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INTRODUCTION |
Sphingolipids are essential components of eucaryotic cells and are
implicated in a growing number of cellular functions including signal
transduction and association with sterols in membranes to form lipid
rafts. To uncover new roles for sphingolipids we have studied
Saccharomyces cerevisiae cells carrying a mutation that
inhibits sphingolipid synthesis at a restrictive temperature. These
studies demonstrate that delivery of the uracil transporter Fur4p to
the plasma membrane requires synthesis of complex sphingolipids, which
most likely reflects the need for de novo formation of lipid rafts, since we find that Fur4p is present in rafts.
Once viewed as experimental artifacts, the existence of lipid rafts is
now generally accepted because they have been identified as important
elements in many cellular functions including trafficking and sorting
of membrane proteins and lipids (for review, see Refs. 1 and 2). More
recently lipid rafts have been recognized as platforms for the
organization and regulation of signal transduction cascades (for
review, see Refs. 3 and 4), as critical components for activating the
mammalian immune system (for review, see Refs. 4-6), and as a key
element in regulating the cellular level of cholesterol (for review,
see Refs. 7 and 8).
Lipid rafts in S. cerevisiae contain ergosterol and complex
sphingolipids (9, 10), whereas the rafts in higher eucaryotes contain
cholesterol and sphingomyelin or glycosphingolipids (1). The complex
sphingolipids in S. cerevisiae are inositol-phosphoceramide (IPC),1
mannose-inositol-phosphoceramide (MIPC), and mannose-(inositol phosphate)2-ceramide (M(IP)2C (11).
Sphingolipid synthesis up through formation of ceramide occurs in the
endoplasmic reticulum, and then ceramide is transported to the Golgi
where the polar head groups are added (11, 12). Lipid raft formation
occurs primarily in the Golgi apparatus in mammals (13), but the
situation is different in yeast. At least for yeast proteins with a
glycophosphatidylinositol anchor, current evidence suggests that they
associate with lipid rafts in the endoplasmic reticulum (10);
presumably such rafts contain ceramides as their sphingolipid
component. Other studies on Pma1p, a proton transporter and the most
abundant protein in the plasma membrane of S. cerevisiae
cells (14), argue that raft association occurs in the Golgi apparatus
(15), where IPC, MIPC, and M(IP)2C would be expected to be
the sphingolipid component of rafts, or alternatively, association
occurs in the endoplasmic reticulum (16).
Our interest in lipid rafts arose from studies on S. cerevisiae cells carrying the lcb1-100 mutation, first
identified as end8
1 because cells are blocked in
endocytosis when shifted to a restrictive temperature (37 °C) (17).
LCB1 encodes a subunit of serine palmitoyltransferase (18),
the enzyme catalyzing the first committed step in sphingolipid
synthesis in which serine is condensed with palmitoyl-CoA to yield the
long chain base 3-ketodihydrosphingosine (for review, see Ref. 11). A
serine palmitoyltransferase molecule containing an Lcb1-100 subunit is
presumably inactivated when cells are shifted to 37 °C. The exact
level of enzyme activity cannot be determined because cell-free
extracts have no measurable activity even when prepared from cells
grown at 25 °C (19). Nonetheless, lcb1-100 cells are
useful because sphingolipid synthesis is reduced rapidly upon shifting
to 37 °C (20) and any process dependent upon sphingolipid
intermediates or complex sphingolipids will be impaired.
Even at a permissive temperature lcb1-100 cells grow slowly
on complex medium and barely grow on defined medium unless it is
supplemented with yeast extract or casamino acids, suggesting that
nutrient uptake is impaired because of reduced amounts of sphingolipids. In the experiments reported here we focus on uracil uptake by the Fur4 protein, the only uracil transporter in S. cerevisiae cells, which has been studied extensively. Fur4p
synthesis begins in the endoplasmic reticulum, where its 12 transmembrane domains are inserted into the membrane (21). During
transit through the secretory pathway it is phosphorylated but is
otherwise unmodified upon reaching the plasma membrane (22). The amount of Fur4p in the plasma membrane is highly regulated, and the protein is
degraded in response to stresses including heat stress and high
concentrations of uracil in the culture medium (22-24). Degradation involves a series of steps including phosphorylation, ubiquitination, endocytosis, and transport to the vacuole where the protein is hydrolyzed (25-27).
As predicted, we find that the steady-state level of the sphingolipid
biosynthetic intermediates dihydrosphingosine (DHS) and
phytosphingosine (PHS) are very low in lcb1-100 cells grown at a permissive temperature, and complex sphingolipids are reduced 50%. When cells are switched to a restrictive temperature, DHS and PHS
do not transiently increase like they do in wild type cells but,
instead, drop to a low level, which explains why complex sphingolipid
synthesis stops. We find that these changes in sphingolipids impact
uracil transport. For example, uracil transport activity is reduced
even in cells grown at the permissive temperature, and after a shift to
a restrictive temperature (a heat shock), activity goes down and is not
restored, whereas in wild type cells, transport activity is restored
within 2 h. To explain these results, we hypothesized that the
block in sphingolipid synthesis prevents raft formation, that Fur4p is
present in lipid rafts, and that rafts are essential for delivery of
newly synthesized Fur4p to the plasma membrane. Our data support these
hypotheses. Furthermore, our data establish that long chain bases are
not required for inactivation of Fur4p uracil uptake activity, but are
required for a later step in Fur4p breakdown.
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MATERIALS AND METHODS |
Strains, Plasmids, and Media--
Strains used in these
experiments are listed in Table
I. Cells were grown in YPD (1% yeast
extract, 2% peptone, 2% glucose), YPAUD (YPD plus 20 µg/ml adenine
sulfate and uracil), complete synthetic medium (SD; 0.34% yeast
nitrogen base (Difco), 1% ammonium sulfate, 2% glucose, 30 mg/liter
adenine and tyrosine, and 20 mg/liter each of histidine, leucine,
lysine, methionine, uracil, and tryptophan) or synthetic media lacking
uracil (SD-Ura) to select cells transformed with a plasmid carrying
URA3. Solid media contained 2% agar. Casamino acids (Difco)
were added to a final concentration of 0.5% to SD medium. Unless
otherwise indicated, transformed cells were grown at 30 °C on
SD-Ura plates, pooled using deionized water, diluted into YPD or YPAUD
medium, and grown overnight at 25 °C. In some experiments Fur4p was
overproduced using cells transformed with YEp352fF (2 µm,
URA3, FUR4, 28), 195gF (2 µm, URA3,
GAL10-FUR4, 23), or pFL38gG-GFP (CEN,
URA3, GAL10-FUR4::GFP, Ref.
29).
Lipid Analysis--
Sphingoid long chain bases (LCBs) and
their phosphorylated species (LCBPs) were extracted from yeast cells
and coupled to 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate, and the derivatized compounds were analyzed by high
performance liquid chromatography (30).
Complex sphingolipids were measured by radiolabeling cells with
myo-[2-3H]inositol (American Radiochemicals
Inc. #116, 20 Ci/ml) as described previously, except that cells were
grown for 16-18 h in the presence of the radioisotope to an
A600 of 1, and then lipids were extracted, deacylated, and chromatographed on thin layer plates (31).
Radioactivity was detected by using a BioScan apparatus, and
sphingolipid standards, co-chromatographed with each radiolabeled
sample, were detected after spraying the thin-layer plate with 10%
copper sulfate in 8% phosphoric acid followed by charring at
160 °C. Radiolabeled sphingolipids were quantified by chromatography
on EDTA-treated silica gel-impregnated paper (Whatman SG81) and
analyzed as described previously (31).
Uracil Uptake Assay--
The assay was based upon published
procedures showing that uptake of uracil requires Fur4p (22, 23). Cells
were grown in YPD medium overnight at 25 °C to an
A600 of 0.3-0.4, and 3 A600 units were harvested by centrifugation at
1900 × g for 3 min at room temperature. Cells were
suspended in 1 ml of YPD broth prewarmed to 30 °C, and 56 nCi of
[14C]uracil (57 mCi/mmol, Sigma) was added. After 10 min
of incubation at 30 °C, the cells were filtered onto a Whatman GF/C
filter and washed 3 times with 2 ml of ice-cold water. Filters were
dried and suspended in 4 ml of Ultima Gold XR scintillation fluid
(Packard; Meriden, CT), and radioactivity was measured in a liquid
scintillation counter. Uptake was linear over 10 min and required Fur4p
(data not shown).
Secretion of Proteins into the Culture Medium--
This
procedure is similar to a published procedure (32). Cells were grown
overnight in YPAUD at 25 °C to an A600 of
0.2-0.3, and 1.5 A600 units were harvested by
centrifugation for 3 min at 1,900 × g at room
temperature. The cell pellet was washed once with 1 ml of SD lacking
methionine and cysteine, then suspended in 350 µl of the same medium
containing 150 µCi of Tran35S-label (ICN Biomedicals,
Inc.), 0.06 mg/ml bovine serum albumin, and 1 mM
phenylmethylsulfonyl fluoride. The reaction was incubated at 25 °C
for 20 min or 37 °C for 15 min and stopped by a 5-s centrifugation in a microcentrifuge to yield cell pellet and medium fractions. The
medium fraction (300 µl) was transferred to a new tube containing 20 µl of both 200 mM NaN3 and 200 mM
NaF and centrifuged for 1 min at 4 °C to remove any remaining cells.
Proteins were precipitated from the medium fraction by a 5-min
incubation on ice in the presence of trichloroacetic acid (final
concentration 6.5%) and deoxycholic acid (final concentration 0.6 mg/ml) and collected by centrifugation for 10 min at 4 °C. The
precipitate was washed twice with 1 ml of acetone (
20 °C), air
dried at room temperature, and dissolved in 30 µl of sample buffer
(50 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 0.1%
bromphenol blue, and 100 mM dithiothreitol). The cell
pellet was suspended in 300 µl of sample buffer and vortexed with
glass beads (0.5 volume) for 4 min at room temperature. Whole cells and
glass beads were removed by centrifugation for 5 s in a
microcentrifuge. The cell-free lysate and the trichloroacetic acid-precipitated proteins were treated for 5 min in a 100 °C boiling water bath and resolved by SDS-PAGE (8% for the media fraction
and 10% for the cell pellet fraction). Radioactivity was quantified by
using a Molecular Dynamics Storm PhosphorImager.
Isolation of Lipid Rafts--
Lipid rafts were isolated
essentially as described previously (10). Cells were grown overnight in
YPD at 25 °C to an A600 nm of 0.3-0.5,
harvested by centrifugation, and washed once with 1 ml of
H2O. The cell pellet was suspended in 350 µl of TNE
buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA) plus protease inhibitors (final concentrations of
0.2 mM 4-[2-aminoethyl]benzenesulfonyl fluoride
hydrochloride, 3 µM E-64, 4 µM pepstatin A,
1 mM 1,10-phenanthroline (Calbiochem), and broken by
vortexing with a 0.5 volume of glass beads for 8 cycles (30 s of
vortexing followed by 30 s on ice). Unbroken cells and beads were
removed by centrifugation for 5 min at 500 × g. The
chilled supernatant fluid (250 µl) was incubated with Triton X-100
(1% final concentration) for 30 min on ice followed by the addition of
Optiprep (Nycomed, Oslo, Norway) to give a final concentration of 40%
(w/v). The sample was placed in a centrifuge tube and overlaid with 1.2 ml of 30% Optiprep in TXNE (TNE plus 0.1% Triton X-100) followed by
200 µl of TXNE and centrifuged for 2 h at 55,000 rpm in a
Beckman TLS55 rotor at 4 °C. Six equal fractions, collected starting
from the top of the gradient, were diluted to 1 ml with
H2O, and proteins were precipitated with trichloroacetic
acid (final concentration 10%) for 30 min on ice. Precipitated
proteins were collected by centrifugation in a microcentrifuge for 20 min at 4 °C, and the pellet was dissolved in 15 µl of 1 M Tris base and 35 µl of dissociation buffer (0.1 M Tris-Cl, pH 6.8, 4 mM EDTA, 4% SDS, 20%
glycerol, 2% 2-mercaptoethanol, 0.02% bromphenol blue). Samples were
heated at 37 °C for 10 min and resolved on 10% SDS-PAGE using a
Tricine buffer system (33), and proteins were detected by immunoblotting.
Immunoblots--
Immunoblotting was performed by transferring
proteins separated on SDS-PAGE to a polyvinylidene fluoride membrane
(Millipore, Bedford, MA) followed by blocking overnight in 2% powdered
skim milk made in TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl). Primary antibodies were rabbit anti-Fur4p
(1/40,000 dilution; Dr. R. Haguenauer-Tsapis), rabbit anti-Pma1p
(1/50,000 dilution; Dr. R. Serrano or A. Chang), and mouse anti-Pgk1p
(1/500 dilution; Molecular Probes). Secondary antibodies were
polyclonal rabbit IgG (1/20,000 dilution; Molecular Probes) and
monoclonal mouse IgG (1/3,000 dilution; Molecular Probes). Bound
antibodies were detected using ECF substrate (Amersham Biosciences), and the fluorescent signal was quantified by using a
Molecular Dynamics Storm PhosphorImagerTM equipped with ImageQuantTM software.
Immunoprecipitation--
Trichloroacetic acid-precipitated
proteins were first neutralized with 20 µl of Tris base then
dissolved in 30 µl of the same dissociation buffer as used for
isolating lipid rafts, except that it lacked 2-mercaptoethanol. Samples
were heated for 10 min at 37 °C, and chilled on ice. TNET buffer
(0.6 ml of 50 mM Tris-Cl, pH 7.4, 150 mM NaCl,
5 mM EDTA, 1% Triton X-00) was added, and insoluble
material was removed by centrifuging at top speed for 30 min in a
microcentrifuge at 4 °C. The supernatant fluid (200 µl plus 400 µl of TNET) was incubated with 40 µl of protein A-Sepharose beads
(10% w/v in TNET) with gentle agitation. The beads were removed by
centrifuging for 3 min at 3000 rpm at 4 °C, and proteins were
immunoprecipitated from the supernatant fluid by adding beads as
mentioned above and 4 µl of anti-GFP antibody (Torrey Pines Biolabs,
San Diego, CA). Immunoprecipitation reactions were incubated overnight
at 4 °C with gentle agitation, and beads were collected as described
above and washed 4 times with 0.5 ml of TNET buffer. Proteins were
eluted from the beads with 30 µl of dissociation buffer and subjected
to heat treatment at 37 °C for 10 min. Proteins were resolved by
SDS-PAGE (10%) in the Tricine buffer system; the gels were dried and
exposed on a Kodak phosphor storage screen. Radioactive proteins were
analyzed by using a PhosphorImager.
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RESULTS |
The Level of Sphingolipid Intermediates and Complex Sphingolipids
Is Reduced in lcb1-100 Cells--
The extreme instability and
temperature sensitivity of serine palmitoyltransferase activity and the
slow growth phenotype of lcb1-100 cells (see below)
suggest that they have a reduced capacity even at a permissive
temperature to make sphingolipid biosynthetic intermediates including
the LCBs DHS and PHS. Because heat induces a transient increase in LCBs
in wild type cells (34-36) it seemed likely that the thermoinstability
of serine palmitoyltransferase in lcb1-100 cells would
prevent the transient increase. This increase regulates the transient
cell cycle arrest in an unknown manner, which occurs shortly after
cells are heat-shocked (19) and also mediates endocytosis and
restoration of the actin cytoskeleton by an unknown mechanism (20, 37).
In addition, we examined LCBPs, which have not been examined previously
in lcb1-100 cells. LCBPs also transiently increase during
heat shock (36, 38) and are thought to mediate some processes that
protect cells against heat stress (for review, see Ref. 11).
LCBs and LCBPs were quantified by tagging them with a fluorescent
reagent after extraction from cells followed by HPLC (30). Parental
RH1800 (LCB1) cells behaved as expected, and all species of
LCBs increased transiently shortly after cells were shifted from 25 to
37 °C (Fig. 1A; maximum
fold increase from time 0: C16-PHS, 1.3-fold;
C18-DHS, 2.5-fold; C18-PHS, 4-fold;
C20-DHS, greater than 30-fold; C20-PHS,
13-fold). RH3809 (lcb1-100) cells behaved very differently.
Even at 25 °C the basal level LCBs was reduced from 2- to 7-fold
compared with parental cells (Fig. 1B). These data differ
from previous data that indicated that lcb1-100 cells grown
at 25 °C contain a normal level of LCBs and that the level did not
change during heat shock (19). Second, the major species of LCBs in
lcb1-100 cells, C16-DHS, C18-DHS,
and C18-PHS, did not increase after heat treatment. Instead
they steadily decreased and reached a plateau after about 30 min.
C20-PHS did increase to about 1 pmol/A600 units of cells, but this concentration
is still only 10% of the level found in parental RH1800 cells, and the
level stayed elevated in mutant cells rather than dropping, as it did
in parental cells. Finally, RH3809 cells did not make any detectable
C20-DHS at any time point.

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Fig. 1.
Changes in LCBs and LCBPs during heat
shock. RH1800 (LCB1) and RH3809 (lcb1-100)
cells were grown to an A600 nm of 0.3-0.4 in
YPD medium at 25 °C, and at time 0 the cultures were shifted to
37 °C. LCBs and LCBPs were extracted, derivatized with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and
analyzed by HPLC. Values are the average for duplicate samples ±S.D.
The LCB values in pmols/A600 units at the zero
time point for RH1800 cells were: C16 DHS = 8.9, C18 PHS = 67, C18 DHS = 20, C20 PHS = 1.6, and C20 DHS = <0.1.
The LCB values at the zero time point for RH3809 cells are:
C16 DHS = 3.4, C18 PHS = 10.3, C18 DHS = 7.0, C20 PHS = <0.1, and
C20 DHS = <0.1. The LCBP values at the zero time
point for RH1800 cells were: C16 DHSP = 4.4, C18 PHSP = 5.9, C18 DHS = 1.3, C20 PHS = <0.1, and C20 DHS = <0.1.
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LCBPs also transiently increase during heat shock, and this is what we
observe for RH1800 (wild type LCB1) cells, where all species
of LCBPs transiently increase, although each species shows different
kinetics (Fig. 1C). Surprisingly, LCBPs were not detected in
lcb1-100 (RH3809) cells, with the limit of detection being 0.1 pmol/A600 unit of cells (data not shown).
The reduced basal level of LCBs in lcb1-100 cells could
cause a reduction in the concentration of IPC, MIPC, and
M(IP)2C. To examine this possibility, sphingolipids were
measured by growing cells for at least 10 generations at 25 °C in
the presence of [3H]inositol. Cells were maintained in
log phase growth during the labeling period to avoid the possibility
that they would differentially take up radiolabel upon entry into
stationary phase. Lipids were extracted, deacylated to remove
glycerophospholipids, and radioactive sphingolipids were analyzed in
two ways. First, they were chromatographed on a high performance
thin-layer plate, and radioactive species were localized by using a
Bioscan apparatus. This approach gives good resolution of IPC, MIPC,
and M(IP)2C and was used to determine which species were
present and what their relative level was. However, quenching within
each band impairs quantification. To avoid quenching, samples were also
chromatographed on SG81 paper, and the chromatogram was cut into
sections and analyzed in a liquid scintillation spectrometer. This
approach does not separate IPC from MIPC.
We observed that IPC plus MIPC were reduced 77% in
lcb1-100 cells and that M(IP)2C was reduced
22% compared with parental RH1800 cells (Table
II). The total reduction in sphingolipid
content is about 50%. This value is based upon the fact that
M(IP)2C has twice the number of radioactive inositol groups
as do IPC and MIPC. [3H]Glycerophosphoinositol, the
deacylation product of phosphatidylinositol, served as an internal
control for radiolabeling. It was present at the same level in both
strains, showing that the reduced radiolabeling of M(IP)2C
and IPC plus MIPC is due to a reduced level of these compounds. From
analysis of the thin-layer chromatogram is was clear that the MIPC band
was reduced in lcb1-100 cells and so was the band
corresponding to IPC with a dihydroxy fatty acid (data not shown). We
conclude that the total concentration of complex sphingolipids is
reduced about 50% in unstressed lcb1-100 cells relative to
wild type cells.
Because the uptake of [3H]inositol drops when
lcb1-100 cells are heat-shocked (data not shown), it was
not possible to measure sphingolipids after heat shock using a
radiolabeling procedure. However, because LCB levels drop in
lcb1-100 cells after heat shock (Fig. 1B), it
seems likely that sphingolipid synthesis also drops, and this would
further impair any cellular process dependent upon continued synthesis
of LCBs, ceramide, or complex sphingolipids.
The Protein Encoded by the lcb1-100 Allele Has Ala-381 Changed to
Thr--
To determine the location of the lcb1-100
mutation, we transformed RH3809 cells with a series of overlapping DNA
fragments and looked for complementation of the mutant allele by
growing cells at 37 °C. A DNA fragment spanning bases 900-1350 of
LCB1 gave rise to cells that grew at 37 °C. This region
of the lcb1-100 chromosomal locus was recovered by gap
repair of a plasmid carrying LCB1, and the DNA sequence of
both strands of several gap-repaired plasmids was determined.
Nucleotide 1141 was found to be mutated from G to A, resulting in
Ala-381 being changed to Thr. Supporting the idea that this mutation
inactivates the gene is the finding that gap-repaired plasmids carrying
the mutation did not restore growth at 37 °C when transformed into
RH3809 cells, whereas the wild type LCB1 allele did restored
growth (data not shown). Using the proposed three-dimensional model for
serine palmitoyltransferase (39), Ala-381 is predicted to be at the
interface between the Lcb1 and Lcb2 subunits, in the vicinity of the
predicted active site. Thus, the Ala-381 to Thr change may affect
catalysis, interaction of the two subunits, or both.
Amino Acid and Uracil Uptake Are Impaired in lcb1-100
Cells--
We observed that cells carrying the lcb1-100
allele grew slower at permissive temperatures than did cells with the
wild type LCB1 allele. For example, strains with the
lcb1-100 allele (RH3809 and RH3804) grew more slowly than
parental RH1800 cells on YPD plates (Fig.
2) and barely grew on defined medium
(SD, Fig. 2), suggesting impaired nutrient uptake. This
hypothesis is supported by the observation that adding 0.5% casamino
acids to SD plates improves growth of lcb1-100 cells
(SD + CA, Fig. 2).

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Fig. 2.
Nutrient uptake limits the growth of
lcb1-100 cells. Strains RH1800 (wild type
(WT)), RH3804 (lcb1-100), and RH3809
(lcb1-100) were streaked onto to agar plates containing
YPD, SD, or SD supplemented with 0.5% casamino acids (SD + CA) and incubated at 30 °C for 4 days.
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We also found that uracil uptake was abnormal and focused on it because
there is only one uracil transporter in S. cerevisiae cells,
Fur4p, and much is known about the regulation of uracil uptake activity
(29). RH3809 (lcb1-100) cells grown at the permissive temperature of 25 °C had 25% less uracil uptake activity as did wild type RH1800 cells (Fig. 3, zero-min
time point). Upon shifting to 37 °C, uracil uptake activity
decreased within 30 min in both strains. These results are similar to
published data for wild type cells subjected to various stresses where
it is known that Fur4p is degraded (23). Within the next 30 min the
activity began to rise in wild type cells, and it returned to about
65% of the starting activity by 150 min. In contrast, mutant RH3809 cells did not restore uptake activity.

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Fig. 3.
Uracil uptake activity is not restored in
lcb1-100 cells after heat shock. Cells were
grown overnight at 25 °C to early log phase, the zero-min sample was
harvested, and then cultures were shifted to 37 °C. Values represent
the average of 2 cultures assayed in duplicate (n = 4)
±S.D. (the asterisk (*) indicates p = 0.012, and the double asterisk (**) indicates
p < 0.001, determined by using Student's t
test).
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Nearly all published studies of Fur4p have used cells overproducing the
protein some 20-40-fold above the endogenous level so that the protein
level could be measured by immunoblotting (Ref. 29 and references
therein). Therefore, we examined lcb1-100 cells carrying
FUR4 on a multicopy plasmid and found that uracil transport
activity behaved the same as it did in cells with only chromosomal
FUR4 (data not shown).
Restoration of Uracil Uptake Activity after Heat Shock Requires
Exocytosis--
To begin to understand why uracil uptake activity is
not restored in lcb1-100 cells after heat shock, we first
determined if restoration requires the secretory pathway. For these
experiments the restoration of uptake activity after heat shock was
compared in wild type and sec6 mutant cells, which have a
temperature-sensitive block in exocytosis. Sec6p is part of the exocyst
complex that is necessary for transport of proteins (40) and
sphingolipids (41) from the Golgi to the plasma membrane. We found that
sec6 cells fail to restore uracil uptake activity after heat
shock (Fig. 4), indicating that
restoration requires a functional secretory pathway.

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Fig. 4.
Restoration of uracil uptake activity after
heat shock requires protein secretion. Uracil uptake activity was
compared in wild type (NY13) and sec6-4 (NY17) cells after
transfer of cultures from 25 to 37 °C. Values represent the average
of 2 cultures assayed in duplicate (n = 4) ±S.D. (the
asterisk (*) indicates p < 0.00001, determined by using Student's t test).
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Restoration of Uracil Uptake Activity after Heat Shock Requires
Sphingolipid Synthesis--
Next we determined if uracil uptake
activity could be restored in lcb1-100 cells if they were
allowed to make sphingolipids at the restrictive temperature.
Sphingolipid synthesis can be restored in lcb1-defective
strains by adding PHS to the culture medium (18). We found that the
addition of 5 µM PHS to the culture medium 15 min before
heat shock restored uracil uptake activity in lcb1-100
cells (Fig. 5) with the same kinetics and
degree of restoration as was seen in wild type cells (Fig. 3). In
contrast, activity in the control cells treated with ethanol, the
carrier for PHS, was not restored (Fig. 5).

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Fig. 5.
Restoration of uracil uptake activity in
heat-shocked lcb1-100 cells requires synthesis of
complex sphingolipids. Uracil uptake activity was compared in
lcb1-100 cells (RH3809) treated with PHS, treated only with
vehicle (EtOH) or treated with PHS and aureobasidin A
(PHS + AbA), which blocks conversion of ceramide to IPC,
MIPC, and M(IP)2C. PHS and AbA stock solutions were
prepared in 95% EtOH and diluted into cultures to give final
concentrations of 5 µM PHS, 1 µg/ml AbA, and 1% EtOH.
Treatments were initiated 15 min before transfer of cultures from 25 to
37 °C. Values represent the average from 4 cultures assayed in
duplicate (n = 8) ±S.D. (the asterisk (*)
indicates p < 0.00001, determined by using Student's
t test).
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Exogenous PHS could restore uracil uptake activity by acting as a
signaling molecule to regulate some step in exocytosis, as has been
suggested (32, 42), or it could act by restoring sphingolipid synthesis
in lcb1-100 cells. To determine which of these
possibilities is correct, lcb1-100 cells were treated with PHS and with aureobasidin A (AbA), which blocks sphingolipid synthesis by inhibiting IPC synthase, the enzyme that transfers inositol phosphate onto ceramide to form IPC (43). Cells were grown in the
presence of increasing concentrations of AbA to find the lowest concentration that inhibited growth under the culture conditions used
(data not shown). Cells treated with PHS and AbA behaved like the
ethanol-treated cells and did not restored uracil uptake (Fig. 5). We
conclude that restoration of uracil uptake activity in heat-shocked
lcb1-100 cells requires de novo synthesis of one or more of the complex sphingolipids, IPC, MIPC, and
M(IP)2C.
Exocytosis Is Selectively Blocked in Heat-shocked lcb1-100
Cells--
We next determined whether exocytosis is completely or
selectively blocked in lcb1-100 cells after heat shock by
examining the secretion of proteins into the culture medium. Cells were pulse-labeled with 35S-labeled amino acids before heat
shock, and radioactive proteins excreted into the culture medium were
examined at the start (0 min) and after 60 and 120 min of heat shock.
At 0 min, the pattern of radioactive proteins secreted by RH3809 cells
is similar but not identical to wild type RH1800 cells (Fig.
6). The primary difference is a higher
concentration of the 120- and 173-kDa bands in the lcb1-100
cell samples. At the 60-min time point secretion of all proteins except
the 173-kDa protein is reduced in mutant RH3809 cells compared with
wild type RH1800 cells. At the 120-min time point secretion of proteins
into the culture medium is mostly restored in RH3809 cells, with the
major missing bands being those of 49 and 111 kDa. As a control to
measure the rate of protein synthesis, radioactivity in the pellet
fractions was quantified by SDS-PAGE and phosphorimage analysis, which
was used to sum all pixels in a lane. At the zero-min time point
lcb1-100 cells incorporated 50% as much radioisotope into
proteins as did the wild type cells, whereas at 60 and 120 min, the
value was 37 and 32%, respectively (data not shown). Even without
correcting for reduced total protein synthesis, it is apparent that
secretion of some but not all proteins is blocked after
lcb1-100 cells are heat-shocked. We conclude from these
data that there is a selective, but not a complete block in the
secretory pathway when lcb1-100 cells are heat-shocked.

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Fig. 6.
Secretion of proteins into the culture medium
is selectively blocked in lcb1-100 cells after heat
shock. Radioactive proteins secreted by RH1800 and RH3809 cells
into the culture medium were measured before heat shock (0 min) and
after 60 and 120 min of heat treatment at 37 °C. For the zero-min
time point cells grown at 25 °C were labeled with
35S-labeled amino acids for 20 min at 25 °C, whereas
cells taken after 60 and 120 min of heat treatment were labeled for 15 min at 37 °C. Proteins excreted into the culture medium were
separated by SDS-PAGE, and radioactive bands were analyzed by using a
PhosphorImager. Molecular size markers (in kDa) are indicated at the
left of the figure. Each time point shows the proteins
secreted into the culture medium for two of the three individual
cultures examined as described under "Materials and Methods." Bands
marked at the right by an open circle ( ) and asterisk (*)
were not secreted by RH3809 cells.
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Fur4p Is Present in Detergent-insoluble Complexes--
It has
recently been shown that some membrane proteins in yeast including
Gas1p and Pma1p are found in detergent-insoluble complexes or lipid
rafts, experimentally identified by insolubility in 1% Triton X-100 at
4 °C and by their low buoyant density in a Optiprep density gradient
(10). Raft association and transport of Pma1p to the plasma membrane
depend upon sphingolipid synthesis (15). Thus, one explanation for the
failure of lcb1-100 cells to restore uracil uptake activity
is that de novo sphingolipid synthesis ceases after heat
shock, thereby preventing new rafts from forming in the Golgi, which
blocks transit of raft-associated proteins to the plasma membrane.
To test this hypothesis we first determined if Fur4p is present in
lipid rafts. Wild type RH1800 and lcb1-100 (RH3809) cells were disrupted and extracted at 4 °C with 1% Triton X-100, and the
extracts were fractionated by centrifugation on an Optiprep density
gradient. The raft-associated control protein Pma1p localized to the
top of the gradient primarily in fractions 1 and 2 in both types of
cells grown either at 25 °C or grown for 120 min at 37 °C (Fig.
7A). Fur4p also localized in
fractions 1 and 2, indicating that it associates with lipid rafts.
Phosphoglycerokinase was used as a control for a soluble, non-raft
protein. It localized in fractions 4, 5, and 6 at the bottom of the
gradient. To verify that we were dealing with detergent-insoluble
complexes, the Triton X-100 extractions were performed at 4 °C and
at 30 °C. Pma1p and Fur4p were only insoluble when the extraction
was performed at 4 °C (data not shown), indicating that the proteins
partition into the detergent-insoluble fraction. In this particular
experiment (Fig. 7A) there appears to be staining by the
Fur4p antibodies in fractions 4-6 of the RH3809 37 °C sample. This
staining was nonspecific because it did not appear in other
experiments, and most importantly, it was not present when Fur4p was
overproduced (Fig. 7B).

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Fig. 7.
Fur4p associates with lipid rafts.
A, RH1800 (wild type (WT)) or RH3809
(lcb1-100) cells were grown overnight in YPD to an
A600 of 0.3 at 25 °C and then shifted to
37 °C for 120 min. Triton X-100-insoluble material was isolated and
fractionated by centrifugation on an Optiprep density gradient. Six
fractions were collected from the gradient and analyzed by Western
blotting using antibodies against Fur4p or against Pma1p, a marker for
raft-associated proteins, and Pgk1, a marker for a non-raft protein.
B, same experiment as in A, except that Fur4p was
overproduced by transforming RH1800 (wild type) or RH3804
(lcb1-100) cells with YEp352fF.
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After 2 h at 37 °C the concentration of Fur4p in wild type
RH1800 cells is less than in the 25 °C sample taken before heat shock (Fig. 7A, top immunoblot fractions 1-3,
compare 25 and 37 °C). This reduction in Fur4p is reflected in
reduced uracil transport activity after 2 h of heat shock (Fig.
3). These results are in agreement with previous studies showing that
Fur4p is rapidly degraded and uracil transport activity is lost in wild
type cells after heat shock, but that both are restored by 2 h
(22, 23). The situation in lcb1-100 cells (RH3809) appears
to be similar but is actually very different. The immunoblot shown in
Fig. 7A indicates that there is slightly less Fur4p in raft
fractions 1 and 2 in the cells grown for 2 h at 37 °C compared
with the 25 °C sample. However, the Fur4p present in fractions 1 and
2 of the 37 °C sample is not newly synthesized protein as it is in
the wild type cells but, instead, represents protein that failed to be
degraded. This explanation is based upon previous reports that Fur4p
fails to be degraded when lcb1-100 cells are heat-shocked (44). Therefore, the uracil transport data (Fig. 3) combined with the
immunoblot data (Fig. 7A) show for the first time that Fur4p
transport activity is inactivated by heat stress in
lcb1-100 cells and that, whatever the inactivation process
is, it does not require a transient increase in long chain bases.
Nearly all published studies of Fur4p have used cells overproducing the
protein, so we determined if the overproduced protein associated with
rafts. Indeed, most of the Fur4p in wild type RH1800 or mutant RH3804
cells transformed with a 2-µm vector carrying FUR4 under
control of its own promoter (YEp352fF) localized to the top two
fractions of an Optiprep gradient just like the raft-bound Pma1 control
protein (Fig. 7B). We conclude from these results that Fur4p
associates with lipid rafts.
Association of Fur4p with lipid rafts provides an explanation for why
lcb1-100 cells do not restore uracil transport activity after heat shock. Heat shock would cause DHS and PHS levels to drop
(Fig. 2), and this change would set off a chain reaction so that
ceramide synthesis in the endoplasmic reticulum and complex sphingolipid synthesis in the Golgi would be greatly reduced. As a
consequence, raft formation either in the endoplasmic reticulum or
Golgi would be impaired, as would raft-dependent processes including transport of newly synthesized Fur4p to the plasma membrane.
We attempted to garner further support for this hypothesis by measuring
the rate at which de novo synthesized Fur4p associates with
rafts. Because the concentration of endogenous Fur4p is low, we used
the galactose-inducible GAL1 promoter to drive
overexpression, as described previously (22). In addition, we used a
FUR4 allele with GFP fused to the C terminus (27) so that
the protein could be immunoprecipitated using a commercially available
antibody. Anti-Fur4p is not commercially available, and our stock was
insufficient for immunoprecipitation experiments. Unfortunately, the
lcb1-100 strains used in our experiments only grow well
with glucose as the carbon source, and this prevented induction of
GAL gene expression. To overcome this impasse we switched to
the W303 strain background. Using established procedures (15, 22) to
pulse label proteins with 35S-labeled amino acids, we
obtained measurable amounts of immunoprecipitated, radiolabeled
Fur4-GFPp before isolation of lipid rafts. However, after raft
isolation by treatment of the sample with 1% Triton X-100,
immunoprecipitable, radioactive Fur4-GFPp was undetectable (data not
shown). Thus, for these technical reasons it was not possible to
directly measure the rate at which de novo synthesized Fur4p
associates with lipid rafts.
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DISCUSSION |
Our results demonstrate that sphingolipid synthesis is essential
for delivery of Fur4p to the plasma membrane (Fig. 5) and that the
protein is present in lipid rafts (Fig. 7). The sphingolipid requirement for delivery to the plasma membrane most likely reflects the need for Fur4p to associate with lipid rafts as they form in the
secretory pathway. We also find that the steady-state level of complex
sphingolipids in lcb1-100 cells is 50% lower than in wild
type cells (Table II). This reduction may be the reason why lcb1-100 cells grow poorly on a defined medium unless it is
supplemented with amino acids (Fig. 2) and why the cells have only
about 75% of the wild type level of uracil transport activity at
permissive temperatures (Fig. 3). Finally, our results establish that
long chain bases are not essential for the process of inactivating Fur4p uracil transport activity but that they are necessary for a later
step in Fur4p breakdown.
Because lipid raft formation requires complex sphingolipids, we
reasoned that the failure of lcb1-100 cells to restore
uracil transport activity after heat shock was due to a block in raft formation. This hypothesis predicts that Fur4p should be associated with lipid rafts, and this is what we found experimentally (Fig. 7).
Another prediction of this hypothesis is that delivery of most proteins
to the plasma membrane or secretion of proteins into the culture medium
should be unaffected in heat-shocked lcb1-100 cells, since
many proteins are not associated with lipid rafts, and their transport
to the plasma membrane should be normal. Indeed, we observed that
secretion of 5 of 7 radioactive proteins into the culture medium was
restored in lcb1-100 cells 120 min after heat shock (Fig.
6). Cells with a complete block in the secretory pathway accumulate
secretory vesicles or enlarge their secretory compartments, which can
be observed by electron microscopy (45). We analyzed
lcb1-100 cells before and after heat shock by electron microscopy and found no differences compared with type cells (data not
shown). Together these data strongly support the hypothesis that
restoration of uracil transport activity requires de novo raft formation, which in turn requires de novo sphingolipid synthesis.
The reported lability of serine palmitoyltransferase activity and other
phenotypes of lcb1-100 cells suggested that they do not
make normal levels of LCBs. Quantification of LCBs in
lcb1-100 cells showed that their basal value was reduced
from 2- to 7-fold compared with parental cells even when cells were
grown at the permissive temperature of 25 °C (Fig. 1). We also found
that lcb1-100 cells had no detectable basal level of LCBPs
nor did heat shock increase LCBPs. The absence of LCBPs may occur
because the concentration of LCBs in lcb1-100 cells is
below the Km for the two lipid kinases, Lcb4p and
Lcb5p, that phosphorylate LCBs to yield LCBPs. Specific functions for
LCBPs have not been identified in S. cerevisiae.
lcb4
lcb5
cells, which lack all detectable
LCBPs, grow normally and only show a very slight reduction in resisting heat stress (Ref. 36 and references therein). So the lack of LCBPs in
lcb1-100 cells seems unlikely to be the cause of the reported phenotypes; the most likely cause is the reduction in LCBs
(Fig. 1) and complex sphingolipids (Table II).
Our observation that lcb1-100 cells have a reduced basal
level of LCBs and that the levels drop rapidly after cells are
heat-shocked at 37 °C, except for C20-PHS (Fig. 1), provide a
chemical basis for explaining why signal transduction pathways
regulated by LCBs are disrupted when lcb1-100 cells are
heat-shocked. DHS and PHS have been shown to regulate the protein
kinases Pkh1p and Pkh2p (46-48), which in turn regulate the kinase
activity of Pkc1p and probably Ypk1p and Ypk2p, which control
endocytosis (47, 48), actin cytoskeletal restoration after heat shock
(47), and the cell integrity pathway (49) in ways that are not entirely
understood. In contrast to our data, it was previously reported (19)
that the basal level of LCBs in lcb1-100 cells was similar
to wild type and dropped very little upon heat shock. The different
results are probably not due to strain differences since their strains and ours originated in Riezman's laboratory and carry the
lcb1-100 allele. Perhaps the differences are due to the way
in which lipids are extracted and processed before HPLC analysis.
The earliest known event in stress-induced degradation of Fur4p is
phosphorylation of serine residues within a PEST sequence mediated in
part by the casein kinases Yck1p and Yck2p (50). Subsequently, the
protein is ubiquitinated, internalized by endocytosis, and degraded in
the vacuole (27, 28). Heat-shocked lcb1-100 cells do not
degrade overproduced Fur4p unless given exogenous PHS (44). Presumably
a transient increase in PHS is needed to facilitate some step in the
degradation pathway, but the exact function of PHS is unknown. Our data
(Fig. 7), obtained by measuring endogenous, not overproduced Fur4p,
confirm this conclusion. In addition, our data show that uracil uptake
activity declines rapidly in lcb1-100 cells after a heat
shock, leading to the important conclusion that PHS (or any other LCB
or LCBP) does not regulate the inactivation of Fur4p uptake activity.
The molecular nature of inactivation is unknown. Future studies will
need to address this question and determine whether PHS regulates
phosphorylation, ubiquitination, protein internalization, or some
subsequent event in the degradation process.
It should now be possible to identify the regions of Fur4p necessary
for raft association and whether or not such association is essential
for uracil transport activity. Rafts are thought to be important
centers for integrating and modulating signal transduction pathways
(3-5), and they may, thus, be critical for regulating breakdown of
Fur4p in response to stresses and other factors that govern uracil
transport activity.