From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225
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ABSTRACT |
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The PGS1 gene of Saccharomyces
cerevisiae encodes phosphatidylglycerophosphate (PG-P) synthase.
PG-P synthase activity is regulated by factors affecting mitochondrial
development and through cross-pathway control by inositol. The
molecular mechanism of this regulation was examined by using a reporter
gene under control of the PGS1 gene promoter
(PPGS1-lacZ). Gene expression subject to carbon
source regulation was monitored both at steady-state level and during
the switch between different carbon sources. Cells grown in a
non-fermentable carbon source had -galactosidase levels 3-fold
higher than those grown in glucose. A shift from glucose to lactate
rapidly raised the level of gene expression, whereas a shift back to
glucose had the opposite effect. In either a pgs1 null
mutant or a rho mutant grown in glucose,
PPGS1-lacZ expression was 30-50% of the level in
wild type cells. Addition of inositol to the growth medium resulted in
a 2-3-fold reduction in gene expression in wild type cells. In
ino2 and ino4 mutants, gene expression was
greatly reduced and was not subject to inositol regulation consistent
with inositol repression being dependent on the INO2 and
INO4 regulatory genes. PPGS1-lacZ
expression was elevated in a cds1 null mutant in the
presence or absence of inositol, indicating that the capacity to
synthesize CDP-diacylglycerol affects gene expression. Lack of
cardiolipin synthesis (cls1 null mutant) had no effect on
reporter gene expression.
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INTRODUCTION |
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Biosynthesis of phosphatidylglycerol (PG)1 and cardiolipin (CL) from the precursor phosphatidic acid involves four sequential steps catalyzed by the enzymes CDP-diacylglycerol (CDP-DAG) synthase, phosphatidylglycerophosphate (PG-P) synthase, PG-P phosphatase, and CL synthase. The committed and rate-limiting step is catalyzed by PG-P synthase (1, 2), which is encoded by the PGS1 gene in Saccharomyces cerevisiae (3). PG and CL are primarily synthesized in the mitochondrial inner membrane and are limited in their distribution to the inner and outer membranes of the mitochondria in yeast (4-6). CL has been postulated to be important for the functioning of several mitochondrial enzymes and the import of proteins into the mitochondria (7-11). PG appears to substitute for potentially critical functions of CL, inasmuch as a 5-fold increase of PG levels can compensate for the loss of CL in mutants lacking CL synthase (cls1 null strains) (12-14), whereas absence of both PG and CL in mutants lacking PG-P synthase results in severe mitochondrial dysfunction (3, 15).
Previous genetic and biochemical studies have indicated that two sets of factors affect PG-P synthase activity: cross-pathway control by inositol and choline and factors affecting mitochondrial development such as carbon source, oxygen, and mutations in mitochondrial DNA (16, 17). Regulation by inositol is unique compared with its regulation of other phospholipid biosynthetic pathways in that a decrease in PG-P synthase activity is sudden and dramatic upon addition of inositol to the growth medium (16). It was speculated that this rapid effect could not be due to repression of gene transcription but, instead, is a result of inactivation and/or degradation of the PG-P synthase. In addition, PG-P synthase activity was found not subject to regulation by the INO2-INO4-OPI1 regulatory genes which are required for cross pathway regulation by inositol (16). The PGS1 gene (also known as the PEL1 gene) encoding PG-P synthase in yeast has been isolated and characterized (3, 18). Sequence analysis revealed that there is at least one putative UASINO element (upstream activating sequence responsive to inositol) 284 base pairs 5' to the putative PGS1 gene start codon, indicating that a common regulatory mechanism responsive to inositol may play a role in PGS1 gene expression (3).
Expression of mitochondrial-encoded genes and mitochondrial biosynthesis is mediated by cis-acting elements and regulatory proteins that respond to the carbon source supporting growth (19). Transcription levels of genes involved are rapidly adapted to changes in the available carbon source. Previous studies have shown there is correlation between mitochondrial development and cellular CL levels (4, 17, 20). Yeast cells growing in a non-fermentable carbon source have higher PG-P synthase activity and a higher CL content than cells grown in glucose. Cells growing in a medium with glucose as the carbon source have two phases of growth. During the fermentative growth phase when ethanol is accumulated at the expense of glucose, CL is maintained at a lower level. After glucose is completely consumed, yeast cells begin to use ethanol as a carbon source, and CL levels almost double within a short period of time (4). Carbon source mainly affects PG-P synthase activity, but not other enzymes in CL biosynthesis, such as PG-P phosphatase or CL synthase (17, 21, 22). The amount of PG-P synthase activity relative to mitochondrial protein is kept at the same level regardless of changes in growth conditions; thus, PG-P synthase activity is regulated coordinately with the amount of mitochondria per cell (2). However, the DNA sequence 5' to the PGS1 gene does not contain any consensus binding sites for regulatory factors involved in carbon source-dependent transcriptional regulation (19).
In this study, we focused on the molecular basis for the regulation of PGS1 expression by factors affecting mitochondrial development, by inositol, and by mutations in the other structural genes necessary for PG and CL synthesis. We demonstrate that PGS1 gene expression is regulated by factors affecting mitochondrial function, by inositol, and by the capacity of cells to synthesize CDP-DAG.
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EXPERIMENTAL PROCEDURES |
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Materials--
All chemicals were reagent grade or better.
o-Nitrophenyl -D-galactopyranoside was from
Sigma. Restriction endonucleases were from New England Biolabs.
Polymerase chain reaction SuperMix was a product of Life Technologies,
Inc. Oligonucleotides were prepared commercially by Genosys
Biotechnologies. YEP broth and synthetic media for yeast growth and
selection were from Bio 101, Inc. Yeast nitrogen base without amino
acids was from Difco. The BCA kit was from Pierce.
Strains, Media, and Growth Conditions-- Yeast strains used in this study are listed in Table I. Yeast cultures were grown at 30 °C in synthetic minimal media (18) containing either 2% glucose, 2% galactose, 2% sodium lactate, or 3% glycerol with 0.95% ethanol as the carbon source. Where indicated, 10 µM or 70 µM inositol was added to growth medium.
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Plasmid Construction-- Plasmid pSD70 contains the putative promoter of the yeast PGS1 gene fused to a lacZ reporter gene. It was created by replacing the EcoRI fragment of plasmid pMA109 (26) with the polymerase chain reaction-amplified region 5' to the PGS1 gene, which includes the putative UASINO element (3). Amplification of this region from yeast chromosomal DNA employed primer 1 (5'-GAAAGGAATTCTAGGTGATATTGC-3') and primer 2 (5'-GAGAATTCTGGAGCAAACGAGTCGTCAT-3'). They were designed according to the DNA sequence in the vicinity of the PGS1 gene (3). Primer 1 begins 331 base pairs 5' to the PGS1 start codon, and primer 2 ends at the 20th base pair in the PGS1 open reading frame. The final fusion plasmid includes a DNA fragment encoding the first 7 amino acids encoded by the PGS1 gene fused in frame with the lacZ gene. Plasmid pSD70 was introduced into yeast cells by transformation of CaCl2-treated cells (25).
Isolation of rho Mutants--
Isolation of
ethidium bromide-induced rho mutants was performed as
described previously (28). Yeast cells were plated on a synthetic
medium containing 10 µg/ml ethidium bromide and 2% glucose and grown
at 30 °C for 48 h. Colonies were screened for by their inability to
grow on a synthetic medium with 3% glycerol as the carbon source.
Although these cells were assumed to be rho mutants, the
only criterion used for their selection was their inability to grow on
a non-fermentable carbon source.
Preparation of Cell Extracts and -Galactosidase
Assay--
Preparation of cell extracts was modified from a previous
report (25). All procedures were carried out at 4 °C. Yeast cells were harvested by centrifugation, washed in 100 mM sodium
phosphate, pH 7.0, and resuspended in the same buffer. The cell
suspension was mixed with an equal volume of prechilled glass beads
(diameter 0.3 mm) and disrupted in a Mini-BeadbeaterTM (Biospec
Products) by four 1-min bursts at 2,800 rpm with a 2-min pause between
bursts. Glass beads and unbroken cells were removed by centrifugation at 1,500 × g for 10 min.
-Galactosidase assays were
modified from a previously described procedure (29). Briefly, 100 µl of cell extract was mixed with 700 µl of 100 mM sodium
phosphate and 200 µl of 2 mg/ml o-nitrophenyl
-D-galactopyranoside, and the mixture was incubated at
30 °C for 30 min to 2 h. The reaction was stopped by adding 1 ml of 1 M Na2CO3, and absorbance
was measured at 420 nm.
-Galactosidase activities reported are equal
to 380 × the optical density at 420 nm produced/min/mg of total
protein in cell extracts. Protein concentration in each cell extract
was determined using a BCA protein assay kit with bovine serum albumin as the standard.
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RESULTS |
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Regulation of PGS1 Gene Expression by Carbon Source--
Carbon
source affects CL content as a result of regulation of PG-P synthase
activity and mitochondrial development. Because PG-P synthase catalyzes
the rate-limiting step in CL biosynthesis, we examined whether carbon
source regulation was mediated via gene expression by monitoring
-galactosidase activity expressed from the
PPGS1-lacZ reporter gene. Because mitochondrial development is more affected in the stationary phase of growth than in
the exponential phase (30), and cellular PG-P synthase activity varies
depending on growth phase (17), we used mid-log phase cells in this
study.
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Effect of Mitochondrial Function on PGS1 Expression-- The pgs1 null mutant strain (YCD4) has severe defects in mitochondrial function (3, 18). In addition to its inability to utilize a non-fermentable carbon source for growth, this mutant also has a petite lethal phenotype (18), characterized by incompatibility with superimposition of extensive lesions in mitochondrial DNA such as in rho mutants. This strain along with a rho mutant served as experimental tools to study the impact of dysfunctional mitochondria on gene expression. The PPGS1-lacZ reporter gene was introduced into strain YCD4, and cells originally grown in glucose-containing medium were suspended in media with different carbon sources for the time indicated (Fig. 1). PPGS1-lacZ reporter gene expression in this mutant was only 30-40% of the wild type level when cells were in glucose-containing media. This activity was even lower when glucose was replaced with lactate or glycerol-ethanol (although no cell growth occurred in these latter carbon sources), suggesting net turnover of existing gene product in the absence of no new synthesis. The results were strikingly similar with that of gene expression in the rho mutant, which also cannot grow on non-fermentable carbon sources. The above result argues that functional mitochondria, and not simply carbon source, are a prerequisite for normal levels of PGS1 gene expression, which may be coordinately regulated with the expression of other mitochondrial proteins.
Regulation of PGS1 Expression by Inositol-- Inositol regulates the expression of enzymes in the major phospholipid biosynthetic pathways and has been shown to repress the level of PG-P synthase activity (1, 2); previous results (16) indicated that PG-P synthase may be regulated by inositol through a mechanism not related to the established modes of inositol regulation. However, sequence examination of the upstream region of the PGS1 gene does show the presence of an UASINO element that could serve as a cis-acting element in inositol-dependent regulation mediated by the INO2-INO4-OPI1 gene circuit. As shown in Fig. 3, PPGS1-lacZ gene expression was derepressed in inositol-free medium and was repressed by the presence of inositol. The degree of repression was not in parallel with inositol concentration, but was in agreement with the PG-P synthase functional assay results measured at steady state (16). The degree of repression was strain dependent with strain YPH102 (data not shown) being more sensitive to repression by 10 µM inositol than strain DL1.
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Expression of the PGS1 Gene in Mutants Affected in CL Biosynthesis-- In addition to PG-P synthase, CDP-DAG synthase (encoded by the cds1 gene) and CL synthase are also directly involved in CL biosynthesis in yeast (1, 2). Alteration of cellular CDP-DAG synthase activity in cds1 mutants affects several major phospholipid biosynthetic enzymes including PS synthase and PI synthase, which catalyze two immediate downstream reactions requiring CDP-DAG (27). Reduction in CDP-DAG synthase activity raises cellular PS synthase levels and causes constitutive expression of inositol-1-P synthase as a result of derepression of CHO1/PSS and INO1 gene expression, respectively (34, 35); this regulation is independent of the INO2 and OPI1 regulatory genes and therefore represents a novel mode of regulation of phospholipid biosynthetic genes independent of inositol (27). Reduction in PI synthase activity in response to low levels of CDP-DAG synthase appears to occur by posttranslational processes (27).
Inasmuch as PG-P synthase catalyzes another immediate step downstream of CDP-DAG synthesis (1, 2), we examined whether low CDP-DAG synthase activity would also affect PGS1 gene expression. The PPGS1-lacZ reporter gene was introduced into the cds1 null mutant bearing a plasmid expressing a human cDNA encoding a CDP-DAG synthase (36), which is sufficient to sustain cell growth if induced from the PGAL1 promoter, but does not correct the other phenotypes associated with low levels of CDP-DAG synthesis (27). PPGS1-lacZ expression was higher in the cds1 null mutant than in the wild type strain, and the degree of repression in the presence of inositol was not as severe in the mutant strain as in the wild type (Fig. 4). This result mimics the results observed for CHO1/PSS gene expression in a cds1 point mutant strain and in cds1 null strains expressing the human cDNA under the above conditions (34, 35). Therefore, PGS1 gene expression also responds to an inositol-independent regulation dependent on the capacity of cells to synthesize CDP-DAG.
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Conclusions-- Previous results had established that PG-P synthase activity in yeast was responsive to cross pathway regulation and mitochondrial function. The results presented here establish that the response to carbon source, mitochondrial function, and inositol as well to the catalytic capacity of the cell to synthesize CDP-DAG is at the level of regulation of PGS1 gene expression. Still unresolved is the mechanism by which inositol induces a rapid decline in PG-P synthase activity as compared with the slower response brought about by repression of gene expression. Future experiments will address the cis- and trans-acting elements that regulate PGS1 gene expression in response to carbon source and mitochondrial function.
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ACKNOWLEDGEMENTS |
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We thank John Lopes for providing plasmid pMA109 and Susan Henry for providing the ino2 and ino4 mutant strains.
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FOOTNOTES |
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* This work was supported in part by Grant GM54273 from the National Institutes of Health (to W. D.).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.
Current address: Mammalian Genetics Laboratory, ABL-Basic Research
Program, NCI-Frederick Cancer Research and Development Center,
Frederick, MD 21702.
§ To whom reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77225. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: wdowhan{at}bmb.med.uth.tmc.edu.
1 The abbreviations used are: PG, phosphatidylglycerol; CL, cardiolipin; PG-P, phosphatidylglycerophosphate; CDP-DAG, CDP-diacylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; UASINO, upstream activating sequence responsive to inositol; PPGS1-lacZ, promoter of PGS1 gene placed 5' to the lacZ open reading frame; PGAL1-hCDS, promoter of GAL1 gene placed 5' to the hCDS open reading frame.
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REFERENCES |
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