From the
The regulation of lipid biosynthesis in the yeast
Saccharomyces cerevisiae by fumonisin B
Cell growth is dependent on the membrane structures in the cell
which compartmentalize various cellular
processes
(1, 2) . Lipids are major membrane components
which play a critical role in the structure and function of membranes.
The major membrane lipids found in eucaryotic cells include
phospholipids, sphingolipids, and neutral lipids
(2) . In
addition to their role as structural components of membranes, lipids
function as cofactors and activators of membrane-associated enzymes
(3) and play a major role in cell signaling
mechanisms
(4, 5, 6) .
A great deal is known
about the synthesis and regulation of phospholipids in the yeast
Saccharomyces
cerevisiae(7, 8, 9, 10, 11) .
Nearly all of the structural genes encoding for the phospholipid
biosynthetic enzymes have been cloned and characterized, and many of
the enzymes have been purified and
characterized
(7, 8, 9, 10, 11) .
The enzymes in the pathway are regulated by both genetic and
biochemical mechanisms. The gene expression of most of the enzymes
responsible for the synthesis of the major membrane phospholipid
PC
In S. cerevisiae, the pathways for the
synthesis of phospholipids, sphingolipids, and neutral lipids share
common lipid intermediates such as DG, CDP-DG, and PI (Fig. 1).
Thus, it is reasonable to question whether overall lipid biosynthesis
is coordinately regulated. Much attention has been paid to the role
sphingoid bases play in lipid metabolism and cell signaling in
mammalian cells
(6, 25) . For example, sphingosine has
been suggested to be a regulator of the PC signaling pathway since
sphingosine activates phospholipase D
(26, 27) and
inhibits PA phosphatase
(28, 29) and protein kinase
C
(6, 30, 31, 32) . Our approach in this
work was to elevate the cellular concentration of sphingoid bases in
S. cerevisiae and examine its effect on lipid biosynthesis.
Sphingoid base levels were elevated in S. cerevisiae by
supplementing cells with fumonisin B
We also examined whether fumonisin B
IPC synthase
(63) and PS synthase
(65) activities were
assayed with their lipid substrates as part of uniform Triton
X-100/lipid-mixed micelles. Since sphingoid bases also form uniform
mixed micelles with Triton X-100 and lipids
(30) , the
concentrations of sphingoid bases were expressed as surface
concentrations in mol %
(24) . Sphinganine and phytosphingosine
inhibited IPC synthase activity in a dose-dependent manner with
IC
The goal of this work was to examine the overall regulation
of lipid biosynthesis in S. cerevisiae by sphingoid bases. Our
rationale was to elevate sphingoid base levels with fumonisin
B
One mechanism for the decrease in
sphingolipid synthesis could be attributed to the regulation of IPC
synthase activity. IPC synthase, which catalyzes the committed step in
sphingolipid synthesis from ceramide and PI
(46) , was inhibited
in vitro by sphinganine and phytosphingosine. Thus, the
elevation of sphingoid bases in vivo caused by fumonisin
B
Fumonisin B
The only major phospholipid whose synthesis and composition
was not decreased by fumonisin B
The addition of
fumonisin B
Fumonisin B
When S. cerevisiae cells enter the stationary phase, TG is
elevated relative to phospholipids
(79) , and ergosterol esters
are elevated relative to ergosterol
(80) . Similar changes in the
lipid composition were observed here when cells were grown in the
presence of fumonisin B
Fumonisin B
This paper is dedicated
to Eugene P. Kennedy on the occasion of his retirement.
was
examined. Fumonisin B
inhibited the growth of yeast cells.
Cells supplemented with fumonisin B
accumulated free
sphinganine and phytosphingosine in a dose-dependent manner. The
cellular concentration of ceramide was reduced in fumonisin
B
-supplemented cells. Ceramide synthase activity was found
in yeast cell membranes and was inhibited by fumonisin B
.
Fumonisin B
inhibited the synthesis of the
inositol-containing sphingo-lipids inositol phosphorylceramide,
mannosylinositol phosphorylceramide, and mannosyldiinositol
phosphorylceramide. Fumonisin B
also caused a decrease in
the synthesis of the major phospholipids synthesized via the
CDP-diacylglycerol-dependent pathway and the synthesis of neutral
lipids. The effects of fumonisin B
and sphingoid bases on
the activities of enzymes in the pathways leading to the synthesis of
sphingolipids, phospholipids, and neutral lipids were also examined.
Other than ceramide synthase, fumonisin B
did not affect
the activities of any of the enzymes examined. However, sphinganine and
phytosphingosine inhibited the activities of inositol
phosphorylceramide synthase, phosphatidylserine synthase, and
phosphatidate phosphatase. These are key enzymes responsible for the
synthesis of lipids in yeast. The data reported here indicated that the
biosynthesis of sphingolipids, phospholipids and neutral lipids was
coordinately regulated by fumonisin B
through the
regulation of lipid biosynthetic enzymes by sphingoid bases.
(
)
is coordinately regulated by the
water-soluble phospholipid precursor
inositol
(7, 8, 9, 10, 11, 12, 13) .
The biochemical mechanisms affecting the activity of phospholipid
biosynthetic enzymes include regulation by inositol
(14) ,
nucleotides
(15, 16) ,
phosphorylation
(17, 18, 19) ,
lipids
(20, 21, 22, 23) , and sphingoid
bases
(24) .
. Fumonisin B
is a neurotoxin
(33) and phytotoxin
(34) which
bears structural similarity to sphingoid bases
(35) (Fig. 2). Fumonisin B
has been shown to
elevate sphingoid base levels in mammalian cells
(36) due to the
inhibition of ceramide synthase activity
(37) . The addition of
fumonisin B
to S. cerevisiae cells resulted in a
decrease in the synthesis of sphingolipids, phospholipids, and neutral
lipids and dramatically affected the overall lipid composition of the
cell. The data reported here were consistent with the conclusion that
the synthesis of the major lipid classes was coordinately regulated by
sphingoid bases. The mechanism of this regulation was due in part to
the inhibition of key lipid biosynthetic enzymes including IPC
synthase, PS synthase, and PA phosphatase by sphingoid bases.
Figure 1:
Lipid biosynthetic pathways in S.
cerevisiae. The pathways shown include the relevant steps
discussed in the text. More comprehensive pathways which include lipid
and water-soluble intermediates may be found in Refs. 7 and 11.
Abbreviations: PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; PtdSer, phosphatidylserine;
CDP-DAG, CDP-diacylglycerol; DAG, diacylglycerol;
PtdOH, phosphatidate; Cho-P, choline phosphate;
CDP-Cho, CDP-choline; PtdIns, phosphatidylinositol;
IPC, inositol phosphorylceramide; MIPC,
mannosylinositol phosphorylceramide;
M(IP)C, mannosyldiinositol
phosphorylceramide; PIPs,
polyphosphoinositides.
Figure 2:
Structures of sphingoid bases and
fumonisin B.
Materials
All chemicals were reagent grade. Triton X-100, Tergitol
(Nonidet P-40), sphingoid bases, ceramide, fumonisin B,
ATP, CTP, inositol, serine, choline, phosphocholine, CDP-choline, and
bovine serum albumin were obtained from Sigma. Phospholipids and
neutral lipids were purchased from Avanti Polar Lipids and Sigma.
CDP-DG was prepared as described previously
(38) . Radiochemicals
and EN
HANCE were purchased from DuPont NEN, and
scintillation counting supplies were from National Diagnostics. Silica
gel-loaded SG81 chromatography paper was from Whatman, Inc., and Silica
Gel 60 thin layer chromatography plates were from EM Science.
Escherichia coli DG kinase was obtained from Lipidex Inc.
Growth medium supplies were purchased from Difco Laboratories.
Methods
Strain and Growth Conditions
Strain
MAT
a ade5(39) , which shows normal
regulation of phospholipid
metabolism
(40, 41, 42, 43) , was used
for analysis of lipids in response to fumonisin B and for
the preparation of enzymes. Cultures were maintained on YEPD medium (1%
yeast extract, 2% peptone, 2% glucose) plates containing 2% Bacto-agar.
Cells were grown in complete synthetic medium
(39) containing
0.5% Tergitol in the absence and presence of the indicated
concentrations of fumonisin B
at 30 °C. Cell numbers
were determined by microscopic examination with a hemacytometer.
Fumonisin B
caused cells to clump. Prior to counting, cell
clumps were dispersed by a brief sonication. Viable cells were
determined by plate counts on YEPD medium.
Mass Analysis of Sphingoid Bases and
Ceramide
Sphingoid bases were extracted from unlabeled cells by
the method of Merrill et al.(44) .
O-Phthalaldehyde derivatives of the sphingoid bases were
prepared and analyzed by high performance liquid chromatography using
C20-sphinganine as an internal standard
(44) . The identity of
the sphingoid bases was determined by comparing its elution profile
with that of authentic standards. For ceramide analysis, lipids were
extracted from unlabeled cells
(45) and subjected to mild
alkaline hydrolysis
(46) to deacylate DG. Ceramide was then
quantified by the method of Bell and co-workers
(47, 48) using E. coli DG kinase
(49) .
Labeling and Analysis of Sphingolipids, Phospholipids,
and Neutral Lipids
Pulse and steady-state labeling of lipids
with [2-H]inositol and
[2-
C]acetate were performed as described
previously
(50, 51, 52, 53, 54) .
Sphingolipids, phospholipids, and neutral lipids were extracted from
labeled cells as by Hanson and Lester
(55) . Sphingolipids were
analyzed by one-dimensional chromatography on silica gel thin layer
plates
(56) . Phospholipids were analyzed by the two-dimensional
chromatography
(57) using Na
EDTA-treated SG81
paper
(58) . Neutral lipids were separated by one-dimensional
chromatography on silica gel thin layer plates
(59) . The
positions of the labeled lipids on chromatograms were determined by
fluorography using EN
HANCE and compared with standard
lipids after exposure to iodine vapor. The amount of each labeled lipid
was determined by liquid scintillation counting of the corresponding
spots on chromatograms.
Preparation of Enzymes
PA phosphatase was purified
to homogeneity as described by Lin and Carman
(60) . IPC synthase
was solubilized from microsomal membranes with 1% Triton X-100 as
described by Fischl and Carman
(61) . Cell extracts
(61) and total membranes
(60) were prepared as described
previously and used for the assay of the indicated enzymes.
Preparation of Labeled
Substrates
[P]PA was synthesized
enzymatically from DG and [
-
P]ATP using
E. coli DG kinase
(60) . [
H]PI was
synthesized from CDP-DG and [2-
H]inositol using
PI synthase purified from S. cerevisiae(61) .
Enzyme Assays
All assays were conducted at 30
°C in a total volume of 0.1 ml unless otherwise indicated. Ceramide
synthase (acyl-CoA:sphinganine (sphingosine)
N-acyltransferase, EC 2.3.1.24) was measured at 37 °C with
25 mM potassium phosphate buffer (pH 7.4), 0.5 mM
dithiothreitol, 40 µM stearoyl-CoA, 3 µM
[H]sphingosine (prepared as a liposome of PC and
sphingosine at a molar ratio of 2:1), and enzyme protein
(62) .
IPC synthase (phosphatidylinositol:ceramide phosphoinositol
transferase) was measured with 50 mM Tris-HCl buffer (pH 7.0),
1 mM MnCl
, 5 mM MgCl
, 5
mM Triton X-100, 0.1 mM ceramide, 0.25 mM
[
H]PI, and enzyme protein
(63) . CDP-DG
synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was
measured with 50 mM Tris-maleate buffer (pH 6.5), 20
mM MgCl
, 15 mM Triton X-100, 0.5
mM phosphatidate, 1.0 mM
[5-
H]CTP, and enzyme protein (64). PI synthase
(CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC
2.7.8.11) was measured with 50 mM Tris-HCl buffer (pH 8.0), 2
mM MnCl
, 3.2 mM Triton X-100, 0.2
mM CDP-DG, 1 mM [2-
H]inositol,
and enzyme protein
(38) . PS synthase
(CDPdiacylglycerol:L-serine
3-O-phosphatidyltransferase, EC 2.7.8.8) was measured with 50
mM Tris-HCl buffer (pH 8.0), 0.6 mM MnCl
,
3.2 mM Triton X-100, 0.2 mM CDP-DG, 0.5 mM
[3-
H]serine, and enzyme protein
(65) . PA
phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4)
was measured with 50 mM Tris-maleate buffer (pH 7.0), 10
mM 2-mercaptoethanol, 2 mM MgCl
, 1
mM Triton X-100, 0.1 mM [
P]PA,
and enzyme protein (66). Choline kinase (EC 2.7.1.32) was measured with
50 mM glycine-NaOH buffer (pH 9.7), 10 mM
MgSO
, 10 mM ATP, 50 µM
[methyl-
C]choline, and enzyme
protein
(67) . Phosphocholine cytidylyltransferase
(CTP:choline-phosphate cytidylyltransferase, EC 2.7.7.15) was measured
with 50 mM Tris-HCl buffer (pH 8.0), 25 mM
MgCl
, 4 mM phosphocholine, 1 mM
[
-
P]CTP, and enzyme protein
(68) .
Cholinephosphotransferase (CDPcholine:1,2-diacylglycerol
cholinephosphotransferase, EC 2.7.8.2) was measured with 50 mM
MOPS-NaOH buffer (pH 7.5), 20 mM MgCl
, 6.5
mM Triton X-100, 1.3 mM DG, 1.3 mM PC, 0.5
mM [methyl-
C]CDP-choline, and
enzyme protein (22). All assays were linear with time and protein
concentration. A unit of enzymatic activity was defined as the amount
of enzyme that catalyzed the formation of 1 nmol of product/min unless
otherwise indicated. Specific activity was defined as units/mg of
protein. Protein concentration was determined by the method of Bradford
(69) using bovine serum albumin as the standard.
Effect of Fumonisin B
The effect of fumonisin Bon Cell
Growth
on cell growth
was examined. In these studies it was necessary to include a low
percentage of the detergent Tergitol to the growth medium to facilitate
fumonisin B
uptake by the cells. Tergitol did not have a
significant effect on the growth of cells grown in the absence of
fumonisin B
. The addition of fumonisin B
to the
growth medium resulted in a dose-dependent inhibition in the growth
rate of cells (Fig. 3). Fumonisin B
also caused an
increase in the incubation time required to reach the stationary phase
of growth (Fig. 3). By the time cells reached the stationary
phase of growth, the final cell densities of the cultures grown in the
presence of fumonisin B
approached the final cell density
of the culture grown in the absence of fumonisin B
(Fig. 3). We questioned whether or not a population of
cells was being selected for that was resistant to fumonisin
B
. To address this question, cells were taken from a
stationary phase culture grown in the presence of 100 µM
fumonisin B
. These cells were washed in fresh growth medium
and inoculated into fresh growth medium with and without fumonisin
B
. These cells responded to fumonisin B
as
described above. Thus, these cells were not resistant to growth
inhibition by fumonisin B
.
Figure 3:
Effect of fumonisin B on cell
growth. Cells were grown in the absence and presence of the indicated
concentrations of fumonisin B
. Cell numbers were determined
by direct microscopic examination. These values were consistent with
the number of viable cells determined by plate counts. The data shown
is representative of three independent growth studies. Concentrations
of fumonisin (µM):
, 0;
, 25;
, 50;
, 100;
, 200.
Effect of Fumonisin B
We previously
demonstrated that free sphinganine and phytosphingosine exist in S.
cerevisiae (24). We examined the cellular concentrations of these
sphingoid bases in cells grown for 30 h in the absence and presence of
fumonisin Bon Cellular
Concentrations of Sphingoid Bases
. Fumonisin B
caused a
dose-dependent increase in the cellular concentrations of sphingoid
bases (Fig. 4). Cells grown in the presence of fumonisin B
accumulated 11- to 50-fold more sphinganine and 22- to 50-fold
more phytosphingosine when compared with control cells. Subsequent
growth studies were performed with a fumonisin B
concentration of 100 µM.
Figure 4:
Effect of fumonisin B on
cellular concentrations of sphingoid bases. Cells were grown for 30 h
in the absence and presence of the indicated concentrations of
fumonisin B
. Sphingoid bases were extracted, and their
O-phthalaldehyde derivatives were prepared and analyzed by
high performance liquid chromatography as described in the text. The
reported values were the average of three determinations.
,
sphinganine;
, phytosphingosine.
To determine if the
accumulation of sphingoid bases was affected by growth phase, the
cellular concentrations of sphinganine and phytosphingosine in
exponential phase cells were determined and found to be 6 to 11
pmol/10 cells (Fig. 5). There was a 2-fold increase
in the cellular concentrations of these sphingoid bases when cells
entered the stationary phase of growth (Fig. 5). Thus, the
cellular levels of sphingoid bases were related to the growth phase.
However, the increase in sphingoid base concentrations due to growth
phase regulation was small when compared with the increase due to
fumonisin B
supplementation. The accumulation of
sphinganine (Fig. 5A) and to a lesser extent
phytosphingosine (Fig. 5B) was highest in exponential
phase cells when compared with stationary phase cells. Thus, the large
accumulation of the sphingoid bases was largely due to fumonisin
B
and not due to growth phase regulation.
Figure 5:
Effect of growth phase on cellular
concentrations of sphingoid bases. Cells were grown in the absence and
presence of 100 µM fumonisin B as indicated.
Cells were harvested in the early exponential (5
10
cells/ml), late exponential (5
10
cells/ml),
and stationary (1.2
10
cells/ml) phases of growth.
Sphingoid bases were extracted, and their O-phthalaldehyde
derivatives were prepared and analyzed by high performance liquid
chromatography as described in the text. The reported values were the
average of three determinations. Exp, exponential.
,
control;
, fumonisin B
.
Effect of Fumonisin B
Sphinganine is a substrate in the reaction
catalyzed by ceramide synthase
(62) . We questioned whether the
mechanism of sphingoid base accumulation in S. cerevisiae cells was due to the inhibition of ceramide synthase activity by
fumonisin Bon Ceramide
Synthase Activity and the Cellular Concentration of
Ceramide
. To examine ceramide synthase activity, we used
the assay system developed for the measurement of this enzyme in
mammalian cells
(62) . Ceramide synthase activity was indeed
found in the total membrane fraction of S. cerevisiae at a
specific activity of 16.5 pmol/min/mg. Addition of 100 µM
fumonisin B
to the assay resulted in a 90% decrease in
ceramide synthase activity (Fig. 6A).
Figure 6:
Effect of fumonisin B on
ceramide synthase activity and the cellular concentration of ceramide.
A, ceramide synthase activity was measured in the absence and
presence of 100 µM fumonisin B
as indicated.
Total membranes were used as the source of ceramide synthase. The
specific activity of ceramide synthase in total membranes was 16.5
pmol/min/mg. B, cells were grown to the late exponential (5
10
cells/ml) phase of growth in the absence and
presence of 100 µM fumonisin B
as indicated.
Ceramide was extracted and quantified using E. coli DG kinase
as described in the text. FB
, fumonisin
B
.
If the
mechanism of sphingoid base accumulation in cells grown with fumonisin
B was due to the inhibition of ceramide synthase activity,
one would expect that the cellular concentration of ceramide would be
reduced. To address this question, cells were grown to the exponential
phase of growth in the absence and presence of 100 µM
fumonisin B
. Ceramide was extracted from cells, and the
cellular concentration was determined using E. coli DG kinase.
The amount of ceramide in cells grown in the presence of fumonisin
B
was 15% of the concentration in the control cells
(Fig. 6B).
Effect of Fumonisin B
Sphingolipids in S.
cerevisiae differ from those of mammalian cells in that they are
structurally less complex and contain phosphoinositol as part of their
polar head groups (70). The major sphingolipids in S. cerevisiae are IPC, MIPC, and M(IP)on Sphingolipid
Synthesis and Composition
C
(70) . These
inositol-containing sphingolipids are composed of phytosphingosine, to
which a long chain fatty acid is linked via an amide bond
(70) .
IPC, MIPC, and M(IP)
C are believed to be synthesized via
the pathway shown in Fig. 1(46, 70) . Since
ceramide is the direct precursor for sphingolipid synthesis, we
examined the effect of fumonisin B
on sphingolipid
synthesis and composition. The phosphoinositol head group of yeast
sphingolipids is derived from the membrane phospholipid PI
(70) .
Since PI is synthesized from inositol (Fig. 1), sphingolipid
synthesis was followed by pulse-labeling cells with
[2-
H]inositol. The amount of
[2-
H]inositol incorporated into each sphingolipid
represented the relative rates of synthesis during the pulse. The
addition of fumonisin B
to the growth medium resulted in a
decreased incorporation of [2-
H]inositol into IPC
(5-fold), MIPC (3-fold), and M(IP)
C (2-fold) when compared
with cells grown in the absence of fumonisin B
(Fig. 7A). The concentration of inositol added to
the growth medium was only 0.1 µM, which is too low to
affect the synthesis of PI or overall phospholipid synthesis (71).
Figure 7:
Effect of fumonisin B on
pulse-labeling of sphingolipids and sphingolipid composition.
A, cells were grown to the exponential (2
10
cells/ml) phase of growth in the absence and presence of 100
µM fumonisin B
as indicated. Cells were then
incubated with [2-
H]inositol (4 µCi/ml) for
30 min to pulse-label sphingolipids. The incorporation of
[2-
H]inositol into sphingolipids during the pulse
was 2,000-4,500 cpm/10
cells. B, cells were
grown to the exponential (2
10
cells/ml) phase of
growth in the absence and presence of 100 µM fumonisin
B
as indicated. The steady-state sphingolipid composition
was determined by labeling cells for five to six generations with
[2-
H]inositol (2 µCi/ml). The incorporation
of [2-
H]inositol into sphingolipids during the
steady-state labeling was 3,000-8,500 cpm/10
cells.
The sphingolipid composition of the cells was determined as described
in the text. The percentages shown for sphingolipids were normalized to
the total lipid composition of cells labeled with
[2-
C]acetate.
, control;
,
fumonisin B
.
Cells were labeled with [2-H]inositol to a
steady-state to analyze the effect of fumonisin B
on
sphingolipid composition. Addition of fumonisin B
to the
growth medium caused a decrease in the steady-state concentrations of
IPC (2.8-fold), MIPC (4.6-fold), and M(IP)
C (2.4-fold)
(Fig. 7B).
Effect of Fumonisin B
When S. cerevisiae cells
are grown in the absence of choline, the major membrane phospholipid PC
is primarily synthesized by a CDP-DG-dependent pathway via the reaction
sequence: PA on Phospholipid
Synthesis and Composition
CDP-DG
PS
PE
PC
(7, 8) (Fig. 1). PI is also synthesized from
CDP-DG
(7, 8) (Fig. 1). The partitioning of CDP-DG
between PI and PS is highly regulated in S.
cerevisiae(7, 14) . Since sphingolipid synthesis is
dependent on the synthesis of PI
(70) , we examined the effect of
fumonisin B
on overall phospholipid synthesis and
composition. Phospholipid synthesis was followed by pulse-labeling with
[2-
C]acetate of cells grown in the absence and
presence of 100 µM fumonisin B
. The amount of
[2-
C]acetate incorporated into each phospholipid
represented the relative rates of synthesis during the pulse. The
presence of fumonisin B
in the growth medium caused a
decrease in the incorporation of label into PA (2-fold), CDP-DG
(3-fold), PS (2-fold), PE (2-fold), and PC (1.5-fold) when compared
with control cells (Fig. 8A). The incorporation of the
label into PI was not inhibited by fumonisin B
. Instead,
the synthesis of PI increased by 1.4-fold (Fig. 8A). As
was seen in the labeling experiments using
[2-
H]inositol, the synthesis of sphingolipids
from [2-
C]acetate was inhibited by fumonisin
B
(Fig. 8A).
Figure 8:
Effect of fumonisin B on
pulse-labeling of phospholipids and phospholipid composition.
A, cells were grown to the exponential (2
10
cells/ml) phase of growth in the absence and presence of 100
µM fumonisin B
as indicated. Cells were then
incubated with [2-
C]acetate (12 µCi/ml) for
30 min to pulse-label phospholipids. The incorporation of
[2-
C]acetate into phospholipids during the pulse
was 15,000-16,000 cpm/10
cells. B, cells
were grown to the exponential (2
10
cells/ml) phase
of growth in the absence and presence of 100 µM fumonisin
B
as indicated. The steady-state phospholipid composition
was determined by labeling cells for five to six generations with
[2-
C]acetate (2 µCi/ml). The incorporation
of [2-
C]acetate into phospholipids during
steady-state labeling was 22,000-26,000 cpm/10
cells.
The phospholipid composition of the cells was determined as described
in the text. The percentages shown for phospholipids were normalized to
the total lipid composition of cells labeled with
[2-
C]acetate. SL, sphingolipids.
, control;
, fumonisin
B
.
The effect of fumonisin
B on the steady-state phospholipid composition is shown in
Fig. 8B. The steady-state concentrations were decreased
for PA (2-fold), CDP-DG (1.7-fold), PS (3-fold), PE (2-fold), and PC
(1.2-fold) in cells supplemented with fumonisin B
when
compared with control cells. Fumonisin B
did not
significantly affect the cellular concentration of PI.
Effect of Fumonisin B
We also examined the effect of
fumonisin Bon Neutral Lipid
Synthesis and Composition
on neutral lipid synthesis by growing cells in
the absence and presence of 100 µM fumonisin B
and pulse-labeling with [2-
C]acetate.
Fumonisin B
decreased in the synthesis of DG (1.2-fold),
monoacylglycerol (2-fold), fatty acids (1.5-fold), fatty alcohols
(2-fold), and ergosterol (2.6-fold) when compared to control cells
(Fig. 9A).
Figure 9:
Effect of fumonisin B on
pulse-labeling of neutral lipids and neutral lipid composition.
A, cells were grown to the exponential (2
10
cells/ml) phase of growth in the absence and presence of 100
µM fumonisin B
as indicated. Cells were then
incubated with [2-
C]acetate (12 µCi/ml) for
30 min to pulse-label neutral lipids. The incorporation of
[2-
C]acetate into neutral lipids during the
pulse was 9,000-16,000 cpm/10
cells. B,
cells were grown to the exponential (2
10
cells/ml)
phase of growth in the absence and presence of 100 µM
fumonisin B
as indicated. The steady-state neutral lipid
composition was determined by labeling cells for five to six
generations with [2-
C]acetate (2 µCi/ml).
The incorporation of [2-
C]acetate into neutral
lipids during steady-state labeling was 16,000-23,000
cpm/10
cells. The neutral lipid composition of the cells
was determined as described in the text. The percentages shown for
neutral lipids were normalized to the total lipid composition of cells
labeled with [2-
C]acetate. TG,
triacylglycerol; MG, monoacylglycerol; FA, fatty
acid; FAL, fatty alcohol; Erg, ergosterol;
ErgE, ergosterol ester.
, control;
, fumonisin
B
.
Steady-state labeling of cells with
[2-C]acetate was performed to analyze neutral
lipid composition (Fig. 9B). Fumonisin B
supplementation increased triacylglycerols (1.8-fold), fatty
acids (2.6-fold), and ergosterol esters (1.7-fold) and decreased
ergosterol (1.2-fold).
Effect of Fumonisin B
The pulse- and
steady-state labeling experiments showed that fumonisin Band Sphingoid Bases
on Lipid Biosynthetic Enzyme Activities
altered the synthesis and composition of sphingolipids,
phospholipids, and neutral lipids. We questioned if the expression of
several key lipid biosynthetic enzyme activities were affected in cells
grown with fumonisin B
. These enzyme activities included
those responsible for sphingolipid synthesis (IPC synthase),
phospholipid synthesis via the CDP-DG-dependent pathway (CDP-DG
synthase, PI synthase, PS synthase) and CDP-choline-dependent pathway
(choline kinase, phosphocholine cytidylyltransferase,
cholinephosphotransferase), and neutral lipid synthesis (PA
phosphatase). We examined enzymes in the CDP-choline-dependent pathway
because this pathway (Fig. 1) contributes to PC synthesis even
when cells are cultured in growth medium lacking
choline
(72, 73) . The choline required for the
CDP-choline-dependent pathway is presumably derived from the turnover
of PC synthesized by the CDP-DG-dependent
pathway
(72, 73) . Cells were grown in the absence and
presence of 100 µM fumonisin B
, cells were
harvested in the exponential phase of growth, cell extracts were
prepared, and the activities of the enzymes were measured. These enzyme
activities were not affected by the addition of fumonisin B
to the cells.
and sphingoid bases had a direct effect on the activities of
these key lipid biosynthetic enzymes. The activity of each enzyme was
measured in the absence and presence of 100 µM fumonisin
B
, 100 µM sphinganine, and 100 µM
phytosphingosine. None of the enzymes examined was affected directly by
fumonisin B
. On the other hand, IPC synthase, PS synthase,
and PA phosphatase activities were inhibited by sphingoid bases (as
will be described below), but the other enzymes were not affected.
values of 3 mol % and 4.3 mol %, respectively
(Fig. 10). PS synthase was also inhibited by sphinganine and
phytosphingosine in a dose-dependent manner with IC
values
of 1.2 mol % and 1.9 mol %, respectively (Fig. 11). IC
values were calculated from plots of the log of the activity
values from Fig. 10and Fig. 11versus the
inhibitor concentrations. Of the two sphingoid base inhibitors,
sphinganine was the more potent inhibitor of IPC synthase and PS
synthase activities. Similar results have been previously reported for
PA phosphatase
(24) . Of these three enzymes, PS synthase
activity was the most sensitive to inhibition by sphingoid bases.
Figure 10:
Effect of sphingoid bases on IPC synthase
activity. IPC synthase activity was measured in the absence and
presence of the indicated surface concentrations of sphinganine
() and phytosphingosine (
). A Triton X-100 extract of
microsomal membranes was used as the source of IPC synthase. The
specific activity of IPC synthase in the Triton X-100 extract was 0.5
nmol/min/mg.
Figure 11:
Effect of sphingoid bases on PS synthase
activity. PS synthase activity was measured in the absence and presence
of the indicated surface concentrations of sphinganine () and
phytosphingosine (
). A cell-free extract was used as the source of
PS synthase. The specific activity of PS synthase in the cell extract
was 0.45 nmol/min/mg.
. Supplementation of S. cerevisiae cells with
fumonisin B
resulted in accumulations in the cellular
levels of free sphinganine and phytosphingosine. It is known that
fumonisin B
inhibits ceramide synthase activity in
mammalian cells
(37) . Ceramide synthase activity in S.
cerevisiae was identified and shown to be inhibited by fumonisin
B
. Moreover, we showed here that fumonisin B
caused a decrease in the cellular concentration of ceramide.
Cells supplemented with fumonisin B
also showed decreases
in the synthesis and steady-state levels of IPC, MIPC, and
M(IP)
C. Taken together, these data indicated that the
reduction in the cellular concentration of ceramide, brought about by
the inhibition of ceramide synthase activity by fumonisin
B
, resulted in a decrease in sphingolipid synthesis and
composition. Inositol-containing sphingolipids play an essential role
in cell growth
(74, 75) . Thus, the inhibition of cell
growth by fumonisin B
must be due in part to the decrease
in sphingolipid synthesis.
supplementation may have led to a decrease in IPC
synthase activity. Another mechanism that may account for the decrease
in sphingolipid synthesis may be the reduction in the cellular
concentration of ceramide as available substrate for the IPC synthase
reaction.
caused a decrease in the synthesis
and composition of phospholipids primarily synthesized by the
CDP-DG-dependent pathway. The mechanism of the inhibition of
phospholipid synthesis was likely to be very complex. At least one
aspect of this complex mechanism may be the regulation of PS synthase
activity, which was inhibited in vitro by sphingoid bases.
This inhibition was consistent with decreased PS synthesis and
composition. Fumonisin B
also caused decreased PA synthesis
and composition. PA is a potent activator of PS synthase
activity
(20) . Thus, the decrease in PA levels may have also
contributed to the decreased synthesis of PS. The decreased synthesis
in PS may in turn be responsible for the decreased synthesis and
composition of PE and PC. These phospholipids are derived from PS in
the CDP-DG-dependent pathway
(7) . Enzymes responsible for PC
synthesis via the CDP-choline-dependent pathway were not affected in
cells supplemented with fumonisin B
nor were their
activities directly affected by fumonisin B
or sphingoid
bases.
was PI. In fact there was
a modest increase in PI synthesis. This was not due to an increase in
the expression of PI synthase activity in fumonisin
B
-supplemented cells or an activation of activity by
sphingoid bases. The increase in PI synthesis may be attributed to the
lack of its utilization as a precursor for the synthesis of
sphingolipids. Furthermore, the increased synthesis in PI may be
attributed to the inhibition of PS synthase activity by sphingoid
bases. PS synthase
(76) and PI synthase
(61) use CDP-DG
as a substrate. Previous work has shown that the partitioning of CDP-DG
between PS and PI is regulated through the inhibition of PS synthase
expression
(41, 42, 77) and activity
(14) by inositol. This inhibition leads to an increase in PI
synthesis at the expense of PS synthesis
(14) . In a similar
manner, the inhibition of PS synthase activity by sphingoid bases may
have contributed to the increase in PI synthesis.
to cells also caused a decrease in the
synthesis of neutral lipids. The effect of fumonisin B
on
DG synthesis was relatively small, but was consistent with the
inhibition of PA phosphatase activity by sphingoid bases
(24) .
The steady-state level of DG was not affected by fumonisin
B
. It should be noted that DG is both a substrate and
product of many reactions in lipid
metabolism
(3, 7, 11) , and, thus, cellular
levels of DG arise from a balance of both synthetic and degradation
reactions. The increase in the steady-state composition of TG in the
fumonisin B
-supplemented cells was just the opposite of
what one would expect if TG levels were only due to the regulation of
PA phosphatase activity by sphingoid bases. Thus, the regulation of TG
synthesis and composition by fumonisin B
are not explained
by any of these analyses.
caused a decrease
in ergosterol synthesis and composition. The mechanism for these
changes was not addressed here. Ergosterol is known to stimulate
glycerophosphate acyltransferase and PE methyltransferase activities in
S. cerevisiae(78) . These enzymes are responsible for
PA synthesis and PC synthesis via the CDP-DG-dependent pathway,
respectively
(7) . Thus, the decrease in ergosterol synthesis in
fumonisin B
-supplemented cells may have contributed to the
decreased synthesis of PA and PC through the regulation of
glycerophosphate acyltransferase and PE methyltransferase activities.
. If sphingoid bases are cellular
signals of growth phase, the fumonisin B
may have affected
yeast growth by simulating the sphingoid base concentrations of
stationary phase cells. This would cause the yeast to enter a
stationary phase-like stage prematurely. This notion was consistent
with the observations in both lipid composition and growth rate of
fumonisin B
-supplemented cells.
was a useful tool to examine the effect of sphingoid bases on
lipid synthesis in S. cerevisiae. The data reported here
showed that the synthesis of the major lipid classes was coordinately
regulated by sphingoid bases. The mechanism of this regulation involved
the direct inhibition of IPC synthase, PS synthase, and PA phosphatase
activities by sphingoid bases. These three enzymes catalyze reactions
which commit to the synthesis of sphingolipids, phospholipids, and TG
(Fig. 1). Thus, these enzyme activities play an important role in
the regulation of overall lipid synthesis. In addition, the expression
of IPC synthase
(63) , PS
synthase
(41, 42, 77) , and PA
phosphatase
(53, 81) are coordinately regulated by
inositol, which plays a major role in lipid synthesis in S.
cerevisiae(7, 11) . The studies reported here
underscore the complexity of the mechanisms which regulate lipid
synthesis in S. cerevisiae.
C,
mannosyldiinositol phosphorylceramide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.