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(Received for publication, February 29, 1996, and in revised form, July 23, 1996)
From the The Saccharomyces cerevisiae PIS1
gene encodes phosphatidylinositol synthase. The amount of
phosphatidylinositol synthase is not affected by the presence of
inositol and choline in the growth medium. This is unusual because the
amounts and/or activities of other phospholipid biosynthetic enzymes
are affected by these precursors, and the promoter of the PIS1
gene contains a sequence resembling the regulatory element that
coordinates the inositol-mediated regulation (UASINO).
We found that transcription of the PIS1 gene was
insensitive to inositol and choline and did not require the putative
UASINO regulatory sequence or the cognate regulatory genes
(INO2 and OPI1).
The PIS1 promoter includes sequences (MCEs) that bind the
Mcm1 protein. Because the Mcm1 protein interacts with both the Sln1 and
the Gal11 regulatory proteins, we examined the effect of mutant alleles
of the MCM1 and SLN1 genes and carbon source on
expression of the PIS1 gene. We found that expression of
the PIS1 gene was reduced when cells were grown in a medium
containing glycerol and increased when grown in a medium containing
galactose relative to cells grown in a glucose medium. The
glycerol-mediated repression of PIS1 gene expression
required both the MCM1 gene and the MCEs, whereas the
SLN1 gene was required for full galactose-mediated
induction of a PIS1-lacZ reporter gene.
Thus, PIS1 gene expression is unique among the phospholipid
biosynthetic structural genes because it is uncoupled from the inositol
response and regulated in response to the carbon source. This is the
first example in yeast of a complete circuit linking a stimulus (carbon
source) to gene regulation (PIS1) using a two-component
regulator (SLN1).
How cells respond to environmental changes has been the subject of
intense investigation. In procaryotes, the two-component regulatory
system is a well defined and recurring mechanism. The two-component
regulatory systems include a sensor and a response regulator that
modulate gene expression in reaction to changes in the environment (1).
Even though two-component systems have been studied in procaryotes for
many years, they have only recently been identified in
Saccharomyces cerevisiae (2, 3, 4, 5). An example of this is the
yeast SLN1 gene product that includes both a sensor and a
response regulator (3). The Sln1p is known to modulate the activity of
the Mcm1 transcriptional regulatory protein (6, 7). Two questions that
need to be addressed are which environmental cues are sensed by this
two-component regulatory system, and which genes are targeted for a
response. Here, we report that regulation of PIS1 gene
expression by carbon sources involves the SLN1 and
MCM1 genes.
The major phospholipids in yeast membranes, phosphatidylinositol
(PI)1 and phosphatidylcholine, are
synthesized by two branches of the phospholipid biosynthetic pathway
that diverge from a common lipid precursor, CDP-diacylglycerol (8, 9, 10).
PI can be synthesized from glucose 6-phosphate, which is converted to
inositol by the soluble enzyme, inositol-1-phosphate synthase (IPS)
(Fig. 1A). Inositol and CDP-diacylglycerol are subsequently
converted to PI by the membrane-associated enzyme PI synthase (PIS)
(11, 12, 13). The yeast PIS1 gene (Fig. 1A) (11, 14,
15) was cloned by its ability to complement a pis mutant
that is conditionally auxotrophic for inositol (11). This
pis mutant strain requires high concentrations of inositol
(greater than 100 µM) for growth because of a lowered
affinity of the PIS enzyme for myo-inositol (16). Disruption
of the genomic PIS1 locus is lethal, establishing that it
encodes an essential function (14).
In large part, our knowledge of the regulation of phospholipid
biosynthetic gene expression has evolved from studies carried out on
the INO1 gene that encodes IPS (Fig. 1A). For
example, IPS activity, IPS subunit amount, and INO1
transcript levels are all reduced when yeast cells are grown in the
presence of inositol and choline (17, 18, 19). Conversely, in the absence
of inositol and choline, the INO1 gene is maximally
expressed (19). This pattern of regulation has also been documented for
other phospholipid biosynthetic genes (CHO1,
CHO2, OPI3, and CKI) that encode
enzymes of the phosphatidylcholine branch of the pathway (20, 21, 22, 23). The
response to inositol is coordinated by a common cis-acting
promoter sequence (5 The PIS enzyme is unique among the phospholipid biosynthetic enzymes
because its activity is not regulated in response to inositol and
choline (13). In addition, immunoblot studies show that the amount of
the 34,000-Da subunit of PIS is unaffected by inositol (13). This lack
of effect on PIS activity and subunit amount appears to be due to a
lack of regulation at the transcriptional level (23). These
observations are surprising because the PIS1 promoter does
contain a potential UASINO element that matches the
consensus UASINO element in 8 of 10 base pairs (see Fig.
1B).
In this study, we found that expression of the
PIS1-lacZ fusion gene was unresponsive to
inositol in a wild type strain and insensitive to ino2 and
opi1 mutations. Instead, expression of the PIS1
and PIS1-lacZ genes was regulated in response to different
carbon sources. This regulation required the MCM1 and
SLN1 genes and two Mcm1p-binding sites (MCE) present in the
PIS1 promoter.
The yeast strains
used in this study are listed in Table I. Yeast cultures
were grown at 30 °C in synthetic media (33) containing 2% glucose
(v/v) and either containing (I+C+) or lacking
(I
Yeast strains used in this study
-Galactosidase Assays
Promoter fusions to the lacZ reporter gene were created by inserting restriction fragments from the PIS1 promoter into YEp357R (34). Each construct used an EcoRI site to fuse the four amino-terminal amino acids from PIS1 (Fig. 1B) in-frame with the lacZ gene. Plasmid pMA109 was constructed using a 960-bp EcoRI-EcoRI restriction fragment from pPI514 (14). Similarly, pMA107 contained a 629-bp HindIII-EcoRI restriction fragment and pMA108 contained a 134-bp XhoI-EcoRI fragment (Fig. 1B). Plasmid pMA101 was generated for the purpose of synthesizing a PIS1 cRNA probe to be used in Northern and slot blot hybridizations. This plasmid was constructed by subcloning a 996-bp EcoRI fragment from pPI514, containing the entire PIS1 coding sequence (14), into pGEM1. RNA AnalysisRNA was isolated from yeast using a glass bead disruption and hot phenol extraction (35). RNA probes (cRNA) for Northern and slot hybridizations were synthesized using the Gemini II core system (Promega) from plasmids linearized with a restriction enzyme and transcribed with an RNA polymerase as follows (plasmid/restriction enzyme/RNA polymerase): pMA101/SalI/T7 (PIS1) and pPLg/BamHI/SP6 (ACT1) (kindly provided by C. Steber and R. E. Esposito, University of Chicago). Northern blot hybridizations were performed as described previously (19), and results were visualized by autoradiography and quantitated by densitometry. Slot blots were performed as described previously (19). However, a more sensitive slot blot procedure, involving formaldehyde denaturation, was used to assay the PIS1 transcript. For this modified slot blot procedure, total cellular RNA was dissolved in 100 µl of a denaturing solution (6% formaldehyde, 30 mM NaPO4, pH 6.8, 1 M NaCl) (36). The mixture was heated to 65 °C for 5 min prior to transferring to Nytran. The UASINO Element in the PIS1 Promoter Is Inactive The presence of a UASINO-like element in the
PIS1 promoter (Fig. 1B) suggested
that PIS1 transcription might be regulated by the cognate
regulatory genes, INO2 and OPI1 (8, 9, 10). To
examine this possibility, PIS1-lacZ expression was examined
using a fusion of 926 bp of the PIS1 promoter to
lacZ (pMA109). The wild type (BRS1001) and opi1
(BRS1021) mutant strains were grown in I Fig. 2. The PIS1 gene is not a member of the inositol-responsive regulon. A, the INO2 and the OPI1 regulatory genes are not required for regulation of PIS1 gene expression. PIS1-lacZ gene expression was assayed in a wild type strain (BRS1001) and strains containing ino2 (BRS2001) and opi1
(BRS1021) mutant alleles. B, deletion analysis of
the PIS1 promoter and schematic representation of
PIS1 promoter fusions to the lacZ reporter gene.
Noted are the positions of the potential UASINO element
(open box), MCEs (hash-marked box), and TATA box
(black box). Plasmids pMA109, pMA107, and pMA108 contained
960, 629, and 134 bp, respectively, of the PIS1 promoter
fused to lacZ. Units of -galactosidase were equal to
1000 × optical density at 420 nm/min/mg of total protein. Each
value represents the mean of three independent trials, and standard
deviations were less than 15% in all cases. Abbreviations:
I or I+, absence or presence of 75 µM inositol, respectively; C or
C+, absence or presence of 1 mM choline,
respectively.
[View Larger Version of this Image (13K GIF file)]
One possibility for the lack of effect of inositol on PIS1 expression is that the function of the UASINO element present in the PIS1 promoter depended on an unorthodox combination of inositol and choline. To address this, we examined the expression of several PIS1-lacZ fusions in response to various combinations of inositol and choline. Expression of lacZ driven by PIS1 promoter constructs, that included the putative UASINO element (pMA109 and pMA107), was unaffected by any combination of inositol and choline (Fig. 2B). Expression of the PIS1-lacZ gene from a deletion construct (pMA108) lacking the UASINO element was increased 2-fold when cells were grown in the presence of inositol and choline (Fig. 2B). These experiments suggest that the putative UASINO-like element present in PIS1 promoter does not function in the same capacity as the UASINO elements present in the promoters of the other phospholipid biosynthetic genes (8, 9, 10). Expression of the PIS1 Gene Is Regulated by Carbon SourceWe
examined if PIS1 gene expression was affected by growth in
media containing different carbon sources. This line of inquiry was
suggested by the observation that the PIS1 promoter binds
Mcm1p (38) and because Mcm1p is a target of the Gal11 regulatory
protein that is involved in carbon source regulation of gene expression
(39). Expression of the PIS1 gene was quantitated by slot
blot hybridization of total cellular RNA (normalized using an
ACT1-specific probe) from a wild type strain (BRS1001). RNA
was purified from this strain grown in complete synthetic media
containing 75 µM inositol and either 2% glucose, 2%
galactose, or 2% glycerol. The amount of the PIS1
transcript increased 51% when cells were grown in galactose and
reduced 53% when grown in glycerol relative to growth in a glucose
medium (Fig. 3).
Fig. 3. Analysis of PIS1 expression in response to carbon source. Transcription of the PIS1 gene was quantitated by slot blot hybridization with a PIS1-specific cRNA probe and normalized for loading variations by hybridization with an ACT1-specific cRNA probe. Wild type cells (BRS1001) were grown in complete synthetic media containing either 2% glucose, 2% galactose, or 2% glycerol. Each value represents the mean of a minimum of three independent trials. [View Larger Version of this Image (51K GIF file)]
MCM1 Regulates PIS1 Gene Expression Since the MCEs in the PIS1 promoter were known to bind Mcm1p (38), this raised the possibility that MCM1 may regulate PIS1 expression in response to the carbon source. To address this, we tested the effect of a temperature-sensitive mcm1 mutant allele on PIS1 gene expression. A strain harboring an mcm1ts mutant allele (C2-110L) (6) and an isogenic wild type strain (C2-2µMU) were grown at 30 °C in media containing either 2% glucose, 2% galactose, or 2% glycerol. Upon reaching midlog phase, half of each culture was maintained at 30 °C (permissive), while the other half was shifted to 37 °C (restrictive). The cultures were allowed to grow for an additional 4 h prior to isolating RNA. Expression of the PIS1 transcript was assayed by quantitative slot blot hybridization and normalized to the ACT1 transcript. In the C2-2µMU wild type strain, PIS1 transcript levels
were similar to those described for the BRS1001 wild type strain
(compare Figs. 3 and 4A). That is, growth in
galactose yielded a slight increase in expression, whereas growth in
glycerol yielded a significant decrease in expression. This pattern of
expression was conserved in the C2-110L strain
(mcm1ts) grown at 30 °C (Fig. 4B).
However, the shift to the restrictive temperature had a significant
impact on PIS1 transcription in the
mcm1ts strain (C2-110L) when grown in glycerol.
Under these conditions, there was a 110% increase in PIS1
transcription relative to cells grown at 30 °C (Fig. 4B).
It should be noted that there was substantially greater standard
deviation observed with the mcm1ts mutant strain
(C2-100L) relative to the wild type strain (C2-2 µMU). This increase
in standard deviation was due to differences between colonies. That is,
the pattern of expression in the three growth conditions (Fig.
4B) was identical for each colony.
Fig. 4. Effect of an mcm1ts mutant allele on PIS1 gene expression. Transcription of the PIS1 gene was quantitated by slot blot hybridization with a PIS1-specific cRNA probe and normalized for loading variations by hybridization with an ACT1-specific cRNA probe. Wild type cells (C2-2 µMU, A) and mcm1ts mutant cells (C2-110L, B) were grown in complete synthetic media containing either 2% glucose (striped bars), 2% galactose (solid bars), or 2% glycerol (stippled bars) at either 30 or 37 °C. Each value represents the mean of three independent trials. [View Larger Version of this Image (21K GIF file)]
SLN1 Regulates PIS1 Gene Expression SLN1 is a
two-component regulator (2, 3, 4) that modulates Mcm1p activity (7). To
determine if expression of the PIS1 gene is a downstream
target in the SLN1 regulatory pathway, we examined the
effect of an sln1 mutant allele on native PIS1
transcription. Expression of the PIS1 gene was quantitated
from wild type (JF819) and sln1 mutant (JF1359) strains
grown in media containing inositol and either 2% glucose, 2%
galactose, or 2% glycerol (Fig. 5). Transcription of
the PIS1 gene was reduced 34% in an sln1 mutant
strain (JF1359) relative to the wild type strain (JF819) when cells
were grown in the glucose medium (Fig. 5). There was also a modest
increase in the level of PIS1 expression in the
sln1 mutant strain when cells were grown in the galactose
medium, but there was no effect when grown in the glycerol medium (Fig.
5).
Fig. 5. Native PIS1 gene expression in an sln1 mutant strain. Transcription of the PIS1 gene was quantitated by slot blot hybridization with a PIS1-specific cRNA probe and normalized for loading variations by hybridization with an ACT1-specific cRNA probe. A wild type strain (JF819) (striped bar) and an sln1 mutant strain (JF1359) (solid bar) were grown in media containing either 2% glucose, 2% galactose, or 2% glycerol. Each value represents the mean of a minimum of three independent trials. [View Larger Version of this Image (37K GIF file)]
We also examined the effect of the sln1 mutant allele on
expression of the PIS1-lacZ fusions (Fig. 6).
The full-length promoter fusion (pMA109), in the wild type strain
(JF819), was expressed at high amounts in the glucose and galactose
media (99 and 235 units, respectively) and reduced to 44 units of
activity in the glycerol medium. In the sln1 mutant strain
(JF1359), expression from the full-length promoter fusion (pMA109) was
decreased 44% when grown in the glucose medium. This result is
consistent with the effect of the sln1 mutant allele on
expression of the native PIS1 gene (Fig. 5). Analysis of the
pMA107 construct, which lacks 331 bp relative to pMA109, yielded an
interesting result. There was a 57% decrease in expression in the
sln1 mutant strain (JF1350) relative to the wild type strain
when cells were grown in the galactose medium (Fig. 6A).
This suggests that the SLN1 gene product is a positive
regulator of PIS1 gene expression in response to galactose.
Deletion of the two MCEs (pMA108) resulted in much higher levels of
expression of the PIS1-lacZ fusion in all three carbon
sources (Fig. 6C). There was an approximately 5-fold
increase in expression when pMA108 transformants were grown in either
glucose or galactose media relative to pMA109 transformants. However,
the sln1 mutation had even more dramatic effect on
lacZ expression from pMA108 (28-fold) when cells were grown
in glycerol medium (Fig. 6C). These results suggest that the
region that contains the MCEs (HindIII to XhoI)
must include a general repressor element as well as a glycerol-specific
repressor element.
Fig. 6. Effect of an sln1 mutation on expression of a PIS1-lacZ fusion. Effect of an sln1 mutation on lacZ expression from pMA109 (A), pMA107 (B), and pMA108 (C). An sln1 mutant strain (JF1359) (solid bars) was transformed with each of the three plasmids (shown in Fig. 2B) and assayed for -galactosidase activity. Activity in
the sln1 mutant strain was compared with that of a wild type
strain (JF819) (striped bars). Cells were grown in media
containing inositol and either 2% glucose, 2% galactose, or 2%
glycerol. Each value represents the mean of a minimum of three
independent trials. Units of -galactosidase are equal to 1000 × optical density at 420 nm/min/mg of total protein.
[View Larger Version of this Image (21K GIF file)]
We report an analysis of the regulation of PIS1 gene expression. The initial experiments examined the function of a potential UASINO element in the PIS1 promoter. The data showed that the UASINO element present in the PIS1 promoter was not functional in its native context (Fig. 2). Expression of the PIS1-lacZ reporter gene (Fig. 2) was unresponsive to inositol and choline supplementation and unaffected by mutations in the INO2 and OPI1 regulatory genes (Fig. 2). It was necessary to examine if PIS1 expression was affected by the ino2 and opi1 mutants because of recent reports of UAS elements that utilize some of these regulatory genes to bring about constitutive gene expression (40, 41, 42). Thus, PIS1 is the only phospholipid biosynthetic gene whose expression is not co-regulated with that of the other genes in the pathway (8, 9, 10). The presence of a nonfunctional UASINO element in the
PIS1 promoter is curious. However, the lack of regulation of
PIS1 gene expression in response to inositol is logical
since the product of the PIS1 gene utilizes inositol as a
substrate (13). The absence of UASINO function may be
explained by the two base deviations relative to the consensus sequence
(5 The most novel finding was that PIS1 gene expression was responsive to the carbon source. The PIS1 gene is the first phospholipid biosynthetic gene shown to be subject to regulation in response to different carbon sources. The particular pattern of regulation is also unusual. For most yeast genes subject to carbon source-mediated regulation, glucose is usually the repressing condition, and galactose is the inducing condition (43). However, in the case of the PIS1 gene, glycerol is the repressing condition (Fig. 3). The only other systems that have been described where gene expression is lowered by growing cells in a nonfermentable carbon source (i.e. glycerol, ethanol, or acetate) are the ribosomal protein genes (44) and genes encoding glycolytic enzymes (45, 46, 47, 48). However, the mechanism for regulation of glycolytic gene and ribosomal protein gene expressions is likely to be different from the mechanism controlling PIS1 gene expression. It has been shown that glycolytic gene expression and ribosomal protein gene expression are controlled by a UAS element that dictates glucose inducibility. The mechanism regulating PIS1 expression is likely to involve a repressor-mediated mechanism since removal of promoter sequences (such as the MCEs) yielded increased expression in the presence of glycerol rather than reduced expression in the presence of glucose (Fig. 6). Regulation of PIS1 expression by carbon sources may coordinate PI biosynthesis with macromolecular synthesis (e.g. ribosome assembly). This has been previously suggested by the fact that yeast cells unable to synthesize PI die (inositol-less death) unless macromolecular synthesis is halted (49). The data show that the MCM1 gene and MCEs are required for
the glycerol-mediated repression of PIS1 gene expression.
Expression of the native PIS1 gene was increased in the
mcm1ts mutant strain grown in glycerol (Fig.
4B). The amount of PIS1 gene expression in
glycerol-grown cells was slightly greater than the amount of expression
in either glucose- or galactose-grown cells. This pattern of expression
was also observed with a PIS1-lacZ fusion lacking the MCEs
(pMA107 in Fig. 6C). The ability of the MCM1 gene
product to function as a repressor has been described previously. It is
known that the Mcm1p functions to repress an a-specific gene
(STE2) in In addition to the MCM1-mediated glycerol repression, there
must also be a general repressor present between the HindIII
and XhoI sites. This is indicated by the fact that the
amount of The carbon source regulation was also affected in an sln1 mutant strain (Fig. 6B). The data suggest that Sln1p may be a positive regulator of PIS1 expression in response to galactose. It is curious that there was no effect of the sln1 mutation on galactose-mediated expression from pMA109 (Fig. 6A) or the native PIS1 gene (Fig. 5). This suggests that there may exist regulatory elements upstream of the HindIII site that interact with Sln1p. It is possible that Sln1p galactose-mediated regulation of native PIS1 transcription occurs only under specific growth conditions. The data also showed a slight decrease in expression of the native PIS1 gene when sln1 mutant cells were grown in glucose media (Fig. 5). However, this effect was observed for all of the promoter constructs in response to almost all carbon sources (Fig. 6). Thus the glucose-dependent effect of the sln1 mutation on PIS1 expression may be an indirect consequence of the mutation. The observation that SLN1 affected expression from the PIS1 promoter in pMA108 is nevertheless significant given that the SLN1 gene encodes a regulatory protein that resembles bacterial two-component regulators (3) and because Sln1p is a regulator of Mcm1p activity (7). Two-component regulators have been identified as a major mechanism by which bacterial cells can transduce an extracellular stimulus into a genetic response (1). However, there were no examples of a yeast two-component system with a recognized external stimulus and a target gene. Thus, the yeast PIS1 gene provides the first such example. * This work was supported in part by a grant from the Potts Foundation (to J. M. L.). 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. § Recipient of a Schmitt Dissertation Fellowship.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biochemistry, Program in Molecular Biology, Loyola
University of Chicago, 2160 S. First Ave., Maywood, IL 60153. Tel.:
708-216-8454; Fax: 708-216-8523.
1 The abbreviations used are: PI, phosphatidylinositol; PIS, PI synthase; IPS, inositol-1-phosphate synthase; bp, base pair(s). We thank John Jackson and Kelly Robinson for insightful discussions and an anonymous reviewer for helpful suggestions. We thank Miriam Greenberg for the suggestion to examine regulation in response to the carbon source and Elizabeth Grayhack for the observation that Mcm1 interacted with the PIS1 promoter. We also thank Jan Fassler and Bik-Kwoon Tye for providing strains and discussing results prior to publication and Camille Steber and Shelley Esposito for providing plasmid pPLg.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc. This article has been cited by other articles:
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