From the Departments of Biochemistry and
** Microbiology and Immunology, MCP Hahnemann University,
Philadelphia, Pennsylvania 19129, the § Departement
d'Enzymologie Cellulaire et Moleculaire, Institut de Botanique,
Strasbourg Cedex, France, the ¶ Department of Veteran Affairs, New
Jersey Medical School, East Orange, New Jersey 07018, and the
Department of Biochemistry, Merck Research Laboratories, Rahway,
New Jersey 07065
Received for publication, January 11, 2001, and in revised form, January 17, 2001
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ABSTRACT |
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A temperature-sensitive
Saccharomyces cerevisiae mutant harboring a lesion in the
ERG26 gene has been isolated. ERG26 encodes 4 The budding yeast Saccharomyces cerevisiae is an
excellent model system for studying sterol biosynthesis and regulation
(1, 2). The sterol biosynthetic pathway in this yeast is highly conserved with its mammalian counterpart. The difference being that
ergosterol is synthesized as the end product sterol rather than
cholesterol. All of the structural genes have been cloned that are
required for the biosynthetic steps necessary to synthesize sterols in
S. cerevisiae (1, 2). In addition, many of the enzyme
activities involved in yeast sterol synthesis have been characterized
with respect to their enzymological parameters (3-7). Moreover, some
physiological functions for yeast sterols have been assigned using
constitutive sterol mutants (2, 8, 9).
One of the later steps in the sterol pathway is the conversion of
4,4-dimethylzymosterol to zymosterol (1). The reaction is carried out
in two sequential demethylation reactions by three separate enzyme
activities. In mammalian and plant cells, the activities required, a
C-4 methyl oxidase, a 4 In S. cerevisiae, the C-4 methyl oxidase, 4 A yeast erg25 mutant has been isolated through a screen
selecting for mutants unable to grow on low iron media (19).
Interestingly, exogenous iron transport activity in this
erg25 mutant is normal, suggesting that the changes in
zymosterol intermediate levels seen in these cells are not affecting
iron uptake but another intracellular process. In mammalian cells, a
cholesterol auxotrophic Chinese hamster ovary cell line has been
isolated that accumulates carboxysterols (20, 21). The lesion
responsible for the sterol metabolic defect is thought to reside within
the gene encoding for 4 In this report, we describe the isolation and initial characterization
of the erg26-1 conditional growth mutant. By using various
analytical and biochemical methods, we demonstrate that erg26-1 cells are defective in 4 Strains, Media, and Miscellaneous Microbial Techniques--
The
yeast strains used in this study are isogenic to W303-1A
(MATa leu2-3,112 trp1-1 ura3-1
his3-11,15 can1-100). Yeast strains were grown in
either YEPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or in
synthetic minimal media containing 0.67% Yeast Nitrogen Base (Difco)
supplemented with the appropriate amino acids, adenine, and uracil.
When required, media was supplemented with 15 µg/ml ergosterol as
described previously (24). 5-FOA plates contained 0.1% 5-FOA.
Yeast transformation was performed using the procedure described by Ito
(25). For routine propagation of plasmids, Escherichia coli
XL1Blue cells were used and grown in LB medium supplemented with
ampicillin (150 µg/ml). Bacterial transformations were carried out by electroporation.
Strain Construction and Plasmids--
Yeast null mutants were
generated by the one-step disruption method of Rothstein (26) using
individual Yeast Integrating plasmid (YIp) deletion constructs (27).
Yeast strains harboring individual deletions were verified by PCR
analysis. The URA3-containing centromeric plasmid pRS414 was
used to construct the various low copy ERG vectors (27).
pGAL-GFP is a LEU2 containing derivative of the
centromeric vector YCpIF3 (28). The GFP sequence contained in this vector harbors an S65A mutation (29). The two hybrid vectors
pBD-Gal4 Cam (CLONTECH) and pACT2 (Stratagene) were
used in the two hybrid studies. All genomic sequences subcloned into the various vectors were obtained by PCR amplification using high fidelity pfu polymerase. All DNA sequences that were
generated by PCR were sequenced and compared with the yeast genome data base. The ERG25 sequence used in our two-hybrid studies
lacked the C-terminal sequences encoding for the consensus ER retention signal. pJR1133-SUT1 and the parent plasmid pJR1133 are 2µ plasmids that carry the URA3 gene. Drs. Chris Beh and Jasper Rine
(University of California, Berkeley, CA) kindly provided them.
Isolation of Sphingolipid Metabolic Mutants--
W303-1A was
mutagenized with ethylmethane sulfunate by standard procedures
(30) and grown on inositol-free synthetic media for 3 days at 25 °C.
Colonies were transferred and grown on filter paper replicas and
labeled with 100 µCi of [3H]inositol, essentially as
described previously (31). After labeling for 40 min at restrictive
temperature (41 °C), filters were washed two times with 5%
trichloroacetic acid, air-dried, and exposed to Kodak BioMax MR film
for 6 days. Following exposure, the filters were treated sequentially
with the following solvents to chemically deacylate and remove the
[3H]inositol incorporated into phosphatidylinositol:0.2
N KOH in methanol/toluene (1:1, v/v) at room temperature
for 60 min, methanol for 10 min, 5% trichloroacetic acid for 10 min,
and twice more in methanol for 10 min each. Filters were dried, sprayed
with EN3HANCE (PerkinElmer Life Sciences), and exposed to
Kodak X-Omat film for 18 h. The signal intensity for each colony
before and after deacylation was compared, and isolates that had
reduced signal after deacylation relative to their initial signal were selected and grown. The entire labeling procedure was repeated on the
selected colonies as described, except radioactivity was quantified on
a Molecular Dynamics PhosphorImager using a tritium-sensitive screen.
Cloning the ERG26 Gene--
Sphingolipid metabolic mutant H21
was transformed with a YCp50-based yeast genomic low copy library (32).
Transformed H21 cells were plated onto synthetic plates lacking uracil,
and plasmids harboring yeast sequences conferring temperature
resistance were selected for at 37 °C. Plasmid YCp50-16-1-1 was
chosen as a representative suppressor plasmid (Fig. 1A)
based on restriction analyses of 12 independent suppressor plasmids.
DNA sequencing analysis and subcloning experiments revealed that the
ERG26 gene was responsible for conferring temperature
resistance to H21. Linkage analysis was used to confirm that the
ERG26 gene was mutated in the H21 mutant strain and that the
mutation was recessive. Several backcrosses were performed on the H21
strain to obtain the haploid segregant designated erg26-1.
The ts and lipid metabolic phenotypes of erg26-1 are identical to that of H21.
Labeling and Analysis of Phospholipids, Neutral Lipids, and
Sterols--
Starting cultures for all labeling experiments were
derived from exponential cells grown at 23 °C in the described
media. For pulse labeling of phospholipids, cells grown in synthetic media were shifted to 23 or 37 °C and incubated with 50 µCi/ml [32P]orthophosphate for 20 min. For steady-state labeling
of phospholipids, 0.05 A600/ml cells grown in
synthetic media were shifted to 23 or 37 °C and incubated with 10 µCi/ml [32P]orthophosphate for at least six
generations. Phospholipids were extracted using the spheroplast method
of Atkinson and Henry (33) and analyzed by one-dimensional TLC as
described previously (34). For pulse and steady-state labeling of
neutral lipids and sterols, cells grown in synthetic media were shifted
to 23 or 37 °C and incubated with 1 µCi/ml
[14C]acetate for 30 min or 4 h, respectively.
Radiolabeled neutral and sterol lipids were extracted using chloroform,
methanol (2:1) and analyzed by one-dimensional TLC using hexane, ethyl
ether, acetic acid (80:20:2) and petroleum ether, diethyl ether, acetic acid (70:30:1), respectively (35). In all cases, radiolabeled lipids
were visualized by x-ray film (Kodak XAR5). The percent value of each
lipid species was determined by densitometry using a Bio-Rad Model
GS-670 Imaging Densitometer and Molecular Analyst Software Version
1.4.1.
Preparation of Microsomes for the 4 4 GC/MS Analysis of Sterols--
Yeast cells were hydrolyzed in 1 N ethanolic NaOH and extracted exhaustively with ethyl
acetate. The extract was treated with diazomethane to form the methyl
esters of any free carboxyl groups and then with Sil-Prep (Supelco) to
form the trimethylsilyl ethers of free hydroxyl substituents. The
sample was then dissolved in hexane and injected onto a 30-m HP-5MS
capillary column (Agilent Technologies) installed in an HP 6890 gas-chromatograph interfaced with an HP 5972A mass selective detector.
After the injection, the column was kept at 100 °C for 2 min then
raised to 265 °C at a rate of 35 °C/min. The carried gas was
helium at a flow rate of ~1 ml/min. For the analysis of the
reaction products formed in the 4 Antifungal Supplementation Assays--
1 × 105
cells were spotted onto YEPD plates lacking or containing the indicated
antifungal compounds. The plates were then incubated at either 23 or
37 °C for 4 days. Alternatively, 2-ml cultures of various yeast
strains were grown to 0.5-1.0 A600 at 23 °C
and subsequently inoculated at 1.0 × 105 cells/ml
into 96-well microtiter dishes containing YEPD and various concentrations of antifungal compounds. Cells were then allowed to grow
at either 23 or 37 °C for several days without shaking, and the cell
number was determined by plate viability assays. The degree of
reduction in the accumulation of sterol intermediates due to antifungal
supplementation was determined by [14C]acetate and TLC analyses.
Isolation and Characterization of a Yeast Sterol Biosynthesis
Mutant Having a Defect in 4
We first analyzed the sterol lipid composition of wild type and
erg26-1 mutants using lipid radiolabeling studies and TLC. Studies were carried out at both the permissive and nonpermissive temperatures for erg26-1 cell growth. Using
[14C] acetate labeling and TLC, we found that
erg26-1 mutant cells accumulated two lipid species that are
not seen in wild type cells (Fig. 1B; ERG26
versus erg26-1). Under the conditions of our analysis, the two species migrated within the first one-third of the TLC plate
(RF 0.06 and 0.21). Both lipids were found to
accumulate at either temperature in erg26-1 cells, but to a
much greater degree at the nonpermissive temperature for growth (Fig.
1, erg26-1, 23° versus
erg26-1, 37°). We found that we
could greatly reduce the accumulation of these lipids in
erg26-1 cells at either temperature by introducing the wild
type ERG26 gene on a low copy plasmid (Fig. 1B;
erg26-1 pRS-ERG26; 37° shown). Thus,
the expression of ERG26 in single copy nearly eliminated the
accumulation of the two aberrant lipid species found in
erg26-1 cells. This result is to be expected, because our
linkage analysis did reveal that a recessive mutation resided within
the ERG26 gene in erg26-1 cells.
As a means to determine the molecular structures of these aberrant
lipid species, GC/MS analyses were performed on total sterol extracts
from wild type and erg26-1 cells grown at the nonpermissive temperature. GC analyses of these extracts revealed the presence of
several lipid peaks, with peaks 1 through 5 being detected in both wild
type and erg26-1 cells (Fig.
2, A and B).
However, there were three additional peaks detected in
erg26-1 cells that were not seen in wild type cells (Fig. 2,
A versus B). The three peaks,
designated 6, 7, and 8, had retention
times of 35.09, 38.73, and 39.32 min, respectively (Table
I). The molecular structures of all of
the peaks detected by GC are outlined in Table I and are based on our
MS analyses. Using the MS data obtained, we identified peaks
1, 2, 4, and 5 as ergosterol,
fecosterol, lanosterol, and 14-methylfecosterol, respectively. We were
unable to predict the molecular structure of peak 3.
The MS spectra of peaks 6 and 7 are shown in Fig.
3. They are consistent with these sterol
intermediates being the methyl esters of a
4-methyl-4-carboxy-diunsaturated sterol (Fig. 3A, peak
7) and a 4-carboxy-diunsaturated sterol (Fig. 3B,
peak 6). Based on the reaction mechanism involved in the
demethylation of 4,4-dimethylzymosterol, we believe that these
intermediates are the methyl esters of the expected substrate for
Erg26p, 4
To begin to ascertain the biochemical basis for the accumulation of
these sterol intermediates in erg26-1 cells, 4
Using GC-MS analysis, we determined the molecular structures of the
ketone products formed by the wild type and mutant Erg26p. Wild type
Erg26p catalyzed the formation of the expected single product,
cholest-7-en-3-one (15). On the other hand, we observed that the mutant
erg26-1p microsomal assay preparation contained additional compounds in
the sterone fraction from the TLC purification. MS spectra analysis of
the sterones in this fraction was consistent with these sterones being
the expected product, cholest-7-en-3-one, as well as other endogenous
sterones, including 4 erg26-1 Cells Have Defects in Neutral Lipid Metabolism--
To
examine in greater detail whether the erg26-1 mutation
caused additional perturbations in lipid metabolism, we determined the
rates of biosynthesis and steady-state levels of neutral lipids and
phospholipids in both wild type and erg26-1 cells.
Experiments were carried out at both the permissive and nonpermissive
temperatures for erg26-1 cell growth. Under these
conditions, erg26-1 cells were found to harbor several
defects in the rate of biosynthesis and steady-state levels of neutral
lipids. First, we found that the rates of biosynthesis (Fig.
4, A and B) and
steady-state levels (Fig. 4, C and D) of
ergosterol and sterol esters were reduced in erg26-1 cells
when compared with wild type cells. The reduction in the levels of
these sterols was seen at both the permissive (Fig. 4, A and
C) and nonpermissive (Fig. 4, B and D)
temperatures for growth. However, the greatest decrease in the rate of
biosynthesis of ergosterol (5-fold) and sterol esters (2.5-fold), and
in the steady-state levels of ergosterol (1.8-fold) and sterol esters (3.0-fold), was seen in temperature-shifted erg26-1 cells
(Fig. 4, B and D). In agreement with these data,
we found a 4-fold decrease in ergosterol content in the microsomes of
erg26-1 cells compared with the wild type microsomes.
Second, we found that erg26-1 cells grown at the
nonpermissive temperature had increased rates of biosynthesis of mono-
(4.0-fold) and diglycerides (3.5-fold), whereas a decreased rate of
biosynthesis of triglycerides (1.7-fold) (Fig. 4, A
versus B). The steady-state levels of these
lipids in erg26-1 cells, however, were found to be
comparable to those of wild type cells (Fig. 4, C
versus D). Finally, two
[14C]acetate-labeled lipids (Fig. 4, double
asterisks) were found to accumulate in erg26-1 cells
under all conditions examined. Their levels were found to drastically
increase in temperature-shifted erg26-1 cells. They were
extracted from the neutral lipid TLC plate and chromatographed under
the conditions used for TLC analysis of sterols. They were found to
comigrate with the 4
We also examined the rates of biosynthesis and steady-state levels of
individual phospholipids under the same conditions as those described
above. The only significant defect in phospholipid metabolism was found
in temperature-shifted erg26-1 cells, where there was an
increase in the rate of biosynthesis of PA (1.9-fold) and a decrease in
the rate of biosynthesis of PI (1.7-fold) (Fig. 5, A versus
B). However, the steady-state levels of these, and of all,
phospholipids detected in erg26-1 cells were found to be
comparable to those seen in wild-type cells (Fig. 5, C
versus D). Under the conditions of our assay, we
did not detect measurable amounts of CDP-DG, phosphatidylglycerol or
cardiolipin. Thus, we cannot say whether the erg26-1
mutation affects the rates of biosynthesis or steady-state levels of
these lipids.
The Increased Accumulation of Toxic Sterol Intermediates Causes the
Loss of Growth of erg26-1 Cells at the Nonpermissive
Temperature--
We explored the physiology underlying the
ts defect in erg26-1cells. Our studies focused on
determining whether the loss of ergosterol biosynthesis or the
increased accumulation of specific zymosterol intermediates was
responsible for the loss of growth at the nonpermissive temperature. To
address this question, we examined whether feeding ergosterol or
certain antifungal compounds to erg26-1 cells resulted in
growth at high temperatures. The antifungal compounds chosen were those
that targeted the sterol pathway, and would, or would not, reduce the
accumulation of the zymosterol intermediates when fed to
erg26-1 cells.
To perform the ergosterol feeding studies, we needed to genetically
manipulate our erg26-1 strain. This is because, under normal
aerobic growth conditions, yeast cells will not take up exogenous
sterols from the media (1). However, they can be made to take up these
lipids by overexpressing the gene, SUT1 (36). Thus,
erg26-1 cells were transformed with the high copy plasmid
pJR1133-SUT1. erg26-1 cells carrying this plasmid
were then examined for their ability to grow at high temperatures in the absence and presence of ergosterol.
Our feeding studies revealed that ergosterol supplementation was unable
to suppress the ts growth defect of our erg26-1
mutant strain. We found that erg26-1 cells incubated on
plates containing15 µg/ml ergosterol were able to grow at 25 °C
but were unable to grow at the nonpermissive temperature for growth,
37 °C (Fig. 6A). The
concentration of ergosterol we used has been shown to be sufficient for
the growth of erg26
In contrast, our antifungal feeding studies showed that certain
antifungal compounds were able to suppress the ts growth
defect of our erg26-1 mutant (Fig. 6B).
Strikingly, the ability of these antifungal compounds to suppress this
defect correlated with their ability to reduce the accumulation of the
zymosterol intermediates (Fig. 6C). Terbinafine is an
allyamine antifungal compound that targets the squalene epoxidase
enzyme encoded for by the ERG9 gene (1). Erg9p function
occurs upstream of Erg26p in the sterol pathway. We found that in the
presence of 1.25 µg/ml terbinafine erg26-1 cells were able
to grow at the nonpermissive growth temperature (Fig. 3B).
We also found that the addition of this concentration of terbinafine
caused a drastic reduction in the levels of the zymosterol
intermediates in erg26-1 cells (Fig. 6C). We
obtained identical results using fluconazole, which is an azole
antifungal that targets the lanosterol C-14 demethylase encoded for by
the ERG11 (1). Erg11p also functions upstream of Erg26p in
the sterol pathway.
In contrast, the addition of 20 ng/ml fenpropimorph, a morpholine
antifungal compound that targets the Erg2p sterol C-8 isomerase enzyme
that is located downstream of Erg26p, did not suppress the
ts growth defect of erg26-1 cells (Fig.
6B). Moreover, the addition of fenpropimorph did not cause a
reduction in the accumulation of the zymosterol sterol intermediates
(Fig. 6C). Thus, we conclude from our results that a
reduction in the accumulation of the zymosterol intermediates in
erg26-1 cells is required and responsible for any
antifungal-dependent ts growth suppression.
Therefore, our results strongly suggest that the accumulation of these
toxic intermediates in erg26-1 cells causes cell death at
high temperatures rather than the loss of ergosterol biosynthesis.
Altering Sphingolipid Biosynthesis Does Not "Bypass" the Cell
Lethality Associated with the Loss of Zymosterol
Biosynthesis--
SUR4/ELO3 and FEN1/GNS1/ELO2
encode for fatty acid elongase enzymes that are required for the
biosynthesis of C26 fatty acids (37). C26 fatty
acids are used exclusively to synthesize complex sphingolipids in yeast
(38). Studies have demonstrated that mutations in SUR4 or
FEN1 cause changes in the sphingolipid composition of cells
(37). Interestingly, previous work also has shown that loss of function
mutations in SUR4 or FEN1 can "bypass" the
essentiality of ERG2 (23), whereas mutations in
FEN1 allow strains lacking the ERG24 gene to
survive (22). The reactions catalyzed by ERG24 and
ERG2 lie just upstream and downstream of the
ERG25-, ERG26-, and ERG27-catalyzed
demethylation reactions, respectively (1). Thus, we wanted to determine
whether altering the sphingolipid composition in strains lacking
ERG25, ERG26, or ERG27 would allow for
growth in the absence of zymosterol biosynthesis.
We tested for sphingolipid-dependent bypass of
ERG25, ERG26, or ERG27 function using
5-FOA sensitivity assays. 5-Fluoroorotic acid is toxic to
URA3 cells, but not ura3
As a control for sphingolipid-dependent suppression, we
first attempted to demonstrate that loss of function of SUR4
allowed strains that lack ERG2 to live (23). However, we
found that, in our W303-1A strain background, erg2 Erg26p and Erg27p Localize to the Endoplasmic Reticulum, and Erg26p
Physically Interacts with Erg25p--
As a means of further
understanding the physiology underlying the ts phenotype of
erg26-1 cells, we determined the subcellular localization of
Erg26p and Erg27p. Plasmids expressing galactose-inducible N-terminal
GFP fusion proteins of Erg26p and Erg27p were transformed into wild
type cells. Cells harboring these vector sequences were grown to
exponential phase in raffinose-containing media, and subsequently
shifted to galactose for 3 h to allow for the induction of fusion
protein expression. The localization of GFP-Erg26p and GFP-Erg27p was
then determined by fluorescence microscopy.
We found that in galactose-induced cells both GFP-Erg26p and GFP-Erg27p
localized to a specific perinuclear organelle in yeast (Fig.
8, C and E). These
localization patterns were not seen in raffinose-grown cells. Nuclear
staining using the DNA-binding dye, 4',6-diamidino-2-phenylindole,
verified that the GFP fluorescence emitted by cells expressing these
fusion proteins surrounded the nucleus and not the vacuolar membrane.
Moreover, the visual localization of these fusion proteins looked
identical to that of the ER-resident GFP-Sur4p (Fig. 8, A
versus C and E). Thus, we conclude
that both Erg26p and Erg27p reside within the ER in yeast.
Erg25p already has been localized to the ER (19). This protein contains
a putative ER retention signal at its C terminus (41). Hydropathy scans
of the amino acid sequences of Erg26p and Erg27p do not reveal the
presence of putative transmembrane domains or ER retention signals.
Based on this information, we hypothesized that Erg26p and Erg27p may
localize to the nucleus through their binding to Erg25p. We used the
yeast two-hybrid assay as a means to test this hypothesis and determine
whether Erg25p could bind Erg26p and/or Erg27p. The yeast two-hybrid
assay uses protein-protein interaction-dependent
transcriptional expression as a means to assay for protein complexation
(42). Thus, we determined the up-regulation of
We found that we could detect a strong interaction between Erg25p
(Table III, row, pACT2-ERG25) and both Erg26p
(column, pBD-GAL4-ERG26; 127 ± 5.6 Miller
units) and Erg27p (column, pBD-GAL4-ERG27;
85.2 ± 2.7 Miller units) but could not detect any interaction
between Erg26p (row, pACT2-ERG26) and Erg27p
(row, pACT2-ERG27) under the two different
conditions tested (7.9 ± 3.3 and 4.8 ± 2.4 Miller units).
When the various ERG genes were expressed alone, the
In a screen designed to isolate cells with defects in sphingolipid
metabolism, we isolated erg26-1, a mutant strain that
harbors a weakened allele of the ERG26 sterol gene. Based on
the recent work of Bammert and Fostel (43), it seems reasonable to
believe that sterol mutants would be isolated in screens aimed at
isolating sphingolipid mutants. These investigators used microarray
technology as a means to examine the effects of several drugs on genome
wide transcription. Included in their study were azole compounds that targeted the sterol pathway. Using these drugs, they found that the
expression levels of genes involved in sphingolipid biosynthesis were
altered in sterol pathway perturbed cells, including the SUR2 gene encoding the hydroxylase required for the
synthesis of the long chain sphingoid base phytosphingosine (44) and
the LCB1 gene encoding for one of the two subunits of the
serine palmitoyltransferase required for the first committed step in
sphingolipid biosynthesis (45). Moreover, they discovered that
azole-treated cells contained altered mRNA levels of genes required
for the synthesis of fatty acids, including ELO1,
OLE1, and FAS1. It is likely that changes in the
expression levels of these genes would have a dramatic impact on
sphingolipid composition. Thus, there is precedent for the coordinate
regulation of sterol and sphingolipid metabolism, at least at the level
of transcription. Interestingly, at least one additional gene thought
to be involved in regulating sterol metabolism has been isolated in the
screen described in this
study.2
By using TLC and GC/MS analyses, we have predicted that the zymosterol
intermediates accumulating in erg26-1 cells are
4 We found that erg26-1 cells harbored defects in neutral
lipid synthesis (Fig. 4). In temperature-shifted erg26-1
cells, the rate of biosynthesis of TG was decreased, whereas DG and MG
synthesis was increased. The most straightforward explanation for these defects is that the accumulation of zymosterol intermediates block TG
synthesis, thus, the accumulation of DG and MG. However, an alternative
explanation is that one, or both, of the zymosterol intermediates are
regulating at some level the individual activities required for TG, DG,
and MG synthesis. Regardless of which hypothesis is true, the type of
regulation exerted by these zymosterol intermediates, whether it is at
the level of transcriptional or post-translation, remains to be resolved.
We also determined that the levels of ergosterol and sterol esters were
decreased in erg26-1 cells. Defects were seen under all
experimental conditions. The decrease in ergosterol most likely is an
indirect effect of weakened 4 The levels of PS, PE, and PC in pulse-labeled erg26-1 cells
grown at either the permissive or nonpermissive temperatures were comparable to wild type cells grown at these same temperatures (Fig. 5,
A versus B). Thus, the biosynthetic
rate of phospholipid synthesis through the CDP-DG pathway (49) does not
seem to be affected in erg26-1 cells. This would suggest
that the levels of CDP-DG in erg26-1 cells and wild type
cells are comparable. On the other hand, the rate of biosynthesis of PA
(1.9-fold increase) was altered in temperature-shifted
erg26-1 cells (Fig. 5, A versus B). How might PA accumulate in temperature-shifted
erg26-1 cells? Quite possibly through
lipid-dependent inhibition of the yeast PA phosphatase
activities responsible for the conversion of PA to DG (50, 51).
Lipid-dependent regulation of the 45- and 104-kDa PA
phosphatase activities has already been shown, because these enzymes
are inhibited by the sphingoid bases dihydrosphingosine and
phytosphingosine (52). erg26-1 cells do harbor defects in sphingolipid biosynthesis.3
Determining whether these long-chain bases accumulate in
erg26-1 cells, and if so, whether they exert an effect on
one or both PA phosphatase activities, is presently being studied in
our laboratory.
In addition, we found that the rate of biosynthesis of PI was down
1.7-fold in temperature-shifted erg26-1 cells (Fig. 5, A versus B). PI is synthesized from
CDP-DG and inositol by PI synthase (53). A large body of work has
pointed to intracellular inositol availability as the primary mode of
regulation of PI synthase (54-56). Inositol is taken up from the media
by the inositol permease encoded for by the ITR1 gene (57).
Inositol uptake may be compromised in temperature-shifted
erg26-1 cells due to the accumulation of zymosterol
intermediates. The reduction in intracellular inositol due to
zymosterol intermediate-dependent inhibition of Itr1p activity
or ITR1 expression would result in a decrease in PI levels.
We must point out, that because CDP-DG levels were not determined in
our studies, we cannot rule out that the decrease in PI levels we see
in temperature-shifted erg26-1 cells is not through a reduction in
CDP-DG levels.
We used fluorescence microscopy to localize GFP-Erg26p and GFP-Erg27p
to the endoplasmic reticulum. When these fusion proteins were expressed
in their respective haploid deletion strains they supported robust
growth.4 Thus, the N-terminal
GFP moiety does not affect protein function. Strikingly, neither Erg26p
nor Erg27p contain any recognizable transmembrane domain segments
within their amino acid sequences. Based on our two hybrid results, we
would hypothesize that Erg26p and Erg27p localize to the ER through
their tethering to Erg25p. Erg25p does contain a consensus ER retention
signal and previously has been localized to the ER (19). Thus, we
believe that Erg25p, Erg26p, and Erg27p form a heterotrimeric complex
within the ER. Faust et al. (46) have suggested that
complexation of these three proteins may be the most efficient way to
catalyze the sequential demethylation reactions, because the removal of
these methyl groups occurs on a single face of the sterol substrate,
4,4-dimethylzymosterol. Work in plant cells suggests that a homologous
protein complex exists in these cells, as well (15).
Recently, it was shown that proper ergosterol levels are critical for
endocytosis in yeast (58). In addition, studies have demonstrated that
sterol-compromised yeast cells have altered uptake of certain cations
and macromolecules (8). In mammalian cells, cholesterol/sphingolipid
rafts play an active role in the trafficking of signaling molecules to
the cell surface (59), whereas cholesterol itself has emerged as an
important messenger lipid involved in proper development (60). However,
it also is well known that a chronic increase in blood cholesterol
levels has been implicated as a progenitor for the plaque formation
that can lead to heart disease and atherosclerosis (61). Thus, an emerging list of the processes requiring sterols for function is
constantly evolving, and will continue to do so as additional studies
examining the physiological functions of sterols are performed. These
types of studies are necessary to understand how sterol metabolites
regulate cell growth.
-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to
zymosterol. Gas chromatography/mass spectrometry analyses of sterols in
this mutant, designated erg26-1, revealed the aberrant
accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well
as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate
of biosynthesis and steady-state levels of mono-, di-, and
triglycerides. Phospholipid radiolabeling studies showed defects in the
rate of biosynthesis of both phosphatidic acid and
phosphatidylinositol. Biochemical studies revealed that microsomes
isolated from erg26-1 cells contained greatly reduced
4
-carboxysterol-C3 dehydrogenase activity when compared with
microsomes from wild type cells. Previous studies have shown that loss
of function mutations in either of the fatty acid elongase genes
SUR4/ELO3 or FEN1/GNS1/ELO2 can
"bypass" the essentiality of certain ERG genes
(Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993)
Lipids 28, 907-912; Silve, S., Leplatois, P., Josse,
A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard,
C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and
Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies
presented here have shown that this
sphingolipid-dependent "bypass" mechanism did not
suppress the essential requirement for zymosterol biosynthesis.
However, studies aimed at understanding the underlying physiology
behind the temperature-sensitive growth defect of erg26-1
cells showed that the addition of several antifungal compounds to the
growth media of erg26-1 cells could suppress the
temperature-sensitive growth defect. Fluorescence microscopic analysis
showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to
the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p,
Erg26p, and Erg27p, which are required for the biosynthesis of
zymosterol, form a complex within the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxysterol-C3 dehydrogenase (C-4
decarboxylase) (4
-CD),1
and a C-3 ketoreductase, have been characterized from partially purified preparations (10-15). These analyses have given an excellent insight into the steps involved in C-4 demethylation. The first step is
initiated by the C-4 methyl oxidase, whereby this enzyme converts the
C-4
methyl group of 4,4-dimethylzymosterol to alcohol and
aldehyde intermediates and then to a carboxylic acid. Next, the
carboxyl group is removed by the 4
-CD, with epimerization of the
4
-methyl group to a 4
stereochemistry and a 3-keto group being
formed during this step. Finally, the 3-keto group is reduced to a
-hydroxy sterol by the 3-ketoreductase. The next round of demethylation is initiated once the second methyl group translocates to
the C-4
position.
-CD, and C-3
ketoreductase required for the demethylation of 4,4-dimethylzymosterol are encoded for by the ERG25 (16), ERG26
(17), and ERG27 (18) genes, respectively. All three genes
are essential under aerobic growth conditions. However, viable single
null mutants have been constructed by genetic means. These strains have
been very useful in determining the changes in sterol composition due
to the individual loss of function of these genes. Yeast strains
lacking the ERG25 gene accumulate the substrate of the
Erg25p, 4,4-dimethylzymosterol (16), whereas strains lacking
ERG26 accumulate the expected 4-methyl carboxy substrate
intermediates (17). On the other hand, mutants lacking ERG27
function accumulate very little cyclic sterols (18). However,
erg27
mutants can be made to accumulate the expected
3-keto sterols if they are fed lanosterol, a sterol intermediate that
is an upstream precursor of the substrate for the Erg27p-directed
3-ketoreductase reaction.
-CD activity, because this activity is almost
completely lacking in these cells.
-CD activity and in
zymosterol biosynthesis, whereby they accumulate aberrant zymosterol
intermediates. Additional lipid metabolic labeling studies show that
erg26-1 cells also harbor defects in neutral lipid and
phospholipid biosynthesis and metabolism. Furthermore, by using
specific pharmacological agents, we demonstrate that the conditional
growth phenotype of erg26-1 cells is due to the toxic
accumulation of zymosterol intermediates at high temperatures rather
than the loss of ergosterol biosynthesis. Further studies show that
this toxic phenotype cannot be suppressed by mutations in sphingolipid
biosynthesis that previously have been shown to suppress ERG
gene essentiality (22, 23). Finally, by using fluorescence microscopy
and two-hybrid analysis, we demonstrate that Erg26p and Erg27p both
localize to the endoplasmic reticulum and that Erg25p physically
associates with Erg26p and Erg27p within the cell.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Carboxysterol-C3
Dehydrogenase Assay--
Yeast cells were disrupted by glass bead
homogenization (0.45-mm diameter) in 100 mM phosphate
buffer (pH 7.5) containing 1.5 mM reduced glutathione and
30 mM nicotinamide for 8 min at 0 °C. The cell extract
was obtained by centrifugation at 10,000 × g for 20 min. Microsomes were then isolated by centrifuging the cell extract
supernatant at 100,000 × g for 90 min. The resulting microsomal pellet was resuspended in 100 mM phosphate
buffer (pH 7.5) containing 3 mM reduced glutathione and
20% glycerol (v/v) using an Elvehjem-Potter homogenizer.
-Carboxysterol-C3 Dehydrogenase Assay--
Microsomes (0.4 ml = 1 mg of protein) were incubated in the presence of 100-200
µM exogenous synthetic
4
-carboxy-5
-cholest-7-en-3
-ol that had been emulsified in 1 g/liter Tween 80, and 200 µM NAD+.
Incubations were continued aerobically at 30 °C with gentle stirring
for 50 min. The reaction was stopped by adding 0.5 ml of 6% KOH/EtOH.
After addition of a known amount of coprostanone (2-10 µg) as an
internal standard, the incubation mixture was extracted three times
with a total volume of 15 ml of n-hexane. After drying with
Na2SO4, the extract was concentrated to dryness and analyzed by TLC on silica gel eluted with
CH2CL2 (two developments). The fraction
migrating as authentic standards of coprostanone and
7-cholestenone and containing the enzymatically produced
7-cholestenone (RF = 0.50-0.70) was
eluted and analyzed by GLC using a fused-silica capillary column DB1
(240-280 °C, 2 °C/min) and hydrogen as the carrier gas. The
amount of
7-cholestenone produced
(tR = 1.11) was calculated from the amount of
coprostanone (tR = 1.0) and the area of their
respective peaks, allowing the rate of the reaction to be determined.
The ketone metabolite and other endogenous sterones (if present) were
identified by capillary GLC and coupled GLC-MS analysis. The ketone
metabolite produced by the reaction was thus unequivocally identified
as cholest-7-en-3-one by coincidental retention time in GLC and by an
electron impact spectrum identical to that of an authentic synthetic
standard. The 4
-carboxy-7-en-3
-ol substrate was synthesized as
described previously (15).
-carboxysterol-C3 dehydrogenase
assay, MS and GLC/MS were determined at 70 eV with a Fisons MD800
spectrometer. The GLC separation was carried out with a Varian 3400 CX
GLC instrument equipped with a flame ionization detector and a
fused-silica capillary column (30-m length; 0.32-mm inner
diameter) × 0.25-µm film coated with DB5 (H2, flow
2 ml/min). The temperature program used included a 30 °C/min rise
from 60 to 240 °C followed by a 2 °C/min rise from 240 to
280 °C. Relative retention times (tR) are
given with respect to cholesterol (tR = 1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Carboxysterol-C3 Dehydrogenase
Activity--
A screen was initiated to isolate yeast mutants having
defects in sphingolipid metabolism. Because S. cerevisiae
cells incorporate inositol into complex sphingolipids, the strategy was
to look for temperature-sensitive (ts) loss of function
mutations that reduced or eliminated the incorporation of
[3H]inositol into the yeast cell membrane. Among the
ts mutants obtained, a sphingolipid biosynthetic mutant was
isolated whose ts phenotype was suppressed in low copy by
the ERG26 gene (Fig. 1A). ERG26 is
believed to encode for the 4
-CD activity in S. cerevisiae
(17). Linkage analysis was performed in this mutant, designated H21,
and showed that the mutation responsible for the ts defect
resided within the ERG26 gene. H21 was backcrossed several times to the parental W303-1A wild-type strain and an
erg26-1 haploid segregant was generated. We used this
erg26-1 strain to examine in greater detail the defects in
sterol lipid biosynthesis brought about by mutating ERG26
and to determine whether neutral lipid and phospholipid biosynthesis
were also perturbed.
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Fig. 1.
Low copy ERG26 suppresses
the ts growth and sterol biosynthetic defects of
erg26-1 cells. A, various
ERG26 strains were streaked onto Ura plates
and grown for 48 h at the indicated temperatures. B,
various ERG26 strains were labeled with
[14C]acetate for several generations. The total lipid
fraction was isolated by chloroform, methanol extraction (2:1). Sterols
were resolved by TLC using the solvent system petroleum ether,
diethylether, acetic acid (70:30:2). Radiolabeled sterols were detected
by autoradiography. In the case of the ERG26 wild type
strain and the erg26-1 strain carrying the plasmid
pRS-ERG26, the levels of sterol intermediates accumulating
at 37 °C are shown.
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Fig. 2.
GC analysis of diazomethane-treated sterol
extracts from erg26-1 cells reveals the presence of
aberrant sterol peaks. Wild type (A) and
erg26-1 (B) cells were grown to exponential phase
at 23 °C. Cells were then shifted to 37 °C for several
generations. Sterol extracts were obtained and the methyl esters of all
sterol intermediates accumulating in cells were synthesized using
diazomethane. GC as described under "Experimental
Procedures" was performed to separate individual derivatized sterol
intermediates.
Sterols detected in wild type and erg26-1 cells
-methyl-4
-carboxy-cholesta-8,24-dien-3
-ol (Fig.
3A), and a novel sterol compound,
4
-carboxy-cholesta-8,24-3
-ol (Fig. 3B). The
4
-methyl-4
-carboxy-cholesta-8,24-dien-3
-ol previously has been
shown to accumulate in strains completely lacking the ERG26
gene (17). We must point out that additional NMR studies will be
required to verify definitively the position of the double bonds and
the stereochemistry of the 4-carboxy group in these sterol derivatives.
However, this is the first identification of a 4-carboxy-diunstaurated
sterol species in yeast. Using the MS data obtained, we have predicted
that peak 8 is an epimer of peak 7.
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Fig. 3.
MS analysis of the GC peaks 6 and 7 found in temperature-shifted
erg26-1 cells. Sterol extracts were obtained and
diazomethane-treated as described in Fig. 2. MS, as described under
"Experimental Procedures" was used to analyze all GC peaks,
including peaks 6 and 7. A, peak
7, 4-methyl-4-carboxy-diunsaturated sterol; B,
peak 6, 4-carboxy-diunsaturated sterol.
-CD
activity was assayed in wild type and erg26-1 microsomal
extracts. The results from these studies revealed that a microsomal
extract obtained from wild type cells grown at the permissive
temperature was able to decarboxylate the 4
-CD substrate,
4
-carboxy-7-en-3
-ol (15) (Table
II). In contrast, the level of 4
-CD
activity in microsomes from erg26-1 cells grown at the
permissive temperature was reduced by 50% compared with that of the
wild type activity. Wild type C-3 sterol dehydrogenase activity could
be restored to erg26-1 cells by introducing the wild type
ERG26 gene on a low copy plasmid (Table I,
erg26-1 pRS-ERG26). Thus, erg26-1 cells do contain weakened 4
-CD activity.
4-Carboxysterol-C3 dehydrogenase activity in wild type and
erg26-1 cells
-Carboxysterol-C3 dehydrogenase activity was measured at 30 °C.
The reaction was performed in 100 mM phosphate buffer (pH
7.5) containing 200 µM
4
-carboxy-5
-cholest-7-en-3
-ol, 200 µM
NAD+, and 1 mg of microsomal protein. The reaction was
terminated after 50 min, and the products of the reaction were
extracted using n-hexane. TLC was used to purify the
reaction products, and their molecular structures were determined by
GLC/MS analysis as described under "Experimental Procedures." 100%
activity corresponds to 215 pmol of sterol decarboxylated/min × mg of protein. Values are the average of two independent experiments.
-methyl-cholest-8,24-dien-3-one and
cholest-8,24-dien-3-one. These aberrant sterones were not seen in any
significant amounts in the assays performed with wild type Erg26p.
Based on these results, we conclude that the accumulation in
erg26-1 cells of the zymosterol intermediates
4
-methyl-4
-carboxy-cholesta-8,24-dien-3
-ol and
4
-carboxy-cholesta-8,24-3
-ol is a direct result of a defect in
4
-CD activity encoded for by the ERG26 gene.
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Fig. 4.
erg26-1 cells have defects in
neutral lipid metabolism. For pulse (A and
B) and steady-state (C and D) labeling
of neutral lipids, cells grown in synthetic media were shifted to
23 °C (A and C) or 37 °C (B and
D) and incubated with [14C]acetate for 30 min
(pulse) or 4 h (steady-state). Radiolabeled
neutral lipids were extracted using chloroform, methanol (2:1) and
analyzed by one-dimensional TLC using the solvent system hexane, ethyl
ether, acetic acid (80:20:2). The percent lipid values are the average
of five independent experiments. Abbreviations used are: TG,
triacylglycerides; DG, diacylglycerides; MG,
monoglycerides; FA, fatty acids; FAA, fatty acid
alcohols; ERG, ergosterol; SE, steryl esters;
**, sterol intermediates.
-methyl-4
-carboxy-cholesta-8,24-dien-3
-ol and 4
-carboxy-cholesta-8,24-3
-ol sterols seen in
erg26-1 cells.
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Fig. 5.
Temperature-shifted erg26-1
cells have defects in the rate of biosynthesis of
phospholipids. For pulse labeling of phospholipids (A
and B), cells grown in synthetic media were shifted to
23 °C (A) or 37 °C (B) and incubated with
[32P]orthophosphate for 20 min. For steady-state labeling
of phospholipids (C and D), cells grown in
synthetic media were shifted to 23 °C (C) or 37 °C
(D) and incubated with [32P]orthophosphate for
at least six generations. Phospholipids were extracted and analyzed by
one-dimensional TLC. The percent lipid values are the average of five
independent experiments. Abbreviations used are: PC,
phosphatidylcholine; PE, phosphatidylethanolamine;
PS, phosphatidylserine; PI, phosphatidylinositol;
PA, phosphatidic acid.
strains (17). We know that the Sut1p
was functioning, because we were able to detect the uptake of
radiolabeled ergosterol in all of the strains overexpressing SUT1. Moreover, we found that the rates of sterol
internalization among the strains were nearly identical. Thus, the lack
of ts suppression by ergosterol was not due to a reduced
rate of uptake in erg26-1 cells.
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Fig. 6.
The accumulation of aberrant zymosterol
intermediates in erg26-1 cells is lethal at high
temperatures. A, wild type cells carrying pJR1133 and
erg26-1 cells carrying pJR1133-SUT1 were spotted
as 10-fold serial dilutions onto Ura plates containing 15 µg/ml ergosterol. The plates were incubated at the indicated
temperatures until wild type cell growth was seen at all dilutions
tested. B, 1 × 107 erg26-1
cells were spotted onto YEPD plates lacking (control) or
containing 1.5 µg/ml terbinafine or 20 ng/ml fenpropimorph. Plates
were incubated at the indicated temperatures for 48 h.
C, erg26-1 cells were incubated in the absence
(control) or presence of 1.5 µg/ml terbinafine or 20 ng/ml
fenpropimorph and [14C]acetate. Sterol extracts were
obtained, and TLC as described in Fig. 1B was used to
analyze the radiolabeled zymosterol intermediate accumulation.
cells (39). Plasmids carrying the URA3 gene are routinely negatively selected for
by streaking cells onto plates containing 5-FOA. With this in mind, we
constructed haploid strains that were deleted for ERG25,
ERG26, or ERG27, and SUR4, and which
harbored a URA3-containing centromeric plasmid (pRS414)
carrying the respective deleted wild type sterol gene. We reasoned
that, if altering sphingolipid biosynthesis in erg25
,
erg26
, or erg27
cells through the loss of
SUR4 bypasses the requirement for zymosterol biosynthesis,
cells lacking each individual sterol gene and SUR4 would be
able to deselect the URA3-containing plasmid and grow on
plates containing 5-FOA. If, on the other hand, the loss of
SUR4 does not suppress the requirement for proper zymosterol
biosynthesis, cells would retain the plasmid and die on the 5-FOA plates.
haploid cells were viable. There have been conflicting reports as to
whether ERG2 actually is essential (23, 40). On the other
hand, we were able to show, for the first time, that deleting
SUR4 allowed strains that lacked ERG24 to grow
(Fig. 7, A and B).
Thus, loss of SUR4 function is able to suppress the
strain-dependent essential requirement for ERG2,
as well as the essential requirement for ERG24 function. In
contrast, we found that deleting SUR4 in strains lacking
ERG25, ERG26, or ERG27 did not bypass
the essential requirement for zymosterol biosynthesis (Fig. 7,
A and B). We obtained similar results when we
examined whether the loss of FEN1 could suppress the need
for proper zymosterol biosynthesis. Moreover, we found that deleting SUR4 in erg26-1 cells harboring
pRS414-URA3-ERG26 did not allow for growth on
5-FOA plates at the nonpermissive temperature for erg26-1
cell growth (Fig. 7, C and D). Taken together,
these results indicate that altering sphingolipid biosynthesis in
erg25
and erg26
cells cannot reduce the
cell toxicity associated with the accumulation of zymosterol
intermediates, or the loss of cyclic sterol biosynthesis in
erg27
cells.
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Fig. 7.
Altering sphingolipid biosynthesis cannot
bypass the essential requirement for proper zymosterol
biosynthesis. Various pRS-containing ERG strains were
streaked onto (A) Ura - and (B)
5-FOA-containing plates. The plates were incubated at 27 °C until
wild type growth was seen (W303-1A, pRS-URA3).
erg26-1 cells harboring pRS-URA3-ERG26 was
streaked out onto 5-FOA plates and incubated at 27 °C (C)
or 37 °C (D).
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Fig. 8.
Erg26p and Erg27p localize to the ER in yeast
cells. Wild type cells harboring pGAL-GFP-ERG26 or
pGAL-GFP-ERG27 were grown in raffinose until cells reached
exponential phase. Cells were then shifted to synthetic media
containing galactose for 3 h. An aliquot of cells was removed and
dispersed onto glass slides using 0.2% low melting agarose. The
localization of individual GFP fusion proteins was determined using a
Leica DM-RBE fluorescence microscope and fluorescein
isothiocyanate optics.
-galactosidase
expression by assaying for activity in cells expressing various
combinations of Erg proteins. The results of the two-hybrid study are
shown in Table III.
Analysis of the two-hybrid interactions between various Erg proteins
-D-galactopyranoside in buffer 1 was added to the
freeze-thawed cells, and the reaction was allowed to proceed for 45 min. The A578 was taken and
-galactosidase activity was
calculated in Miller units. The Miller units presented are the average
of three independent experiments.
-galactosidase activity levels were similar to the background value
obtained from expressing both empty vectors together (column, pBD-GAL4 Cam). Thus, the values that we obtained demonstrating the interactions between Erg25p and both Erg26p and Erg27p are not due to the
self-activation of
-galactosidase expression by single
ERG genes alone. We conclude from these data that Erg26p and
Erg27p are most likely tethered to the ER through their association
with the ER-resident Erg25p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methyl-4
-carboxy-cholesta-8, 24-dien-3
-ol, and
4
-carboxy-cholesta-8,24-3
-ol. A previous study has shown that
4
-methyl-4
-carboxy-cholesta-8,24-dien-3
-ol accumulates in
cells lacking the ERG26 gene (17). This result was based on
GC/MS analyses. The 4
-carboxy-cholesta-8,24-3
-ol species was not
detected in these same cells. Based on the enzymatic mechanism for the
demethylation of 4,4-dimethylzymosterol (46), 4
-carboxy-cholesta-8,24-3
-ol would be the second round
carboxysterol substrate for C-3 sterol dehydrogenase. Thus, one would
predict that 4
-carboxy-cholesta-8,24-3
-ol can only accumulate in
cells possessing some degree of 4
-CD activity. This activity would have to be compromised to allow for 4
-carboxy-cholesta-8,24-3
-ol intermediate accumulation. This is borne out by our biochemical assays
of 4
-CD activity in erg26-1 cells.
-CD activity rather than the regulation
of the ERG4-encoded sterol C-24 reductase (1). If Erg4p activity was
down-regulated, the accumulation of the ergosterol precursor
ergosta-5,7,22,24(28)-tetraenol should have been detected in
erg26-1 cells, because a sterol containing a C-24(28) double bond has been shown to accumulate in erg4 cells (47). The
reduction in sterol ester levels may be a combined result of a
reduction in ergosterol and the inability of the yeast
acyl-CoA:cholesterol O-acyltransferase enzymes (48) to
utilize the accumulated zymosterol intermediates as substrates.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge A. Hoeft for performing the GC-MS analysis on microsomal reactions. We thank Drs. Chris Beh and Jasper Rine for various plasmids. We thank members of the Bill Bergman and Tom Edlind laboratories for many helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Mid-Atlantic American Heart Association Grants 0051102U and 9805529U (to J. N.) and by the March of Dimes Foundation Basil O'Connor Starter Scholarship Grant FY99-277 (to J. N.).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.
To whom correspondence should be addressed: 245 N. 15th St.,
Philadelphia, PA 19102. Tel.: 215-762-1941; Fax: 215-762-4452; E-mail: Joseph.Nickels@drexel.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M100274200
2 S. Mandala, personal communication.
3 K. Baudry, E. Swain, M. Germann, J. Allegood, A. Merill, S. Mandala, M. Kurtz, and J. T. Nickels, submitted, manuscript in preparation.
4 K. Baudry and J. T. Nickels, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
4-CD, 4
-carboxysterol-C3 dehydrogenase;
5-FOA, 5-fluoroorotic acid;
PCR, polymerase chain reaction;
YIp, Yeast Integrating plasmid;
GC/MS, gas
chromatography/mass spectrometry;
CDP-DG, CDP-diacylglycerol;
ts, temperature-sensitive;
ER, endoplasmic reticulum;
GFP, green
fluorescence protein;
DG, diacylglycerides;
MG, monoglycerides;
TG, triglycerides;
PI, phosphatidylinositol;
PA, phosphatidic
acid.
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REFERENCES |
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