of these mutants resulted in the finding that sphingolipids are
necessary for the yeast heat stress response (9, 11).
The induction of de novo synthesis of free sphingoid bases
and yeast ceramides upon heat stress have suggested a role for these
lipids as possible signaling moieties. Already, two such functions have
been attributed to sphingolipids in the yeast heat stress response.
First, addition of exogenous sphingolipids was shown to induce a
reporter gene attached to a stress response element (10). Second, it
was shown that sphingoid bases mediate a
ubiquitin-dependent degradation of nutrient permeases in
response to heat stress (15).
The current study examines the role of sphingolipids in the
heat-induced transient cell cycle arrest. An initial role for sphingolipids was disclosed by experiments on the sphingolipid deficient strain 7R4. Also, the temperature-sensitive serine
palmitoyltransferase strain lcb1-100 was found to be defective in the
transient cell cycle arrest, indicating a requirement for de
novo generated sphingolipids in this effect. The role of specific
sphingolipids in the cell cycle arrest was investigated using knockouts
of sphingolipid enzymes, and these results implicate the free sphingoid
bases as the bioactive species. A yeast strain containing the
hyperstable Cln3-1 showed this cyclin to act downstream of the
sphingoid base-induced arrest. Finally, a role of the sphingoid base
kinases in cell cycle recovery was determined, and is suggested to
likely be through clearance of the sphingoid bases rather than the
formation of the phosphorylated sphingoid bases per se.
These data show that de novo synthesis of sphingoid base is
involved in the transient cell cycle arrest likely through Cln3 and
that recovery from the cell cycle arrest is partially mediated by the
sphingoid base kinases.
Chemicals, Compounds, and Yeast
Strains--
D-erythro-Sphingosine,
phytosphingosine, RNase, proteinase K, and stearylamine were obtained
from Sigma. Both L-erythro- and D-erythro-dihydrosphingosine were from Avanti.
Tritiated palmitate and tritiated palmitoyl-CoA were obtained
from American Radiolabeled Chemicals. Propidium iodide was from
Molecular Probes. ENHANCE was from PerkinElmer Life Sciences.
L-threo-Dihydrosphingosine was synthesized as
previously noted (16). Yeast strains used in this study are listed in
Table I with genotype and source.
Budding Determination--
The percentage of budded cells upon
heat stress was determined by seeding 1-2 × 107
cells/ml in 5 ml of yeast extract, bactopeptone and dextrose (YEPD)1 media. Cells were
rested for 6 h to allow for growth to mid log phase and then grown
at 30 or 39 °C. At the given time points, cells were pelleted at
4 °C and resuspended in 100 µl of ice-cold water. The resuspended
cells were then put on microscope slides and imaged. Representative
fields were photographed at 100× magnification, and photographs were
subsequently scored for budding.
Serine Palmitoyltransferase Assay--
Protein was extracted
from a 5-ml mid log phase culture. The cells were pelleted, washed in
ice-cold water, and repelleted. Cells were then resuspended in ~0.5
ml of extraction buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 1 mM EDTA, 1 mM EGTA,
0.1% 2-mercaptoethanol, and a 5 µg/ml concentration of chymotrypsin, leupeptin, aprotinin, and pepstatin). Glass beads were added and cells
lysed by ten cycles of vortexing for 30 s. Beads and large cellular debris were pelleted, and the supernatant was recovered. An
aliquot was used for the Bio-Rad Bradford protein determination with
bovine serum albumin as standard. Serine palmitoyltransferase activity
of the protein samples was measured under the following conditions. The
reaction buffer consisted of 0.1 M HEPES, 5 mM dithiothreitol, 2.5 mM EDTA (pH7.4), 50 µM
pyridoxal 5'-phosphate, 0.2 mM palmitoyl-CoA, 5 mM serine, and 200 µg of protein. The reaction was
started with the addition of tritiated palmitoyl-CoA (with 0.2 mM cold as carrier) and run for 30 min at 30 °C. The reaction was stopped with 1.5 ml of chloroform:methanol (1:2, v/v).
Dihydrosphingosine (50 µg) was added as carrier. To induce the phase
break, 1 ml of chloroform and 1 ml of 0.5 N ammonia hydroxide were added and the solution was vortexed. The aqueous phase
was discarded, and the organic phase was dried down. The organic
extract was then resuspended in chloroform, spotted on a plate for thin
layer chromatography (TLC), and developed in a solvent system of
chloroform:methanol:2 N ammonia hydroxide (40:10:1, v/v).
The plate was then sprayed with EnHance and exposed to film. The spot
comigrating with keto-dihydrosphingosine was scraped and counted. A
blank of no protein was extracted as background.
Labeling Cells with Palmitate--
Two milliliters of YEPD media
was seeded with 5 × 106 cells/ml and rested for
2 h. Tritiated palmitate (1 µCi/ml) was added, and the cells
were incubated for 6 h. Cells were pelleted, washed with water,
and extracted as described (17). The lipids were dried down and
base-hydrolyzed as described previously (18). The resulting lipids were
dried down and resuspended in 75 µl of chloroform:methanol:water
(1:2:0.1, v/v) and spotted onto a TLC. The TLC plate was developed in a
solvent system of chloroform:methanol:4.2 N ammonia
hydroxide (9:7:2, v/v). Results were imaged by spraying with ENHANCE
and exposing to film for 3 days.
Extraction and Analysis of Sphingoid Bases--
A total of
2 × 109 cells were taken from an overnight YEPD
culture. Extraction was done as described previously via a modified Bligh and Dyer extraction (9, 19). Lipids were then dried down,
and two-thirds were used for sphingoid measurement with the other
one-third used for organic phosphate measurement. The lipids for
sphingoid base measurements were derivatized with
ortho-phthaldialdehyde and separated over a reverse phase
C18 column with detection by a Shimadzu fluorescent detector
essentially as described (20). Samples were compared by use of the
nonendogenous sphingoid base L-threo-dihydrosphingosine as an internal
standard added before the lipid extraction.
Cell Cycle Studies--
Yeast cells were seeded at 5 × 105 to 2 × 106 cells/ml in YEPD media and
rested 6-8 h at 30 °C. Yeast cells were then treated under the
given conditions of temperature and or lipid treatment. At the given
time points a control and treated sample were taken and centrifuged.
The cell pellet was washed twice with ice cold water and then fixed in
1 ml of 70% ethanol, all at 4 °C. After fixing overnight, the cells
were again pelleted, washed with 5 ml of 50 mM sodium
citrate, and then resuspended in 1 ml of sodium citrate. RNase was then
added and incubated for 1 h at 50 °C, and then proteinase K was
added and incubated under the same conditions. Finally, 0.85 ml of
sodium citrate and 0.15 ml of propidium iodide solutions were added.
Cells were kept 24 or more h in the dark at 4 °C. Cells (15,000)
were analyzed per histogram using a Becton Dickinson
fluorescence-activated cell analyzer. Data were modeled using the
program ModFit LT.
Requirement for Sphingolipids in Heat-induced Transient Cell Cycle
Arrest--
The role of sphingolipids in the heat-induced transient
cell cycle arrest was investigated in the sphingolipid-deficient
strain, 7R4, and its isogenic wild type 7R4-LCB1. Strain 7R4-LCB1
showed the expected arrest at 1 h for 39 °C as evaluated by
budding (Fig. 1A). Budded
7R4-LCB1 cells decreased from 66% in the asynchronous samples to 24%
at 1 h of heat stress and recovered to 62% budded by 2 h of
heat stress. In contrast, the sphingolipid-deficient strain 7R4 did not
arrest. Strain 7R4 had 58% budded cells in an asynchronous culture and
upon 1 h at 39 °C maintained 60% budded cells, with 55% of
cells still budded at 2 h (Fig. 1B). Because the only
difference between these two strains is the presence or absence of
sphingolipids, these results show that lack of sphingolipids results in
an inability to transiently arrest upon heat stress.
de Novo Formation of Sphingolipids through Serine
Palmitoyltransferase Is Necessary for Heat-induced Transient
G0/G1 Arrest--
To investigate the role of
de novo sphingolipid synthesis in the heat-induced arrest,
strain lcb1-100 was characterized in terms of serine
palmitoyltransferase activity, production of sphingolipids at 30 °C,
and increased de novo synthesis of sphingoid bases upon heat
stress. This temperature-sensitive strain, lcb1-100, was isolated as a
secretory mutant (end8-1) containing a point mutation in the
LCB1 gene (21), which is a necessary component of
serine palmitoyltransferase (22). Proteins from the strains lcb1-100, RH406, 7R4-LCB1, and 7R4 were extracted to compare the in
vitro serine palmitoyltransferase activity. Protein extract from
strain 7R4-LCB1 produced 2.88 nmol of keto-dihydrosphingosine per
milligram of protein, and protein extract from strain RH406 produced
1.52 nmol of keto-dihydrosphingosine per milligram of protein (Fig. 2A). Protein extract from the
LCB1 deletion strain 7R4 showed minimal activity (0.064 nmol/mg of protein), as did the lcb1-100 (0.082 nmol/mg of protein)
protein extract (Fig. 2A). Thus, protein extracted from
lcb1-100 had minimal in vitro serine palmitoyltransferase activity compared with the wild type protein extracts. To determine if
in vitro inactivity of serine palmitoyltransferase from
lcb1-100 affected the steady-state sphingolipid synthesis in
vivo, we labeled both wild type and the lcb1-100 strains with
tritiated palmitate for 6 h at 30 °C. Palmitate is converted to
palmitoyl-CoA by the cells and then condensed with serine, by serine
palmitoyltransferase, to form keto-dihydrosphingosine (Fig. 4).
Keto-dihydrosphingosine is then further metabolized to the complex
sphingolipids. The base-hydrolyzed organic extract of strain lcb1-100
had labeled bands corresponding to sphingoid bases and complex
sphingolipids comparable to the levels seen in the RH406 extract
(Fig. 2B). Bands corresponding to dihydrosphingosine,
phytosphingosine, inositol phosphoceramide, mannose inositol
phosphoceramide, and mannose (inositol-phosphate)2 ceramide
were present in both extracts (Fig. 2B). Furthermore, high
pressure liquid chromatographic analysis of Bligh and Dyer extracts of
the lcb1-100 strain were found to contain normal levels of the expected
four species of sphingoid bases found in yeast (data not shown).
Therefore, despite minimal in vitro enzyme activity,
lcb1-100 was found able to produce sphingolipid levels comparable to
those of the isogenic wild type at 30 °C. These results suggest that
the point mutation in LCB1 renders the enzyme activity
labile in cell extracts but normal function is maintained in
vivo.
Heat stress has been shown previously to induce a transient increase in
sphingoid bases of wild type strains. Therefore, the increased
production of sphingoid bases upon heat stress was examined in the wild
type yeast strain RH406 (Fig. 2C) and heat-sensitive serine
palmitoyltransferase strain lcb1-100 (Fig. 2D). Lipid
extracts from RH406 showed increases in C18 phytosphingosine (252%),
C20 phytosphingosine (435%), C18 dihydrosphingosine (238%), and C20 dihydrosphingosine (289%) by 15 min at 39 °C. In contrast the lcb1-100 strain lipid extracts showed no increases in any of the four
measured sphingoid bases (Fig. 2D). Therefore, lcb1-100
yeast cells have normal steady-state levels of sphingolipids, but were unable to produce an increase of the sphingoid bases upon heat stress.
Next strains lcb1-100 and RH406 were evaluated for the transient cell
cycle arrest by both budding and cell cycle analysis. The RH406 cells
showed a large arrest at 1 h of heat stress with only 24% of the
cells budded compared with 64% of cells budded in the asynchronous
culture. The cell cycle analysis of RH406 showed large
G0/G1 and G2/M peaks with an
intervening S phase area (Fig. 3) and
some post G2/M cells. The cell cycle plot showed a
significant loss of S phase area at the 1 h of 39 °C time point corresponding to the loss in budding. Therefore, the transient G0/G1 arrest seen at 1 h of heat stress by
budding appeared as a loss of S phase area in the cell cycle analysis.
In contrast to the RH406 budding loss, the percentage of budded
lcb1-100 cells did not change with 1 h (67%) of heat stress as
compared with asynchronous cells at 30 °C (67%). The cell cycle of
lcb1-100 displayed a pre-G0/G1 peak of debris
and then peaks for G0/G1 and G2/M
with an intervening S phase area. The asynchronous cell cycle profile
of lcb1-100 was maintained (Fig. 3) at 1 h of heat stress,
corresponding to the lack of change in the percentage of budded cells.
These data demonstrate a necessary role for the de novo
induction of sphingolipids via serine palmitoyltransferase activity in
the heat-induced transient G0/G1 arrest.
Epistasis Analysis of Sphingolipid Mutants Suggests That Free
Sphingoid Bases Are the Likely Active Species in the Cell Cycle Arrest
Response to Heat Stress--
The recent cloning of many of the genes
involved in the sphingolipid synthesis and breakdown pathways (Fig.
4) now allows for the analysis of
bioactive species by utilizing key mutants in these pathways.
Production of sphingoid base phosphates has been shown to occur mainly
through Lcb4 and minimally through Lcb5 (23). Table II shows that each of these knockouts
arrested and recovered upon heat stress as measured by the percentage
of S phase. Moreover, the double-knockout of both LCB4 and
LCB5 also arrested (S phase not detectable). However, the
double deletion of LCB4 and LCB5 did not recover by 2 h of heat
stress as shown by no detectable cells in the S phase area. The
isogenic wild type strain JK93d showed no detectable cells S phase area
at 1 h of 39 °C and recovered to 34% S phase by 2 h of
39 °C. These data show that the two sphingoid base kinases are not
required for the transient arrest but are required for the recovery
phase.
Ceramide production has been found to be greatly decreased in the
ELO2 deletion mutant, probably as a result of a deficiency in the very long chain fatty acids that are normally acylated at the
amino group of the sphingoid bases (24). The knockout of
ELO2 was arrested (S phase not detectable) and recovered
(38%) as well as the isogenic wild type JK93d. Therefore, ceramide
production is not likely to play a role in the transient arrest induced
at 39 °C.
Also tested were the knockout of YSR2, a sphingoid
base phosphate phosphatase (18) and the knockout of DPL1,
the dihydrosphingosine phosphate lyase (25) (Table II). The knockout of
YSR2 became arrested (2% S phase) and recovered (33% S
phase) upon heat stress. The knockout strain of DPL1
also arrested with 4% S phase at 1 h at 39 °C. By 2 h at
39 °C, the knockout strain of DPL1 recovered to 13% of cells in S
phase. These data provide further evidence that the bioactive species
are not the sphingoid base phosphates.
Finally, the knockout strain of SYR2, the dihydrosphingosine
hydroxylase, which catalyzes conversion of dihydrosphingosine to
phytosphingosine (26), was analyzed for cell cycle upon heat stress.
This strain arrested at 1 h (2% S phase) and recovered by 2 h (13% S phase) at 39 °C. Similar results were obtained using the
isogenic wild type W303 (Table II). This indicates that
phytosphingosine is not needed for arrest, but still could be
sufficient to induce arrest (see below). Overall, the epistasis
analysis indicates that sphingoid bases themselves are the best
candidates for the active molecules responsible for mediating the cell
cycle arrest.
Exogenous Sphingoid Bases Cause a Transient Cell Cycle
Arrest--
The conclusion from the epistasis analysis that the
sphingoid bases are the bioactive agents in the cell cycle arrest led us to investigate the effects of exogenous sphingoid bases. Despite being found not to be necessary for the cell cycle arrest by the aforementioned genetic analysis, phytosphingosine was found to induce
arrest as measured by a decrease in S phase. Thus exogenous phytosphingosine was found to cause an arrest at 10 µM or
higher (Fig. 5A) by 30 min of
treatment. The arrest by phytosphingosine was very rapid in that S
phase was significantly decreased by 20 min to 11% compared with an
asynchronous baseline of 30% and caused a nearly complete arrest (4%
S phase) at 30 min (Fig. 5B). Treatment with 20 and 40 µM phytosphingosine showed a large decrease in S phase at
20 min and maintained a full arrest for up to 60 min (data not shown).
Treatment of 10 µM phytosphingosine showed a rapid
recovery starting at forty 5 min (22% S phase), whereas higher
concentrations were slowed in recovery of S phase.
The specificity of exogenous sphingoid bases to induce cell cycle
arrest was next examined. Phytosphingosine,
D-erythro-sphingosine, and
D-erythro-dihydrosphingosine were found to be
nearly equally effective (Table III) at
treatments of 20 µM for 30 min. The percentage of cells
in S phase was 2% for phytosphingosine, 3% for
D-erythro-dihydrosphingosine and 3% for
D-erythro-sphingosine at this dose and time
point. Next, the chemically similar lipid stearylamine was tested and found not to induce an arrest at 30 min (22% S phase). Finally, stereospecificity was evaluated by use of
L-erythro-dihydrosphingosine versus
D-erythro-dihydrosphingosine.
L-erythro-Dihydrosphingosine induced a partial
arrest at 30 min (7.50% S phase) but was not as effective as the
natural D-erythro-dihydrosphingosine (3% S phase).
Short chain ceramides were not used as exogenous treatment, because
they are quickly taken up and, once taken up, are rapidly broken down
to their sphingoid base
precursors.2 Overall, these
data show significant specificity to the naturally occurring sphingoid bases.
Cln3 Acts Downstream of the Sphingoid Bases in the Cell Cycle
Arrest Pathway--
Cyclins have been shown to be key players in the
heat stress-induced (7) and alpha factor-induced
G0/G1 arrest (27). Furthermore, previous
studies have shown that a hyperstable Cln3 (CLN3-1) is
resistant to cell cycle arrest upon heat stress (7). Therefore, it
became of interest to determine whether cyclins function downstream of
the sphingoid bases. Strain LDW6A (CLN3-1) and its isogenic
wild type (GR2) were tested in response to heat stress. As expected,
LDW6A was found to lack the heat stress-induced cell cycle arrest seen
in the wild type GR2 (7) (data not shown). The effect of treatment with
20 µM phytosphingosine on the cell cycles of strains GR2
and LDW6A was next evaluated. Strain GR2 showed a loss of the S phase
area in its cell cycle profile at 30 min, with a recovered S phase area
by 120 min (Fig. 6A). The Cln3-1-containing strain LDW6A did not show any loss of S phase area
(Fig. 6B) over the time course of phytosphingosine
treatment. Therefore, Cln3 functions downstream of the sphingoid bases
to prevent their effect on inducing a transient cell cycle arrest.
Possible regulators of Cln3 were also evaluated to determine whether
they are required in the heat-induced cell cycle arrest pathway. As
cyclin Cln3 has been shown to be also regulated by the alpha
factor/MAPK pathway (27), the knockout strain of the MEKK
(STE11) of this pathway was tested with heat stress.
Heat-stressed
Cyclin Cln3 has also been shown to be ubiquitinated and subsequently
degraded (28), suggesting a possible mechanism of regulation. Also, it
was previously shown that de novo synthesis of sphingolipids regulates a ubiquitin pathway through Doa1 (15). Therefore, the
knockout strain of a regulatory component of the proteasome pathway,
DOA1, was evaluated and found to arrest (Table IV) at 45 min
of heat stress with 1% of the cells in S phase. These data indicate
that the ubiquitination and degradation pathway is not required for the
heat-induced cell cycle arrest.
The Sphingoid Base Kinases Likely Mediate Cell Cycle Recovery by
Sphingoid Base Clearance--
The aforementioned epistasis analysis
led to the finding that the double-knockout of the sphingoid base
kinases was deficient in the recovery from the heat-induced cell cycle.
Therefore, the
The sphingoid base kinases have been shown to act on phytosphingosine,
sphingosine, and dihydrosphingosine (23). However, the yeast
biosynthetic pathway through ceramide synthase appears to be able to
incorporate only the endogenous sphingoid bases, phytosphingosine and
dihydrosphingosine, and not sphingosine.2 Therefore,
sphingosine appears to go through the kinases for clearance and or
conversion to other products. Therefore, we first tested the effect of
phytosphingosine on the kinase double-knockout strain. Treatment of the
The need for sphingolipids in the heat stress response has been
established with several studies, which show that sphingolipids provide
a distinct advantage in growth of yeast at higher temperatures (9-11,
15). The questions now being addressed in current research are 3-fold.
First, the source of the sphingolipids generated in response to heat
stress are being studied as well as the mechanisms involved in
regulating sphingolipid metabolism. Second, the various sphingolipids
generated are being examined for possible roles as bioactive molecules.
Finally, the mechanisms by which sphingolipids mediate their roles in
the heat stress response are being evaluated. Recent research has shown
large increases in the sphingoid bases and, subsequently, the ceramides
upon heat stress (9, 11). The primary source of the increased levels of
sphingoid bases has been shown to be by de novo synthesis
(9, 11). The data presented in this study and other studies are
beginning to define key roles for the de novo production of
sphingoid bases through serine palmitoyltransferase.
The current study defines a vital role for de novo
production of sphingolipids as needed for the transient cell cycle
arrest, which occurs upon an upshift in temperature from 30 to
39 °C. Using the strain with a suppressor of the LCB1
knockout, 7R4, we showed that sphingolipids are needed for the
aforementioned arrest. Strain lcb1-100 was characterized and found to
make the normal complement of sphingolipids at 30 °C but to lack the
increased de novo synthesis of sphingoid bases upon heat
stress. Importantly, this lcb1-100 strain was found to be deficient in
the transient cell cycle arrest seen after heat stress. Therefore, the
immediate conclusion from this study is that this cell cycle arrest is
dependent upon the de novo production of sphingolipids.
To address the question of which sphingolipid(s) is (are) a candidate
for regulating this process, we performed an analysis of the key
mutants of the sphingolipid biosynthetic and breakdown pathways in
terms of the heat-induced transient cell cycle arrest. These data
showed that the active species was most likely the free sphingoid
bases. This finding was furthered by studies showing that exogenous
treatment with sphingoid bases was able to induce the transient arrest
in the cell cycle, thus reproducing the effects of heat stress.
The transient G0/G1 arrest caused by heat
stress has been shown to be blocked by the hyperstable Cln3-1 (7). In
this study, it was found that a strain containing the hyperstable Cln3
was found to not arrest upon treatment with 20 µM
phytosphingosine. Therefore, Cln3 acts downstream of sphingoid bases in
the pathway leading to cell cycle arrest. To try and further define
these pathways, we tested three likely candidates. Two MAPK pathways thought or known to regulate Cln3 are the alpha factor pathway and
stress-induced pathway (27). However, in the case of heat stress-induced cell cycle arrest, the two MAPK pathways tested were
found to not be involved.
Next, the ubiquitin pathway was tested because Cln3 has been shown to
be phosphorylated, then ubiquitinated, and finally degraded (28). This
form of regulation was further suggested by the recent finding of
sphingolipid regulation of ubiquitinization and proteolysis of nutrient
permeases (15). However, regulation by this mechanism was shown to not
be involved in the cell cycle arrest. Thus, our research opens the
possibility of a novel mechanism of regulation by sphingoid bases of
Cln3-mediated cell cycle arrest.
On the other hand, recovery from the heat-induced cell cycle arrest was
found to be mediated through the sphingoid base kinases (LCB4, LCB5). We considered two possible roles
for the kinases in this effect. Either the sphingoid base phosphates,
the products of the sphingoid base kinases, are needed for recovery of
an asynchronous cell cycle, or the kinases are required for the
clearance of the sphingoid bases to remove the signal of arrest. We
showed that clearance is the more likely mechanism, because the double
knockout of the sphingoid base kinases recovered from
phytosphingosine-induced arrest but not from sphingosine arrest; the
latter required specifically the action of the kinases for metabolic
clearance. Also, the knockout of DPL1, the
dihydrosphingosine phosphate lyase, was shown to have a slowed recovery
of S phase (14%) compared with its isogenic wild type (34%) (Table
II). These data are also consistent with this mechanism, because the
loss of the ability to clear sphingoid base phosphates affects the
overall clearance of the sphingoid bases, thus affecting recovery from
arrest. Clearly, the roles of kinases and the lyase are important and
need further study in the heat stress response.
The study of sphingolipids in the yeast heat stress response has
rapidly progressed. Now, one can explore the roles of the various
sphingolipids, especially sphingoid bases, because they have been
implicated in the trehalose response, possibly gene regulation through
stress response elements (10), ubiquitination pathway (15), and now the
transient cell cycle arrest response. Furthermore, yeast sphingoid
bases have been implicated in the regulation of the internalization
step of endocytosis likely through protein phosphorylation (29, 30).
Also, the study of the regulation of the enzymes involved in the
production of the sphingolipids will prove to be exciting, especially
the regulation of serine palmitoyltransferase upon heat stress and
possibly other responses. Finally, how and through what pathways the
sphingolipids mediate responses to various stimuli can now be studied.
Overall, sphingolipids are emerging as key molecules in the yeast
heat stress response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Percent budding of strains 7R4 and 7R4-LCB1
upon heat stress. Yeast cells of strain 7R4-LCB1 (A)
and 7R4 (B) were pelleted, resuspended, and placed on
slides. A representative field was photographed at 100× and scored for
budding. Each bar is the average of two separate
measurements shown with their range.
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Fig. 2.
Analysis of serine palmitoyltransferase
activity, labeled sphingolipids, and levels of heat-stressed sphingoid
bases. A, protein was extracted from strains 7R4,
7R4-LCB1, RH406, and lcb1-100 and measured in vitro for
serine palmitoyltransferase activity at 30 °C. A graph of production
of keto-dihydrosphingosine (Keto-dhsph) by given protein
extracts is shown. Each bar is the average of two
separate measurements shown with their range. B, film of the
TLC plate spotted with base-hydrolyzed tritiated palmitate-labeled
lipid extracts from strains RH406 and lcb1-100. Lipids labeled are
dihydrosphingosine (dhsph), phytosphingosine
(psph), inositol phosphoceramides (IPC),
lyso-phosphoinositol (lyso-PI), mannose inositol
phosphoceramide (MIPC), and mannose (inositol
phosphate)2 ceramide
(M(IP)2C). C and D,
percentage increase of sphingoid bases upon heat stress. Lipids were
extracted, base-hydrolyzed, derivatized, and analyzed by high
performance liquid chromatography. Data are represented as percentage
of time-matched controls, with each point being the average of
duplicate measurements shown with their range.
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Fig. 3.
Budding and cell cycle analysis upon heat
stress of strains RH406 and lcb1-100. Budding was evaluated as
described previously in Fig. 1. Yeast cells were seeded at 2 × 106 cells/ml, rested 6 h, and then treated as shown.
Cells were then pelleted, fixed, treated with RNase and Proteinase K,
and stained with propidium iodide. Stained cells were analyzed by flow
cytometry and analyzed by ModFit LT.
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Fig. 4.
Representation of sphingolipid biosynthesis
and breakdown in S. cerevisiae. Scheme shows the
enzymes of sphingolipid biosynthesis and breakdown. SPT, serine
palmitoyltransferase; KeDHS, keto-dihydrosphingosine; LCBK,
sphingoid base kinase; YPC1, yeast phytoceramidase; YDC1,
yeast dihydroceramidase; IPC, inositol phosphoceramide; MIPC,
mannose inositol phosphoceramide; M(IP)2C, mannose
(inositol phosphate)2 ceramide; LCBPP, sphingoid base
phosphate phosphatase; DHSH, dihydrosphingosine hydroxylase; and
CS, ceramide synthase. Figure courtesy of Dr. Cungui Mao.
Deletions used in Table II are underlined in the
scheme.
Heat-induced transient cell cycle arrest analysis of sphingolipid
mutants
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Fig. 5.
Dose and time dependence of
phytosphingosine-induced arrest. Cells of the yeast strain
7R4-LCB1 were treated with phytosphingosine (psph) as
denoted and run through flow cytometry and modeled by ModFit LT.
A, graph represents percentage S phase at 30 min of
treatment with the given concentrations of phytosphingosine.
B, graph shows the time course of arrest using 10 µM phytosphingosine.
Specificity of sphingoid bases in causing a transient cell cycle arrest
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Fig. 6.
Cell cycle analysis of strains GR2 and LDW6A
(CLN3-1) after treatment with 20 µM phytosphingosine. Log phase
cultures of yeast strains GR2 and LDW6A were treated with 20 µM phytosphingosine (psph), and the resultant
cell cycle was determined by flow cytometry at the given time
points.
ste11 cells showed 1.11% of cells in S
phase at 45 min. The time point of 45 min was used, because this was
the optimal time when the isogenic wild type strain 4741 was arrested
(Table IV). Therefore, inactivation of
the alpha factor-activated MAPK pathway by STE11 knockout
did not affect the ability of heat stress to arrest the cell cycle. We
further evaluated other potential regulators of Cln3 by using a
knockout strain of the MEKK (BCK1) of the stress-induced MAPK pathway (27). The
bck1 cells were still able to
arrest (2% S phase) at 45 min of heat stress (Table IV). Thus,
inactivation of the stress-induced MAPK pathway by BCK1
knockout did not affect the heat stress-induced cell cycle arrest.
These data show that neither the alpha factor or stress-induced MAPK
pathways are required for heat stress-induced cell cycle arrest.
Analysis of possible regulators of Cln3 via mutants
lcb4,5 strain was tested over a longer
time course of heat stress (Fig.
7A). The
lcb4,5
strain showed an arrest at 1 h with the expected large decrease in
the S phase area. By 2 h, the G2/M phase had increased
to a large broad-shouldered peak (77% of cells). The 3- and 4-h heat
stress time points confirmed this finding, because the majority of
cells were found in the G2/M peak (87 and 79%,
respectively). However, by 4 h of heat stress the double-knockout strain had some S phase (3%) and an increased
G0/G1 peak compared with the 3-h time point
(7% compared with 18%) indicating some recovery of a normal cell
cycle. These data indicate two possibilities for the sphingoid base
kinases to play. One possibility is that the sphingoid base phosphates
play a role in the recovery of the normal cell cycle upon heat stress.
The other possibility is that clearance of the sphingoid bases via the
kinase pathway removes the arrest signal and thus allows cell cycle
recovery.
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Fig. 7.
Cell cycle of strain lcb4,
lcb5 after heat stress and treatment with sphingosine and
phytosphingosine. Log phase cultures of strain
lcb4, lcb5 were
treated as denoted, and the resultant cell cycle was determined by flow
cytometry at the given time points. A, cells were upshifted
from 30 to 39 °C. B, cells were treated with 20 µM phytosphingosine (psph). C,
cells were treated with 20 µM sphingosine
(sph). Graphs were generated by ModFit LT.
lcb4,5 strain with 20 µM phytosphingosine resulted in a loss of S phase area by 1 h (Fig. 7B).
The S phase area of the double-knockout strain was partially recovered
by the 2-h treatment and completely recovered by the 4-h time point. Ergo, recovery was not dependent upon the making of phosphorylated phytosphingosine through the sphingoid base kinases. Next, the kinase
double knockout was treated with 20 µM sphingosine.
Sphingosine treatment also caused the expected cell cycle arrest by
1 h; however, the arrest was not reversed over the 4 h of
treatment tested (Fig. 7C). The 4-h time point of 20 µM sphingosine had no detectable S phase. Therefore, the
inability of the double knockout of the sphingoid base kinases to
either metabolize sphingosine in the yeast sphingolipid biosynthetic
pathway or through phosphorylation by the kinases led to a prolonged
arrest of its cell cycle. Taken together these results suggest that the
clearance hypothesis of alleviating the cell cycle arrest is the more
likely explanation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Candace Enocksen, Dr. Gerry Johnston, Dr. Cungui Mao, and Dr. Hannie Siestma for their help.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-43825.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) .
¶ To whom correspondence should be addressed: Dept. Biochemistry and Molecular Biology, 114 Doughty St., P. O. Box 250780, Medical University of South Carolina, Charleston, SC 29482. Tel.: 843-792-4321; Fax: 843-876-5172; E-mail: hannun@musc.edu.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M007425200
2 G. Jenkins, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: YEPD, yeast extract, bactopeptone and dextrose; MAPK, mitogen-activated protein kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase.
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