Department of Molecular and Cell Biology,
University of California at Berkeley, Berkeley, California 94720
Sterol levels affect the expression of many genes in yeast and
humans. We found that the paralogous transcription factors Upc2p and
Ecm22p of yeast were sterol regulatory element (SRE) binding proteins
(SREBPs) responsible for regulating transcription of the sterol
biosynthetic genes ERG2 and ERG3. We
defined a 7-bp SRE common to these and other genes, including many
genes involved in sterol biosynthesis. Upc2p and Ecm22p activated
ERG2 expression by binding directly to this element in
the ERG2 promoter. Upc2p and Ecm22p may thereby
coordinately regulate genes involved in sterol homeostasis in yeast.
Ecm22p and Upc2p are members of the fungus-specific Zn[2]-Cys[6]
binuclear cluster family of transcription factors and share no homology
to the analogous proteins, SREBPs, that are responsible for
transcriptional regulation by sterols in humans. These results suggest
that Saccharomyces cerevisiae and human cells regulate
sterol synthesis by different mechanisms.
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INTRODUCTION |
Sterols, essential for life in most,
if not all, eukaryotes, are synthesized by a pathway known variously as
the mevalonate pathway, the isoprenoid pathway, or the sterol
biosynthetic pathway. Although sterols (which include molecules ranging
from cholesterol to steroid hormones) are the major products of the
sterol biosynthetic pathway, this pathway also produces many important
nonsterol compounds that are necessary for cellular processes such as
respiration, glycosylation, protein prenylation and photosynthesis. In
the heavily studied mammalian sterol biosynthetic pathway, regulation occurs at both the transcriptional and the posttranscriptional levels.
At present, the best-understood aspect of this regulation is the
transcriptional response to changes in sterol levels. Sterol depletion
in mammalian cells causes activation of the transcription factors known
as sterol regulatory element (SRE) binding proteins (SREBPs)
(8), encoded by the homologous genes SREBP-1 and SREBP-2. When sterols are abundant, the SREBPs are inactive, tethered to the
endoplasmic reticulum (ER) membrane by two intrinsic transmembrane helices. When sterol levels drop, regulated proteolysis releases the
transcriptional activation domains of the SREBPs from the membrane
tether, allowing the activation domains to translocate to the nucleus.
Once in the nucleus, these domains activate transcription of genes
involved in sterol and fatty acid homeostasis.
The SREBPs bind the same DNA sequences in vitro (21). In
vivo, the SREBPs activate an overlapping set of target genes but may
have slightly different DNA binding specificities and/or activities (8, 22). Hence, differential activation and synthesis of the SREBPs may make it possible for mammalian cells to respond to a
demand for different levels of sterol and nonsterol products.
Many genes in the sterol biosynthetic pathway in Saccharomyces
cerevisiae are transcriptionally regulated in response to changes in sterol levels (6, 13). However, less is known about
this regulatory mechanism in yeast. Also, yeast lack convincing
homologues of the mammalian SREBPs. This work describes the
identification of a yeast SRE shared by many genes involved in sterol
biosynthesis and the identification of two SREBPs, Upc2p and Ecm22p,
which regulate transcription of the sterol biosynthetic genes. Both Upc2p and Ecm22p are members of the Zn[22]-Cys[6] binuclear cluster family of fungal transcription factors (31).
UPC2 was originally identified as a semidominant allele that
allows sterol uptake under aerobic conditions (11). Upc2p
is also involved in the anaerobic activation of expression of the
DAN/TIR mannoproteins (1). The ECM22 gene was
identified as a mutant sensitive to the cell wall perturbing agent
calcofluor white (20). Our data indicated that both Ecm22p
and Upc2p were involved in regulation of sterol biosynthesis and
identified ERG2 and ERG3 as targets of both proteins.
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MATERIALS AND METHODS |
Strains and media.
All yeast strains used were isogenic with
W303-1a except as noted in Table 1. Gene
deletions were made by a PCR-based gene disruption method
(2) such that the entire open reading frame (ORF) was
replaced by marker sequences from pRS403 (HIS3), pRS404 (TRP1), pRS405 (LEU2), or pRS406
(URA3) (29). The Escherichia coli
strain used was ER2508 from New England Biolabs.
Yeast cells were grown in minimal medium (YM; 0.67% Difco yeast
nitrogen base with 5% ammonium sulfate without amino acids) containing
2% glucose and required supplements. Solid media contained 2% agar
(Bacto). A 25-mg/ml stock solution of lovastatin (a generous gift from
J. Bergstrom, Merck) was made as previously described (12). Lovastatin, Geneticin (Gibco), and amphotericin B
(Sigma) were used in solid media at final concentrations of 40 µg/ml, 1 mg/ml, and 100 ng/ml, respectively. All drugs were added after autoclaving. Lovastatin plates were incubated at 24°C, Geneticin plates at 30°C and amphotericin B plates at 37°C. Amphotericin B
plates were used within 12 h of preparation. Bacteria were grown in LB Amp (1% tryptone, 0.5% NaCl, 0.5% yeast extract, 100 µg of
ampicillin/ml).
Plasmids.
The plasmid constructions of this work made
extensive use of the pRS vector series (29) as well as the
Univector series (19). pJR2325 was an ERG2-lacZ
reporter in an integrating TRP1 plasmid based on pRS414. The
CEN ARS fragment of pRS414 was removed by cutting with
Eco0109I and religated. The plasmid contained the
ERG2 promoter fragment from 
751 to +12 relative to the ATG initiation codon and an XbaI site fused to the second amino
acid of lacZ. The PGK terminator was subcloned 3'
of lacZ.
pJR2326 was an ERG2-KanMX reporter in an integrating
LEU2 plasmid based on YIplac128 (14). pJR2326
was made by a three-fragment ligation of the
SphI-XbaI fragment (containing the
ERG2 promoter) of pJR2325, a KanMX fragment
amplified by PCR using primers av100 (5'-TAAACATCTAGAGGTAAGGAAAAGACTCACGTTTCG-3') and solig191
(5'-GAAAACAAGAATTCTTTTTATTGTCAGTAC-3') cut with
XbaI and EcoRI and the YIplac128 vector backbone
cut with SphI and EcoRI.
pJR2307 was a 2µm URA3-marked plasmid based on pLacZi
(GenBank accession no. U89671) from Clontech. The 2µm origin was PCR
amplified from pRS426 using primers av249
(5'-TTTCCCCGAAAAGTGCCTGCAGAACGAAGCATCTGTGCTTC-3') and av250
(5'-CATGATAATAATGGTTCTGCAGTATGATCCAATATCAAAGGAAATG-3') and
subcloned as a PstI fragment into the NsiI site
of pLacZi to make pJR2307. Plasmids pJR2308-2315 and pJR2327-2329 were
made by subcloning a phosphorylated double-stranded oligonucleotide into the EcoRI and XhoI sites of pJR2307. The
oligonucleotides were designed to have single-stranded overhangs on
either end to facilitate cloning into the EcoRI and
XhoI sites. The design of the oligonucleotides was such that
the EcoRI site was followed by an EcoRV site (to
allow for easy identification of plasmids with inserts), followed by
the relevant sequence from the ERG2 promoter and an
XhoI site. pJR2316 was made from pJR2307 by subcloning a
fragment of the ERG2 promoter PCR-amplified using primers
av134 (5'-AAGAGAGTCGACGGTACCCATTTCGGCACTAAAATC-3') and av67
(5'-TCTTGTCGACCATGGCGCTGCAGATCTATC-3') into the
EcoRI and XhoI sites of pJR2307. pJR2317 was made
similarly as pJR2316 but used pJR2320 as a template for the PCR. The
ERG2 promoter in this plasmid therefore had a mutated SRE.
pJR2318 was an ERG3-lacZ reporter made by
subcloning an ERG3 promoter fragment as an
EcoRI-XhoI fragment into the same sites in pLacZi
(integrating plasmid carrying URA3) (GenBank accession no.
U89671) from Clontech. The ERG3 promoter fragment was
generated by PCR amplification from genomic DNA using primers av127
(5'-ATTCCCGAATTCGTCCTGCTTTGAGTCGTTTTC-3') and av128
(5'-CTCTAACTCGAGCCGCGATGTTTCTTTCGACC-3').
pJR2320 contained the ERG2 promoter from 751 to +198 as an
EcoRI-XhoI fragment in pRS406 (integrating
plasmid carrying URA3). The ERG2 SRE was mutated
(from 5'-CTCGTATAAG-3' to 5'-ACGATATCTA-3') by using a PCR sewing
technique (2) using genomic DNA from W303-1a as a template
and the primers av134
(5'-GTACTGGAATTCCTGCATTATCATCCCGATTGCT-3'), av194
(5'-AACCACGGCCACGATATCTACCGCAAGGAAAACTACCGGT-3'), av195 (5'-TTCCTTGCGGTAGATATCGTGGCCGTGGTTCGATTCTGCC-3'), and av308
(5'-CCCCGTAACTCGAGTTAAGTGCGTCTCTGACATCCTG-3'). pJR2321 was
similar to pJR2320 but had a wild-type SRE. The insert was generated by
PCR using the primers av134 and av308 and genomic DNA from W303-1a as a template.
pJR2330 was a 2µm LEU2 plasmid containing a 3.6-kb
fragment containing ECM22. The ECM22 fragment was
PCR amplified using primers av357
(5'-GACGTTGGATCCGGTAGAGTGGCGCTAGCATAG-3') and av358
(5'-GCATTTAGGATCCAACGTATACCGGGTGTACCTC-3'), and the
resulting fragment was subcloned into the BamHI site of pRS425. pJR2331 contained ECM22 on a 3.6-kb BamHI
fragment (same as in pJR2330) subcloned into the BamHI site
of pJR2332. pJR2332 was pRS426 (2µm, URA3), cut with
EcoRI and SalI and resealed by blunt-end
ligation, such that the EcoRI-SalI fragment in
the multiple cloning site was missing.
pJR2322 was made from pJR2333 by cre-lox site-specific
recombination with pHB2 as previously described (19),
resulting in a GST-Ecm22p1-497 fusion protein
expressed from the tac promoter. pJR2333 was made by
subcloning a PCR-amplified fragment carrying ECM22 (codons 1 to 497 with a STOP codon inserted after codon 497) as an
XhoI-SacI fragment into the same sites in pUNI10 (19). The PCR fragment was generated by using DynaZyme EXT
(Finnzymes) with primers av352
(5'-CTCCATCTCTCGAGAAATGACATCCGATGATGGGAATG-3') and av390
(5'-CAAATTCAAGAGCTCTTATCTAGACAGCCCGACTAATTCAG-3') and plasmid pJR2330 as a template.
pJR2323 was made by subcloning a PCR-amplified fragment containing
Upc2p1-395 (amino acids 1 through 395) as an
XhoI-NotI fragment into the bacterial expression
vector pBAT4 (23). The PCR fragment was generated using
primers av570 (5'-ACAGTTCCTCGAGATGAGCGAAGTCGGTATACAGAATC-3') and av571
(5'-AGTAGTTGCGGCCGCTTATGATTGTAAAGGCCCTTCCTTTACC-3').
2µm overexpression screen.
The diploid host strain
(JRY7184) was transformed with a 2µm URA3 genomic
(YEp24-based) library (9) using a lithium acetate-based transformation protocol (17) and allowed to recover for
6 h in liquid minimal medium lacking uracil, selecting only for
uracil prototrophy provided by the plasmid. Transformants were
subsequently plated on minimal medium lacking uracil and containing
Geneticin (1 mg/ml) to select for transformants with elevated
expression of ERG2-KanMX. From an estimated 100,000 initial
transformants, 70 Geneticin-resistant colonies were selected, and 41 of
these isolates also showed increased expression of
ERG2-lacZ. Thirteen plasmids were recovered that conferred
increased expression of ERG2-lacZ upon retransformation.
Eleven of these contained overlapping genomic fragments that included
the gene ECM22. A 2µm plasmid with a 3.6-kb PCR fragment
containing only the ECM22 gene (pJR2330) was sufficient to
increase the expression of ERG2.
RNA blots.
Cultures for RNA preparation were grown in
minimal medium containing all necessary supplements. Cultures grown
under inducing conditions contained 40 µg of lovastatin/ml. All
cultures were inoculated from a fresh stationary-phase culture (grown
overnight) and grown for 16 h to an optical density at 600 nm
(OD600) of 0.2 to 1.0. Then, 50 ml of these
cultures was harvested by centrifugation, and the cell pellet was
frozen at 80°C. Total RNA purification and RNA blot hybridization
were performed as previously described (7) with 10 µg of
total RNA used in all lanes.
-Galactosidase assays.
-Galactosidase liquid assays
and filter lifts were performed as described previously (2,
33).
Mobility shift assays.
Mobility shift assays were performed
essentially as described previously (4). The wild-type
probe used was a 60-bp double-stranded DNA made by annealing av401
(5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAACTACCGGTGCTATCGTTCTCGTTTGGA-3') to av402
(5'-AGGATCCAAACGAGAACGA TAGCACCGG TAG T T T TCC T TGCGGC T TATACGAGGGCCGTG-3').
The 4-bp overhangs (indicated by underscores) were then filled in by
using Klenow fragment (New England Biolabs) and radiolabeled dCTP
(NEN). The probe was purified on a 3% agarose gel, recovered on a DEAE
membrane (NA45; Schleicher & Schuell), eluted in DEAE elution buffer
(10 mM Tris-Cl [pH 7.9], 1 mM EDTA [pH 8.0], 1 M NaCl), ethanol
precipitated, and resuspended in Tris-EDTA (pH 7.6). The extent of
labeling was determined by scintillation counting. A total of 20,000 cpm were used in each reaction, which corresponded to ca. 3 ng of
probe. The probe with the 10-bp SRE mutation was made in the same way
by using oligonucleotides av403 (5'-CGACCA C GG CCACG ATATC TAC C G CA AG G AAAAC T AC C GG TGC TAT CG T TCTCGTTTGGA-3')
and av404
(5'-AGGATCCAAACGAGAACGATAGCACCGGTAGTTTTCCTTGCGGTAGATATCGTGGCCGTG-3'). Specific competitors were made by annealing two complementary oligonucleotides (without single-strand overhangs). The sequences of
the oligonucleotides used were
5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAA-3' and
5'-TTTTCCTTGCGGCTTATACGAGGGCCGTGGTCG-3', except for the
point mutations shown in the figure. Sheared salmon sperm DNA was used as nonspecific competitor in all experiments.
Bacterial extracts were made as follows. A 1-ml quantity overnight in
LB Amp of the bacterial strain ER2508 expressing either glutathione
S-transferase (GST) alone (pHB2), the
GST-Ecm22p1-497 fusion protein (pJR2322), or
Upc2p1-395 (pJR2323) or carrying the plasmid
pBAT4 (23) was diluted into 100 ml of LB Amp culture and
grown at 37°C to an OD595 of 0.5. Expression of
the various fusion proteins was then induced by adding 100 µl of 4 M
IPTG (isopropyl--D-thiogalactopyranoside) and
growing the culture for an additional 4 h. Cells were harvested by
centrifugation at 3,000 × g for 5 min, and the cell
pellet was frozen at 80°C. A whole-cell bacterial extract was made
by adding 1 ml of buffer (2 mM Tris-Cl [pH 7.5], 10% glycerol, 2 mM
MgCl2, 10 µM
Zn2SO4, 125 µg of sheared
salmon sperm DNA/ml) to the frozen pellet. Lysis was achieved by six
15-s sonications, with a 30-s rest on ice between each sonication. The
extract was cleared by centrifugation in an Eppendorf centrifuge for 15 min at 2,000 × g. Protein concentrations were
determined by Bio-Rad protein assays (Bio-Rad). We used 25 µg of
total protein in each reaction. The protein extracts made from strains
carrying plasmids pJR2322 and pHB2 differed from those made from
strains carrying plasmids pBAT4 and pJR2323 by the addition of 75 mM NaCl.
Transmembrane helix prediction.
Transmembrane helix
predictions were obtained using the following programs: PHDhtm
(http://www.embl-heidelberg.de /predictprotein/predictprotein.html), TMAP (http://130.237.130.31/tmap/single.html) (24,
25), TMpred (http://www.ch.embnet.org/software/TMPRED_form.html),
PSORT (http://psort.nibb.ac.jp/), TMHMM
(http://www.cbs.dtu.dk/services /TMHMM-2.0/), and HMMTOP (http://www.enzim.hu/hmmtop/) (32). TMAP and TMpred
predicted four transmembrane helices in both proteins. PHDhtm predicted two transmembrane helices in Ecm22p and one in Upc2p, PSORT predicted one transmembrane helix in both proteins, and TMHMM and HMMTOP both
predicted no transmembrane helix in either protein.
| |
RESULTS |
Overexpression of ECM22 and UPC2
increased expression of ERG2
ERG2,
which encodes 
8-7 sterol isomerase, is one of many genes in the
yeast sterol biosynthetic pathway that are transcriptionally regulated
in response to changes in sterol levels (6, 13, 30). The
proteins responsible for mediating this regulation have not been
described. A genetic selection was performed to identify transcription
factors responsible for the sterol-mediated regulation of
ERG2 expression. Specifically, an overexpression strategy was used to select for genes that, when overexpressed, led to
increased expression of ERG2. ERG2 expression was
monitored by a pair of integrated reporter genes consisting of
ERG2 regulatory sequences fused to the coding regions of
KanMX (ERG2-KanMX, pJR2326) and of
ERG2 regulatory sequences fused to LacZ
(ERG2-lacZ, pJR2325). These reporters allowed detection
of increased ERG2 expression by increased resistance to
the translational inhibitor Geneticin (conferred by
KanMX) and by increased -galactosidase activity (encoded by lacZ). Yeast transformation is itself
mutagenic, and mutations at most steps of ergosterol biosynthesis can
lead to elevated reporter expression due to disruption of feedback
repression. Because mutations in ergosterol biosynthesis would
primarily be recessive, a diploid strain was used to avoid this
substantial background.
A diploid strain (JRY7184) homozygous for both ERG2-lacZ and
ERG2-KanMX was transformed with a high-copy (2µm, YEp24)
genomic library (9) and then plated on minimal medium
lacking uracil, selecting for the plasmid-borne URA3 gene.
The medium also contained Geneticin to select for transformants with
elevated expression of ERG2-KanMX. Eleven plasmids were
recovered from this screen that had overlapping genomic fragments
containing the gene ECM22. A plasmid with a 3.6-kb fragment
containing only the ECM22 gene (pJR2330) was sufficient to
increase expression of both ERG2 reporters.
Ecm22p is a member of the Zn[2]-Cys[6] binuclear cluster family of
fungal transcription factors (31). The deduced Ecm22p protein sequence is 45% identical to that encoded by UPC2
(Proteome, Inc.
[http://www.proteome.com/databases /index.html]), a member of
the same family of transcription factors. The sequence similarity between Ecm22p and Upc2p was particularly high in the carboxyl-terminal region (76% identity, 238 of 314), as well as in the amino-terminal region (70% identity, 56 of 80), which contained the presumptive DNA
binding domain (77% identity, 30 of 39) (Fig.
1A). No plasmids containing
UPC2 were isolated in the initial screen. Nevertheless, overexpression of UPC2 also led to increased expression of
an ERG2-lacZ reporter (pJR2316) (data not shown). Thus,
overexpression of either ECM22 or UPC2 affected
expression of ERG2.
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FIG. 1.
(A) Ecm22p and Upc2p show strong sequence similarity.
They are 77% identical in the DNA-binding domain (DBD) and 76%
identical in the carboxyl-terminal region. (B) ECM22 and
UPC2 had overlapping functions. The phenotypes of
wild-type (wt; W303-1a), ecm22 (JRY7180),
upc2 (JRY7179), and ecm22 upc2
(JRY7181) strains were compared by spotting 10-fold serial dilutions of
each strain onto minimal medium plates containing 100 ng of
amphotericin B or 40 µg of lovastatin/ml.
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ECM22 and UPC2 had overlapping
functions.
The strong sequence similarity between ECM22
and UPC2 and their shared ability to activate
ERG2 expression implied that these genes might have similar
or overlapping functions. Evidence of overlapping functions of Ecm22p
and Upc2p was also recently reported by Shianna et al.
(28). The sensitivity of each of the single null mutants
as well as the double null mutant to two sterol-related drugs,
lovastatin and amphotericin B, was tested (Fig. 1B). These two
inhibitors provide useful measures of the overall expression level of
the sterol biosynthetic pathway: reduced flux through the pathway
results in lovastatin sensitivity and reduced ergosterol levels in the
plasma membrane result in amphotericin B resistance. The ecm22
upc2 double mutant (JRY7181) was much more resistant to
amphotericin B than either of the single mutants (JRY7179 and JRY7180)
or the wild-type strain (W303-1a), indicating that the double mutant
had reduced levels of ergosterol. The double mutant was also much more
sensitive to lovastatin than either of the single mutants or the wild
type. Qualitatively, phenotypes such as these might result from a loss
of expression of ERG2 alone (as shown below). However, a
deletion of the SRE in the ERG2 promoter, to which both
Ecm22p and Upc2p bound (described below), did not result in increased
resistance to amphotericin B or sensitivity to lovastatin. Furthermore,
whereas erg2 is viable, a recent report shows that
ecm22 and upc2 are synthetically lethal in some strain backgrounds (28). This result, together with
the resistance to amphotericin B and the sensitivity to lovastatin of
our ecm22 upc2 double mutant relative to either single
mutant, suggested that the two genes had overlapping functions that
extended beyond activation of ERG2, as confirmed below.
Lovastatin is an inhibitor of sterol biosynthesis and causes a decrease
in the production of both sterols and a variety of nonsterol products
made by this pathway. The sensitivity of the ecm22
upc2 double mutant might reflect either the consequences of a
lack of sterols or a lack of some other product of the pathway. We
distinguished between these two possibilities by inhibiting sterol
synthesis after the last branch point of the pathway. A sterol-specific
block can be achieved with an erg2 null mutation. ERG2 encodes one of several nonessential enzymes in the
latter steps of the sterol biosynthetic pathway that modify the basic sterol structure. In the absence of ERG2, cells make a
sterol adequate for growth but not the preferred ergosterol. We
attempted to generate an erg2 ecm22 upc2 triple
mutant by crossing an ecm22 upc2 mutant (JRY7181) to
an erg2 ecm22 mutant strain (JRY7189). All possible
double mutant segregants from this cross (erg2 ecm22
[2], erg2 upc2 [2], and
ecm22 upc2 [5]) were viable. However,
all five erg2 ecm22 upc2 triple mutant segregants
died shortly after spore germination, generating microcolonies of <100
cells (data not shown). The synthetic lethality of the erg2
ecm22 upc2 triple mutant was further evaluated by generating an erg2 ecm22 upc2 triple mutant (JRY7188) kept
alive by a URA3-marked plasmid containing a wild-type copy
of ECM22 (pJR2331), as evidenced by the inability of the
strain to grow on medium containing 5-fluoroorotic acid (5-FOA), which
selects against URA3 (Fig. 2).
If the lovastatin sensitivity of the ecm22 upc2 double
mutant were due to depletion of a nonsterol product of the pathway, the
triple mutant should still be viable but lovastatin sensitive. In
contrast, if the lovastatin sensitivity reflected an inability to
respond to depletion of late sterol intermediates or ergosterol, the
triple mutant should mimic the ecm22 upc2 double
mutant in the presence of lovastatin and be dead. The synthetic lethality of the erg2 ecm22 upc2 triple mutant
therefore suggested that the viable ecm22 upc2 double
mutant was deficient in a regulatory response to sterol depletion.
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FIG. 2.
erg2 was synthetically lethal with
ecm22 upc2. An erg2 (JRY7187)
strain, an ecm22 upc2 (JRY7181) strain, and an
ecm22 upc2 erg2 (JRY7188) strain, all carrying
the plasmid pJR2331 (ECM22, URA3), were grown on 5-FOA.
Only the ecm22 upc2 erg2 strain was unable to
grow.
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Ecm22p and Upc2p regulated transcription of ERG2 and
ERG3 in response to sterol levels.
To establish
whether or not Ecm22p and Upc2p played a role in sterol-mediated
regulation of ERG2 transcription, ERG2 expression levels were measured in an ecm22 (JRY7180), an
upc2 (JRY7179), and an ecm22 upc2
(JRY7181) mutant grown under inducing and noninducing conditions (Fig.
3A). Inducing conditions differed from
noninducing by inclusion of lovastatin in the growth medium. ERG2 expression was measured using a set of ERG2
reporters containing ERG2 regulatory sequences fused to the
lacZ coding sequence as described below (see Fig. 5). Either
ECM22 or UPC2 was sufficient to confer
sterol-mediated regulation to a reporter containing the whole
ERG2 promoter. In the absence of both ECM22 and
UPC2, no ERG2 induction was detected in response
to lovastatin. This regulation was in large part dependent on the
presence of an 11-bp SRE (see below) in the ERG2 promoter.
Furthermore, Upc2p was capable of activating expression through the
11-bp SRE, indicating that the SRE alone was sufficient for
sterol-mediated regulation by Upc2p.
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FIG. 3.
(A) UPC2 and ECM22 were
necessary for sterol-mediated regulation of ERG2-lacZ.
ERG2-lacZ reporters were used to determine the contribution of
each of the genes to the regulation of ERG2. "Full
promoter" refers to a reporter having ERG2 promoter
sequences from 751 to 93 controlling transcription of
lacZ. "SRE mutated" has the same promoter except
that 10 bp of the SRE have been mutated. "SRE only" has only the
11-bp SRE controlling lacZ. The plasmids were
transformed into wild-type (wt; W303-1a), upc2
(JRY7179), ecm22 (JRY7180), and ecm22
upc2 (JRY7181) strains. -Galactosidase assays were
performed on the transformants after they were grown for 16 h
either in minimal medium (uninduced) or in minimal medium containing 40 µg of lovastatin/ml (induced). The assay values represent the average
of two determinations. (B) UPC2 and ECM22
were necessary for sterol-mediated regulation of
ERG3-lacZ. An ERG3-lacZ reporter was
integrated at the URA3 locus of wild-type (JRY7190),
upc2 (JRY7191), ecm22 (JRY7192),
and upc2 ecm22 (JRY7193) strains.
-Galactosidase assays were performed on the transformants following
growth for 16 h in conventional medium (uninduced) or in the
presence of 40 µg of lovastatin/ml (induced). The assay values
represent the average of two determinations.
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The effect of Ecm22p and Upc2p on sterol-mediated regulation of
ERG2 was further investigated by measuring ERG2
mRNA levels in ecm22, upc2, and
ecm22 upc2 mutants (JRY7180, JRY7179, and JRY7181)
grown under inducing and noninducing conditions (Fig. 4). Under noninducing conditions, the
level of ERG2 mRNA in the upc2 mutant was
comparable to the level in wild-type cells. Thus, UPC2 was
not required for expression of ERG2 per se. In contrast, the
upc2 strain did not induce ERG2 transcription
when grown in medium containing lovastatin, whereas the wild-type
strain did. Therefore, UPC2 was necessary for
ERG2 induction under low-sterol conditions. The
ERG2 mRNA levels in the ecm22 mutant were
similar to the levels in wild type, under both noninducing and inducing conditions. Thus, ECM22 was not required for uninduced
expression of ERG2 and also was not necessary for
sterol-mediated regulation of ERG2, at least at the time
point assayed. The discrepancy between the mRNA and the
-galactosidase measurements of ERG2 expression in this
mutant may simply reflect the higher stability of -galactosidase (5) compared to ERG2 mRNA. The
-galactosidase measurements integrated most or all of
ERG2 induction over the 16-hour induction, whereas the mRNA
levels captured a regulatory snapshot at 16 h. Together, these
data indicated that either Upc2p or Ecm22p was capable of mediating
sterol regulation of ERG2 transcription and suggested that
Ecm22p may have a more transient effect on ERG2 transcription in response to sterol limitation. Furthermore, because ERG2 expression under noninducing conditions was reduced in
the ecm22 upc2 double mutant compared to either of the
single mutants and wild type, both proteins contributed to the
uninduced level of ERG2 transcription.
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FIG. 4.
UPC2 was necessary for sterol-mediated regulation of
ERG2. ERG2 transcript levels were monitored by RNA blot
hybridization. Total RNA was prepared from wild-type (wt; W303-1a),
ecm22 (JRY7180), upc2 (JRY7179),
and ecm22 upc2 (JRY7181) strains grown for 16 h in minimal medium (uninduced) or in the presence of 40 µg of
lovastatin/ml (induced). ACT1 served as the loading
control.
|
|
A similar experiment established that both Ecm22p and Upc2p were
involved in the sterol-mediated regulation of the ERG3
gene. As discussed below, ERG3 is one of several sterol
biosynthetic genes that had regulatory sequences identical to the
ERG2 SRE (Table 2). We used an
ERG3 reporter with ERG3 regulatory sequences fused to lacZ (pJR2318) integrated at URA3
(JRY7190, JRY7191, JRY7192, and JRY7193) to study the effect of Ecm22p
and Upc2p on ERG3 expression. No induction of
ERG3 occurred in response to lovastatin treatment in the
ecm22 upc2 double mutant. Both ECM22 and
UPC2 individually were able to confer sterol-mediated regulation of ERG3 transcription (Fig. 3B).
An 11-bp SRE in the ERG2 promoter conferred
regulation by sterol levels.
Analysis of the ERG2
promoter led to the identification of an SRE. In these experiments,
fragments 5' to the ERG2 coding region were subcloned into a
reporter, consisting of the CYC1 promoter directing
expression of lacZ (pJR2307). This context allows a regulatory sequence from another gene to be recognized by its ability
to activate and/or regulate transcription from the start site provided
by the CYC1 promoter. An 11-bp (5'-CTCGTATAAGC-3') SRE,
positioned between bp 186 and 176 relative to the ERG2 ORF, conferred sterol-mediated regulation to the CYC1-lacZ
reporter (Fig. 5).
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FIG. 5.
An SRE in the ERG2 promoter was
sufficient to confer sterol-mediated regulation of expression.
Fragments of the ERG2 promoter were subcloned into a
CYC1-lacZ reporter. The inserts are represented
schematically on the left: black bars indicate inserts that conferred
sterol-mediated regulation, and gray bars indicate fragments that did
not. pJR2316 had the whole ERG2 promoter from 751 to
93 (numbers refer to base pairs 5' of the A of the initiation codon).
Other sequences inserted were as follows: pJR2308
(5'-GATATCGCACATTCCTGCCCTTACGCTCCAGGGCAGAATCGAACCACGGCCCT-3'),
pJR2309
(5'-GATATCGTATAAGCCGCAAGGAAAACTACCGGTGCTATCGTTCTCGTTTGGAT-3'),
pJR2310
(5'-GATATCGATTTTCAGTATGGAAGAATTTGGATAGATCTGCAGCGCCATGG-3'),
pJR2311
(5'-GATATCTCCAGGGCAGAATCGAACCACGGCCCTCGTATAAGCCGCAAGGAAAA-3'),
pJR2312
(5'-GATATCTACCGGTGCTATCGTTCTCGTTTGGATGATTTTCAGTATGGAAGAAT-3'),
pJR2313 (5'-GATATCTCGTATAAGCCGCAAGGAAAAC-3'), pJR2314
(5'-GATATCTCGTATAAG-3'), pJR2327
(5'-GATATCGCAAGGAAAACTACCGGTGCTATCGTTCTCGTTTGGAT-3'),
pJR2328 (5'-GATATCACTACCGGTGCTATCGTTCTCGTTTGGAT-3'), pJR2329
(5'-GATATCGCAAGGAAAACTACCGGTG-3'), and pJR2315
(5'-GATATCGATACGATT-3'). pJR2307 had no inserted
sequences. The resulting plasmids were transformed into the
wild-type strain W303-1a. -Galactosidase assays were performed on
the transformants after they were grown for 16 h in conventional
minimal medium (uninduced) or in the presence of 40 µg of lovastatin
per ml (induced). The assay values represent the average of 2 determinations.
|
|
The ERG2 SRE was necessary for induced and uninduced
expression but not for basal expression.
The RNA analysis in Fig.
4 indicated that Ecm22p and Upc2p contributed to the uninduced level of
ERG2 expression. To determine whether the SRE was necessary
either for basal expression of ERG2 or just for regulation
of ERG2 expression, a 10-bp mutation in the SRE in the
native ERG2 promoter was constructed. This mutation blocked
induction of ERG2 mRNA by lovastatin (Fig.
6A). However, the uninduced level of
expression of ERG2 in both mutant and wild-type strains was
quite low, raising the possibility that the SRE was required for basal
expression, as well as for uninduced and induced expression. Indeed,
the lacZ data in Fig. 3A indicated that the SRE contributed
to the uninduced level of expression. In the absence of expression,
induction would not be reliably measured. To address this possibility,
we tested whether the phenotypes of a strain with a deletion of the SRE
in the ERG2 promoter (JRY7182) were equivalent to those of a
strain with a deletion of the ERG2 gene itself (JRY7187). If
the two phenotypes differed, the ERG2 gene must be expressed
at some level in the absence of a SRE. The erg2 strain
was significantly more amphotericin B resistant and lovastatin sensitive than the strain with the SRE deletion (Fig. 6B). Therefore, deletion of the SRE did not eliminate expression of ERG2.
Hence, the SRE was a mediator of ERG2 regulation and not of
its basal expression. However, the higher levels of expression in the
presence of the SRE than in its absence under noninducing conditions
(Fig. 3A) implied that a fraction of Ecm22p or Upc2p molecules were active under noninducing conditions.
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FIG. 6.
(A) The SRE was necessary for sterol-mediated regulation
of ERG2. ERG2 transcript levels were monitored by RNA
blot analysis. Total RNA was prepared from strains that had a mutated
sre (JRY7182) and wild-type SRE
(JRY7183) grown for 16 h in minimal medium (uninduced) or in the
presence of 40 µg of lovastatin/ml (induced). ACT1
served as the loading control. (B) The SRE was not necessary for basal
expression of ERG2. The phenotypes of strains
sre (JRY7182), wild-type (wt) SRE (JRY7183), and
erg2 (JRY7187) were compared by spotting 10-fold
serial dilutions of each strain onto minimal medium plates containing
100 ng of amphotericin B or 40 µg of lovastatin/ml.
|
|
Ecm22p and Upc2p bound the ERG2 SRE directly.
The deduced sequence of Ecm22p and Upc2p indicated that they were both
members of the Zn[2]-Cys[6] binuclear cluster family of fungal
transcription factors (31), raising the possibility that
they were transcriptional activators that bound directly to the SRE. To
test this prediction, we developed a mobility shift assay using a
radioactively labeled 60-bp probe containing sequences from the
ERG2 promoter that included the SRE. A protein extract made
from bacteria expressing a truncated version of Upc2p (amino acids 1 through 395) (pJR2323) produced a mobility shift that was dependent on
the presence of the SRE (Fig. 7A). The
shifted band could be competed away by an excess of an unlabeled
double-stranded oligonucleotide that contained the SRE but not by one
lacking it. A probe in which the 11 bp in the SRE were replaced by a
random sequence was not shifted under the same conditions. Thus, Upc2p bound DNA directly in an SRE-dependent manner, as expected of a
transcription factor controlling the expression of ERG2.
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FIG. 7.
(A) Upc2p bound directly to the ERG2
promoter in a SRE-dependent manner. Mobility shift assays were
performed by using a radiolabeled double-stranded DNA probe containing
sequences from the ERG2 promoter overlapping the SRE and
25 µg of bacterial protein extract from a strain expressing a
truncated Upc2p1-395 protein (amino acids 1 through 395)
(pJR2323) or carrying the vector plasmid pBAT4 (23).
Arrows indicate shifted bands. Lanes where the probe contained a
wild-type SRE are labeled "+"
(5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAAC TACCGG TGC TATCG T TC TCGTTTGGA-3'),
and lanes where 10 bp of the SRE were replaced by random sequence
(underlined) are labeled ""
(5'-CGACCACGGCCACGATATC TACCGCAAGGAAAAC TACCGGTGC TATCGTTCTCGTTTGGA-3').
"None" indicates no competitor other than salmon sperm DNA was
used. Unlabeled double-stranded oligonucleotides
(5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAA-3'
is labeled "+";
5'-CGACCACGGCCACGATATCTACCGCAAGGAAAA-3' is
labeled "") were used as specific competitors, where indicated,
at a 250-fold molar excess to the radioactively labeled probe. (B)
Ecm22p also bound directly to the ERG2 promoter in a
SRE-dependent manner. The experiment was performed as for Upc2p, but
the protein extract was made from bacteria expressing either GST alone
(pHB2) or GST-Ecm22p1-497 (pJR2322).
|
|
A similar experiment showed that Ecm22p also bound directly to the
ERG2 promoter. A protein extract made from bacteria
expressing a truncated version of Ecm22p (amino acids 1 through 497) as
a GST fusion protein (pJR2322) produced a mobility shift that was dependent on the presence of the SRE. Hence, Ecm22p, as well as Upc2p,
bound DNA directly in a SRE-dependent manner (Fig. 7B).
Using this mobility shift assay, 7 of the 11 bp in the SRE were
identified to be critical for binding by Upc2p. A series of double-stranded oligonucleotides were constructed with consecutive transversions of each of the 11 bp of the SRE. These unlabeled oligonucleotides were used as specific competitors in mobility shift
assays with the radioactively labeled 60-bp probe that contained a
wild-type SRE. Only oligonucleotides that could not compete with the
labeled probe for Upc2p binding would allow visualization of a shifted
band. The critical base pairs in the SRE were the 7-bp sequence TCGTATA
(Fig. 8A). In a similar experiment
testing the DNA binding specificity of Ecm22p, the same 7-bp sequence was critical for the binding by Ecm22p (Fig. 8B). Therefore, both Upc2
and Ecm22p bound the same 7-bp TCGTATA sequence.
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FIG. 8.
(A) The 7-bp sequence TCGTATA was necessary for Upc2p
binding to the ERG2 promoter. Mobility shift assays were
performed using 25 µg of protein from a bacterial extract expressing
a truncated Upc2p1-395 protein (pJR2323) and a
radioactively labeled double-stranded DNA probe containing sequences
from the ERG2 promoter with a wild-type SRE as described
for Fig. 7. Fourteen different specific double-stranded competitors
were used. One of the competitors contained a wild-type SRE (wt SRE)
and had the sequence
5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAA-3', one had the
entire SRE mutated
(5'-CGACCACGGCCACGATATCTACCGCAAGGAAAA-3', labeled "mutant
SRE"), one had a wild-type SRE but all other sequences were mutated
(labeled "wt SRE, mut. flank."), and the rest contained a series of
consecutive transversions in each of the base pairs of the SRE. The
sequence of these competitors was
5'-CGACCACGGCCCTCGTATAAGCCGCAAGGAAAA-3' except for the
mutations noted in the figure. The competitors were used at a 250-fold
excess compared to the probe. (B) Ecm22p bound the same 7-bp sequence
(TCGTATA) as did Upc2p. The experiment was performed as for Upc2p
except that the protein extract was made from bacteria expressing
GST-Ecm22p1-497 (pJR2322).
|
|
| |
DISCUSSION |
The present study identified an SRE and two SREBPs required for
the regulation of the sterol biosynthetic genes ERG2 and
ERG3. Upc2p and Ecm22p were necessary for the regulation of
ERG2 expression in response to changes in sterol levels, and
both bound directly to the ERG2 promoter. Phenotypes of the
ecm22 upc2 double mutant further supported a role for
ECM22 and UPC2 in sterol homeostasis.
The SRE in the ERG2 promoter was both necessary and
sufficient to confer regulated transcription in response to sterol
depletion by lovastatin. Seven base pairs in the ERG2 SRE
were necessary for the binding of Upc2p and Ecm22p to DNA. These 7 bp
had a perfect match in a 24-bp upstream activating sequence (UAS) found
in the ERG3 promoter that is necessary for the regulation of
ERG3 in response to sterols (3).
ECM22 and UPC2 were also necessary for the sterol
regulation of ERG3. Furthermore, the same 7-bp sequence
element was found in the promoters of other yeast genes that are
regulated by sterols, suggesting that these genes may be coordinately
regulated by Upc2p and Ecm22p through their SREs (Table 2).
Sterols and anaerobic growth.
Sterol biosynthesis is dependent
on oxygen and can take place only during aerobic growth. Unlike
aerobically growing cells, anaerobically growing cells can acquire
sterols by taking them up from the growth medium. UPC2 was
first identified in a screen for mutants that take up sterols under
aerobic conditions (11). The sterol uptake phenotype is
due to a semidominant allele containing a point mutation in the
carboxyl-terminal part of UPC2. Recently, UPC2
was shown to be necessary for the anaerobic induction of the DAN/TIR
genes (1). Due to the oxygen requirement of sterol production, this effect could in principle reflect either a response to
oxygen or a response to sterol levels.
Lovastatin inhibits an early step in sterol biosynthesis. Thus,
lovastatin treatment will reduce levels of early, nonsterol, products
of the pathway in addition to reducing sterol levels. However, the
erg2 null mutant blocks sterol synthesis specifically, leaving early products of the pathway unaffected. The synthetic lethality of erg2 in combination with the ecm22
upc2 double mutant therefore suggested that a sterol-specific
block generated a signal to activate Ecm22p and Upc2p. When this signal
failed to upregulate sterol biosynthesis, because the transcription
factors responsible for the effect had been deleted, the cells died.
This conclusion is supported by the induction of ERG2
expression in an erg24 mutant (30).
Moreover, ERG3-lacZ expression is induced in an
erg2 mutant (3). Both of these observations
pinpoint a late sterol product as the key product whose level is
monitored by Ecm22p and Upc2p. We therefore propose that the principal
function of Ecm22p and Upc2p is to regulate genes in response to sterol levels.
Effect of ECM22 and UPC2 on sterol
biosynthesis.
Upc2p and Ecm22p activated ERG2
transcription in response to the need for more sterols by binding
directly to a 7-bp SRE in the ERG2 promoter. The presence of
identical sequence elements in the promoters of several other genes
involved in sterol biosynthesis in yeast indicated that Upc2p and
Ecm22p may regulate multiple genes in this pathway (Table 2). Sequence
elements identical to the ERG2 SRE are within 500 bp 5' of
the coding region of ERG1, ERG2, ERG3, ERG6, ERG8, ERG13,
and ERG25, and within the first 1,000 bp 5' of
ERG11 and ERG12. With the exception of
ERG13, all of these genes have been shown to be
transcriptionally regulated by sterols (3, 6, 13, 18, 30).
It therefore seems likely that Upc2p and Ecm22p are involved in the
sterol regulation of genes other than ERG2 and
ERG3. Hence, Upc2p and Ecm22p may be major transcriptional
regulators of sterol-responsive genes in the sterol biosynthetic
pathway of yeast.
The SRE motif is found in the promoters of a number of other
sterol-regulated genes in yeast. Some of these genes are involved in
sphingolipid biosynthesis. Both LCB1 and SUR2 are
regulated by sterols (6), and LCB1 contains an
SRE within the first 500 bp of the 5' untranslated region. This raises
the possibility that sphingolipid biosynthesis in yeast may be
coordinated with sterol biosynthesis, with Upc2p and Ecm22p providing
the coordination.
Our data indicated that Upc2p and Ecm22p were transcriptional
activators of ERG2 and other genes and that the activities
of the proteins were influenced by sterol levels. The glutamine-rich domain in the middle of both Upc2p and Ecm22p was consistent with this
hypothesis. However, at least three of the genes with a SRE in the
promoter (CYC7, BAP2, and DIP5) are
repressed, rather than induced, by the lack of sterols
(6). This paradoxical response of SRE-containing genes
raises the formal possibility that Upc2p and Ecm22p may also be
transcriptional repressors or recruit a repressor under certain
conditions. In fact, such a dual effect is seen in the case of the
transcriptional repressor Ume6p, another member of the Zn[2]-Cys[6]
binuclear cluster family of transcription factors. During mitosis Ume6p
recruits the Sin3p-Rpd3p histone deacetylase, resulting in repression
of adjacent genes, whereas in meiosis Ume6p recruits the
transcriptional activator Ime1p (27), thereby activating
transcription. However, if Upc2p and Ecm22p were activators as well as
repressors, the nature of their transcriptional effect would have to
depend on other DNA-binding proteins in the vicinity of the SRE or upon
the precise position of the SRE in the promoter. Alternatively, other
proteins in yeast may mediate repression in response to sterol depletion.
Numerous genes shown to be regulated in response to changes in sterol
levels (6, 13) lack promoter elements with similarity to
the ERG2 SRE. It is possible that Upc2p and Ecm22p could
indirectly regulate a broader set of genes than those that have SREs in
their promoters by regulating other transcription factors.
Alternatively, Upc2p and Ecm22p may bind additional sequence elements
different from the SRE identified here. Hap1p, another member of the
Zn[2]-Cys[6] binuclear cluster family, can bind two unrelated
sequences (15, 26). Moreover, in identifying the 7 bp of
the SRE necessary for binding, we tested only one point mutation (a
transversion) in each position. In fact, the anaerobic response element
(AR1; TCGT TYAG) (10) through which Upc2p induces
anaerobically expressed genes (1) differs from the SRE by
2 bp. It is still not known whether or not Upc2p binds directly to AR1.
It is therefore possible that further mutations of the binding site may
be functional. Hence, it is conceivable that Upc2p and Ecm22p could
directly regulate genes lacking a SRE.
Why have two genes for the same function?
Upc2p and Ecm22p had
closely overlapping functions, and both proteins were capable of
conferring sterol-mediated regulation to ERG2 and
ERG3 by binding directly to the same 7-bp regulatory sequence. The major difference between UPC2 and
ECM22 in our experiments was that Ecm22p failed to
activate transcription from the 11-bp SRE alone, although the effect of
Ecm22p on the whole promoter was SRE dependent. Ecm22p binding and/or
activation therefore may be dependent on other factors that bound the
ERG2 promoter. Based on -galactosidase assays, the
effect of Ecm22p was somewhat weaker than that of Upc2p. Furthermore,
Ecm22p seemed to have a more transient effect on ERG2
expression than Upc2p. In a situation where Ecm22p and Upc2p may
compete for binding to the same site, different promoters with
different relative affinities for Ecm22p and Upc2p may provide a range
of sterol-mediated regulatory responses.
Activation of Upc2p and Ecm22p.
The central unanswered
question is the mechanism by which sterols influence the activity of
Upc2p and Ecm22p. Recent work by Abramova et al. (1)
showed that UPC2 transcription, although expressed
aerobically, is anaerobically induced, and that the semidominant
UPC2-1 mutant, which has a point mutation in the carboxyl-terminal domain, causes constitutive aerobic expression of
anaerobically induced genes. Hence, the carboxyl-terminal domain may
repress transcriptional activity. Interestingly, the carboxyl-terminal domains of both Ecm22p and Upc2p were predicted to have one or more
transmembrane helices, making it a possibility that Ecm22p and Upc2p
directly measure sterol levels in membranes. However, the number of
transmembrane helices predicted varied considerably depending upon
which program was used to make the prediction.
Nevertheless, if Upc2p and Ecm22p are integral membrane proteins, then
Upc2p and Ecm22p, much like the mammalian SREBPs, may be released from
the membrane in response to the need for more sterols. Regulated
proteolysis as a mechanism for activating a membrane-bound
transcription factor has been described in yeast (16).
Sterols may regulate proteolytic activation by influencing properties
of the ER membrane.
Another possibility is that the carboxyl-terminal domain of Ecm22p and
Upc2p is a direct sterol sensor, even though it has no similarity at
the protein sequence level to any currently known sterol binding
domains. Clearly, determining where the Ecm22p and Upc2p proteins
reside in the cells under noninducing conditions and whether they are
structurally altered upon induction will be decisive in testing these
and other models of activation.
The Zn[2]-Cys[6] binuclear cluster family of transcription factors
is fungus specific (31) and, as expected, we have not found any proteins with similarity to the amino-terminal DNA binding region of Upc2p and Ecm22p in any nonfungal organism. Perhaps more
surprisingly, we have also found no proteins with homology to the
carboxyl-terminal domains of either protein outside of fungi.
Therefore, proteins homologous to Ecm22p and Upc2p are probably absent
from mammals. Consequently, despite seemingly parallel sterol
regulatory circuits in yeast and mammals, the transcription factors
involved share no sequence similarity and are clearly not homologous
proteins. Understanding the mechanism of activation of Ecm22p and Upc2p
may therefore reveal a new dimension to how organisms regulate sterol synthesis.
We thank J. Zupicich for assistance with TMH predictions, J. Gin
for technical assistance, and S. Okamura and V. Boyartchuk for comments
on the manuscript.
A.V. was supported by a doctoral fellowship from the Norwegian Research
Council (grant 70429/410). Further research support was provided by a
grant from the National Institutes of Health (GM35827), with core
support from an NIEHS Mutagenesis Center grant (ESO1896).
| 1.
|
Abramova, N. E.,
B. D. Cohen,
O. Sertil,
R. Kapoor,
K. J. A. Davies, and C. V. Lowry.
2001.
Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae.
Genetics
157:1169-1177[Abstract/Free Full Text].
|
| 2.
|
Adams, A.,
D. E. Gottschling,
C. A. Kaiser, and T. Stearns.
1997.
Methods in yeast genetics.
Cold Spring Harbor Laboratory Press, New York, N.Y.
|
| 3.
|
Arthington-Skaggs, B. A.,
D. N. Crowell,
H. Yang,
S. L. Sturley, and M. Bard.
1996.
Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast ergosterol pathway.
FEBS Lett.
392:161-165[CrossRef][Medline].
|
| 4.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl.
1997.
Current protocols in molecular biology.
John Wiley & Sons, Inc., New York, N.Y.
|
| 5.
|
Bachmair, A.,
D. Finley, and A. Varshavsky.
1986.
In vivo half-life of a protein is a function of its amino-terminal residue.
Science
234:179-186[Abstract/Free Full Text].
|
| 6.
|
Bammert, G. F., and J. M. Fostel.
2000.
Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol.
Antimicrob. Agents Chemother.
44:1255-1265[Abstract/Free Full Text].
|
| 7.
|
Boeck, R.,
B. Lapeyre,
C. E. Brown, and A. B. Sachs.
1998.
Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant.
Mol. Cell. Biol.
18:5062-5072[Abstract/Free Full Text].
|
| 8.
|
Brown, M. S., and J. L. Goldstein.
1999.
A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
Proc. Natl. Acad. Sci. USA
96:11041-11048[Abstract/Free Full Text].
|
| 9.
|
Carlson, M., and D. Botstein.
1982.
Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase.
Cell
28:145-154[CrossRef][Medline].
|
| 10.
|
Cohen, B. D.,
O. Sertil,
N. E. Abramova,
K. J. A. Davies, and C. V. Lowry.
2001.
Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites.
Nucleic Acids Res.
29:799-808[Abstract/Free Full Text].
|
| 11.
|
Crowley, J. H.,
F. W. Leak, Jr.,
K. V. Shianna,
S. Tove, and L. W. Parks.
1998.
A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae.
J. Bacteriol.
180:4177-4183[Abstract/Free Full Text].
|
| 12.
|
Dimster-Denk, D.,
M. K. Thorsness, and J. Rine.
1994.
Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in Saccharomyces cerevisiae.
Mol. Biol. Cell
5:655-665[Abstract].
|
| 13.
|
Dimster-Denk, D.,
J. Rine,
J. Phillips,
S. Scherer,
P. Cundiff,
K. DeBord,
D. Gilliland,
S. Hickman,
A. Jarvis,
L. Tong, and M. Ashby.
1999.
Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the genome reporter matrix.
J. Lipid Res.
40:850-860[Abstract/Free Full Text].
|
| 14.
|
Gietz, R. D., and A. Sugino.
1988.
New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.
Gene
74:527-534[CrossRef][Medline].
|
| 15.
|
Hach, A.,
T. Hon, and L. Zhang.
1999.
A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.
Mol. Cell. Biol.
19:4324-4333[Abstract/Free Full Text].
|
| 16.
|
Hoppe, T.,
K. Matuschewski,
M. Rape,
S. Sclenker,
H. D. Ulrich, and S. Jentsch.
2000.
Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
Cell
102:577-586[CrossRef][Medline].
|
| 17.
|
Ito, H.,
Y. Fukuda,
K. Murata, and A. Kimura.
1983.
Transformation of intact yeast cells treated with alkali cations.
J. Bacteriol.
153:163-168[Abstract/Free Full Text].
|
| 18.
|
Leber, R.,
R. Zenz,
K. Schrottner,
S. Fuchsbichler,
B. Puhringer, and F. Turnowsky.
2001.
A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene Saccharomyces cerevisiae.
Eur. J. Biochem.
268:914-924[Medline].
|
| 19.
|
Liu, Q.,
Z. L. Mamie,
D. Leibham,
D. Cortez, and S. J. Elledge.
1998.
The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.
Curr. Biol.
8:1300-1309[CrossRef][Medline].
|
| 20.
|
Lussier, M.,
A. M. White,
J. Sheraton,
T. di Paolo, et al.
1997.
Large-scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.
Genetics
147:435-450[Abstract].
|
| 21.
|
Magana, M. M., and T. F. Osborne.
1996.
Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter.
J. Biol. Chem.
271:32689-32694[Abstract/Free Full Text].
|
| 22.
|
Pai, J. T.,
O. Guryev,
M. S. Brown, and J. L. Goldstein.
1998.
Differential stimulation of cholesterol and unsaturated fatty acid biosynthesis in cells expressing individual nuclear sterol regulatory element-binding proteins.
J. Biol. Chem.
273:26138-26148[Abstract/Free Full Text].
|
| 23.
|
Peranen, J.,
M. Rikkonen,
M. Hyvonen, and L. Kaariainen.
1996.
T7 vectors with a modified T7lac promoter for expression of proteins in Escherichia coli.
Anal. Biochem.
236:371-373[CrossRef][Medline].
|
| 24.
|
Persson, B., and P. Argos.
1994.
Prediction of transmembrane segments in proteins utilising multiple sequence alignments.
J. Mol. Biol.
237:182-192[CrossRef][Medline].
|
| 25.
|
Persson, B., and P. Argos.
1996.
Topology prediction of membrane proteins.
Prot. Sci.
5:363-371[Abstract].
|
| 26.
|
Pfeifer, K.,
B. Arcangioli, and L. Guarente.
1987.
Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene.
Cell
49:9-18[CrossRef][Medline].
|
| 27.
|
Rubin-Bejerano, I.,
S. Mandel,
K. Robzyk, and Y. Kassir.
1996.
Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1.
Mol. Cell. Biol.
16:2518-2526[Abstract].
|
| 28.
|
Shianna, K. V.,
W. D. Dotson,
S. Tove, and L. W. Parks.
2001.
Identification of a UPC2 homolog in Saccharomyces cerevisiae and its involvement in aerobic sterol uptake.
J. Bacteriol.
183:830-834[Abstract/Free Full Text].
|
| 29.
|
Sikorski, R. S., and P. Hieter.
1989.
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122:19-27[Abstract/Free Full Text].
|
| 30.
|
Soustre, I.,
P. H. Dupuy,
S. Silve,
F. Karst, and G. Loison.
2000.
Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae.
FEBS Lett.
470:102-106[CrossRef][Medline].
|
| 31.
|
Todd, R. B., and A. Andrianopoulos.
1997.
Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif.
Fungal Genet. Biol.
21:388-405[CrossRef][Medline].
|
| 32.
|
Tusnády, G. E., and I. Simon.
1998.
Principles governing amino acid composition of integral membrane proteins: applications to topology prediction.
J. Mol. Biol.
283:489-506[CrossRef][Medline].
|
| 33.
|
Xie, K.,
E. J. Lambie, and M. Snyder.
1993.
Nuclear dot antigens may specify transcriptional domains in the nucleus.
Mol. Cell. Biol.
13:6170-6179[Abstract/Free Full Text].
|
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Abstract
Introduction
