Regulation of Phosphatidylglycerophosphate Synthase by Inositol in Saccharomyces cerevisiae Is Not at the Level of PGS1 mRNA Abundance*

Phosphatidylglycerophosphate synthase catalyzes the committed step in the synthesis of the mitochondrial phospholipid cardiolipin. We showed previously that phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae is increased in conditions favoring mitochondrial development and during growth in the absence of inositol. Interestingly, the regulatory effects of inositol were not altered in ino2, ino4, or opi1 mutants suggesting that regulation in response to inositol is not at the level of gene transcription. We report here that steady state mRNA levels of the PGS1 gene, which encodes phosphatidylglycerophosphate synthase, were not altered by inositol or choline. Growth in the presence of the inositol-depleting drug valproate led to an increase in phosphatidylglycerophosphate synthase activity unaccompanied by increased PGS1 mRNA. PGS1 mRNA abundance was not decreased in ino2 or ino4 mutants and was unaffected in an opi1 mutant. Therefore, regulation of phosphatidylglycerophosphate synthase by inositol is not mediated at the level of mRNA abundance and does not require the INO2-INO4-OPI1 regulatory circuit. PGS1 was increased in glycerol/ethanol compared with glucose media and was maximally expressed as cells entered the stationary phase. Deletion of the mitochondrial genome did not affect PGS1 expression. Thus, whereas inositol controls phosphatidylglycerophosphate synthase activity, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.

Cardiolipin (CL), 1 a unique phospholipid with dimeric structure, is ubiquitous in eukaryotes and predominantly found in the mitochondrial inner membrane (1). It plays a role in mitochondrial bioenergetics by optimizing activities of enzymes in the oxidative phosphorylation pathway, including complexes I, III, IV, and V (2)(3)(4)(5), and the ADP-ATP carrier (6 -8). It is also involved in mitochondrial biogenesis possibly via assisting protein import into mitochondria (8) and maintaining optimal mitochondrial internal structure (9). As a reflection of the importance of CL, its synthesis is highly regulated in response to various factors (1). Fully understanding the regulation of CL biosynthesis will provide important insight into the function of CL in cellular processes.
Phospholipid biosynthetic pathways have been extensively characterized in Saccharomyces cerevisiae. Phospholipid synthesis proceeds via a three-branched pathway from the central intermediate CDP-DAG. A general regulation pattern has been observed for most structural genes encoding the enzymes in the PC and inositol biosynthetic pathways including INO1, CHO1, PSD1, CHO2, and OPI3 (10,11). The central feature of this regulation is repression by inositol and choline. A consensus sequence inositol choline-responsive element, also termed UAS INO , was found in the promoter regions of genes coordinately regulated by inositol. In the absence of inositol, heterodimers of the positive transcriptional regulators Ino2p and Ino4p bind to the UAS INO to activate transcription. In the presence of inositol, the OPI1 gene product acts to repress transcription.
Transcriptional control of UAS INO -containing genes is uniquely affected by the growth phase. Expression is maximal during logarithmic growth and is reduced as cells enter the stationary phase (11). Repression of these genes in the stationary phase may be correlated with the repression mechanism involving the two major phospholipid precursors, inositol and choline, because both mechanisms of regulation share two common characteristics (12). First, they require a functional UAS INO sequence in the promoter region of targeted genes, which may serve as the cis-element for the activation of transcription. Second, ongoing PC synthesis is essential for the repression of gene expression.
The biosynthesis of CL is conserved in all eukaryotic organisms. It occurs via three enzymatic reactions (1). PGPS catalyzes the committed step of CL synthesis, formation of PGP from CDP-DAG and glycerol-3-P. PGP phosphatase dephosphorylates PGP to PG. CL synthase catalyzes the final step of CL synthesis, condensation of CDP-DAG and PG to form CL. In contrast to the phosphatidylinositol and PC branches, biosynthesis of CL occurs solely in the mitochondria. Therefore, regulation of CL synthesis may be expected to respond not only to cross-pathway control by inositol and choline but also to cues affecting mitochondrial development.
Like many enzymes in the phosphatidylinositol and PC branches, PGPS, the committed enzyme of CL synthesis, is subject to coordinate control by the phospholipid precursors inositol and choline (13). However, the mechanism of regulation at the level of gene expression has not been elucidated. Previous studies suggested that regulation of the CL pathway by inositol occurs via a mechanism that differs from that of the PC and inositol biosynthetic pathways. Activity of the first pathway enzyme, PGPS, is reduced ϳ70% when cells are grown in the presence of exogenous inositol in S. cerevisiae. However, enzyme activity is not affected in ino2, ino4, or opi1 mutants, suggesting that control by inositol is not mediated at the level of PGS1 transcription (13). Furthermore, in contrast to enzymes encoded by UAS INO -containing genes, PGPS activity increases 2-4-fold in the early stationary phase (14). These observations are consistent with the lack of a functional UAS INO sequence in the PGS1 promoter. Nevertheless, in an analysis of PGS1 expression using a plasmid-borne lacZ reporter construct under control of the putative PGS1 promoter, ␤-galactosidase activity was decreased 2-3-fold during growth in the presence of inositol. This regulation was attributed to a potential UAS INO element 284 bp 5Ј to the PGS1 open reading frame (15). However, this element has an UAS INO that has an A in place of a T in the critical third position, which was shown to be functionally inactive (16). It is likely that the reporter construct on a multicopy plasmid does not accurately reflect PGS1 regulation as it occurs in the genome.
Regulation of the CL biosynthetic pathway by inositol has been observed in higher eukaryotic organisms as well as yeast. Addition of inositol to lung microsomes inhibited PG synthesis up to 94% (17), and PG levels increased more than 10-fold during inositol starvation in two independently isolated inositol-auxotroph Chinese hamster ovary cell lines (18,19). However, the underlying mechanisms that mediate such regulation remain elusive.
Identification of the S. cerevisiae structural genes PGS1 (20,21) and CRD1 (22)(23)(24) coding for enzymes in the CL biosynthetic pathway enables the molecular analyses of the regulation of CL biosynthesis. To determine how PGS1 expression is regulated we measured PGS1 mRNA abundance directly. Our studies revealed that regulation of PGPS activity by inositol is not mediated at the level of mRNA abundance, and it does not require the INO2-INO4-OPI1 regulatory circuit. Rather, transcriptional regulation of PGS1 occurs primarily by derepression during the stationary growth phase when mitochondrial development is increased.

Materials
All chemicals used were reagent grade or better. [␣-32 P]UTP was purchased from PerkinElmer Life Sciences. CDP-DAG was obtained from Life Science Resources, and L-[2-3 H]glycerol 3-phosphate (20 Ci/ mmol) was purchased from American Radiolabeled Chemicals. The PCR was performed using the MasterTaq kit from Eppendorf. RT-PCR was performed using the Access RT-PCR system from Promega. The Wizard Plus Miniprep DNA purification system, the pGEM-T Easy Vector System, and the Riboprobe System kit were from Promega. All other buffers and enzymes were purchased from Sigma. Glucose, yeast extract, and peptone were purchased from Difco.
Disruption of PGS1-The S. cerevisiae PGS1 gene was replaced by TRP1 using a one-step gene replacement strategy in which 1220 bp from the center of the open reading frame beginning 263 bp after the start codon was replaced with 862 bp of the TRP1 gene. Transformants of diploid cells were selected on Trp Ϫ drop out synthetic medium. Dissection of tetrads produced haploid strains that contain only one copy of the disrupted PGS1 gene. Disruption of PGS1 was confirmed by PCR using primers against the TRP1 sequence (Trp5Ј, 5Ј-CG-CAAACTTTCACCAATGGA-3Ј) and the sequence 3Ј to the PGS1 coding sequence (PGS23193Ј, 5Ј-AGGGCATTTCCATTACTTCCA-3Ј).
Isolation of rho 0 Mutants-Isolation of ethidium bromide-induced rho 0 mutants was performed as described previously (25). Briefly, cells were grown to saturation in synthetic minimal medium with 2% glucose and 25 g/ml ethidium bromide (filter-sterilized). A second culture was inoculated from the first in the same medium and grown to saturation. Single colonies were streaked on YPD. Essentially all single colonies were rho 0 mutants. The resulting strains were crossed with a series of rho Ϫ tester strains 104, 105, 106, and 107. Failure to complement any of the tester strains confirmed the loss of mitochondrial genome.
Quantitative Reverse Transcriptase-PCR (RT-PCR)-Yeast strains were grown to the early stationary growth phase in synthetic medium FIG. 1. Identification of PGS1 mRNA. The pgs1⌬ (QZY4) and isogenic wild-type (FGY3) strains were grown in synthetic medium. Northern analysis (A) was performed with total RNA from cells harvested at the early stationary phase and hybridized with 32 P-labeled riboprobes specific for PGS1 and the internal standard gene TCM1 as described under "Experimental Procedures." RT-PCR (B) was performed with total RNA as described under "Experimental Procedures." with 2% sucrose. Total RNA was isolated by hot phenol extraction (26) and treated with DNase to avoid DNA contamination. RT-PCR was performed using the RT-PCR Access System from Promega. PGS1 mRNA was amplified using primers RTpgs15Ј (5Ј-AAGCGAGACT-GAGTTGGTGG-3Ј) and RTpgs13Ј (5Ј-ATGAGACGCCCTTTGATTGG-3Ј). The internal control ACT1 was amplified using primers RTact15Ј (5Ј-TGAGGTTGCTGGTTTGGTTA-3Ј) and RTact13Ј (5Ј-TTGTGGT-GAACGATAGATGGA-3Ј). Standard reactions used 30 cycles, 50 pmol of each primer, and 100 ng/l RNA in 50 l. RT-PCR products were separated by agarose electrophoresis. The intensity of DNA products was analyzed using Adobe Photoshop software. The products of PGS1 from each RNA preparation were normalized to ACT1. Northern Analysis-Cells were grown in minimal synthetic medium with either glucose or glycerol/ethanol as a carbon source. Inositol, choline, or valproate was added when indicated. Cells were harvested at the indicated times; RNA was isolated by hot phenol extraction (26) and fractionated on an agarose gel and then transferred to a nylon membrane. The blots were hybridized with 32 P-labeled PGS1 and INO1 riboprobes followed by a riboprobe for the constitutively expressed ribosomal protein gene TCM1 to normalize for loading variation. RNA probes for Northern analysis were synthesized using the Promega Riboprobe System and from plasmids linearized with restriction enzymes as follows (plasmid, restriction enzyme, RNA polymerase, respectively): pGEM-PGS1, ClaI, T7 (PGS1); pJH310, HindIII, T7 (INO1) (27); pAB309, EcoRI, SP6 (TCM1) (27); pPLG, BamHI (ACT1) (28). The results were quantitated by phosphorimaging.
Preparation of Mitochondrial Extracts-Mitochondrial extracts were prepared as described previously (13). Briefly, cells were harvested by centrifugation at 4°C and washed once with a buffer containing 50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 300 mM sucrose, and 10 mM ␤-mercaptoethanol (Buffer 1). Pellets were resuspended in Buffer 1 to a concentration of 1 g/ml (wet weight), and cells were broken by vortexing with glass beads for five 1-min intervals, with cooling of the cells on ice between intervals. Extracts were centrifuged in a Sorvall SS34 rotor at 5000 rpm for 5 min, and supernatants were pelleted by centrifugation for 10 min at 15,000 rpm in a Sorvall SS34 rotor. The mitochondrial pellets were washed twice with Buffer 1, suspended in 50 mM Tris-HCl (pH 7.5), 20% glucose, 10 mM ␤-mercaptoethanol (Buffer 2) to a concentration of 2.5 mg/ml (wet weight), and stored at Ϫ80°C.
Analysis of Protein Concentration and PGPS Enzyme Activity-Mitochondrial extracts were assayed for protein concentration by the method of Bradford with a protein assay kit from Bio-Rad using bovine serum albumin as the standard. PGPS activity was assayed at 30°C by quantitating the incorporation of 0.5 mM [2-3 H]glycerol 3-phosphate (4,000 cpm/nmol) into chloroform-soluble material as described previously (13). The reaction mixture contained 50 mM MES HCl (pH 7.0), 0.1 mM MnCl 2 , 0.2 mM CDP-DAG, 1 mM Triton X-100, and mitochondrial extract corresponding to 100 g of protein in a total volume of 0.1 ml. The reaction mixture was incubated at 30°C for 20 min immediately following the addition of labeled substrate. The reaction was stopped by adding 0.5 ml of 0.1 N HCl/methanol. Chloroform (1 ml) and 1 M MgCl 2 (1.5 ml) were added to each assay tube. The tubes were then vortexed and the phases were separated by brief centrifugation. Aliquots of 0.5 ml of the chloroform layer were transferred into scintillation vials and then dried under a nitrogen stream. Organic counting scintillant (3 ml) was added, and the radioactivity in each sample was determined with an LS-3801 liquid scintillation counter. Triplicate samples were assayed for each point, and a blank tube containing crude extract that was inactivated before the reaction was included as a control for each sample. Specific activities were defined as units/mg of protein, where 1 unit is the amount of enzyme that catalyzes the formation of 1 nmol of product/min at 30°C.

RESULTS
Identification of PGS1 Transcripts-The PGS1 transcript was identified by comparison of RNA from wild-type and pgs1⌬ strains. Total RNA from pgs1⌬ and isogenic wild-type strains was isolated and hybridized with a PGS1 riboprobe. A 1.7-kb PGS1 mRNA was observed only in RNA from the wild-type cells (Fig. 1A). RT-PCR analysis of total RNA isolated from the wild-type and pgs1⌬ mutant confirmed this result. As seen in Fig. 1B, PGS1 was amplified only from wild-type RNA.
PGS1 Expression Peaks in the Early Stationary Phase-Maximum activities of CDP-DAG synthase, PS synthase, and the phospholipid N-methyltransferases were found in the exponential growth phase in cells grown in complete synthetic medium (29). Transcription of the structural genes coding for those enzymes showed a similar expression pattern. In contrast, PGPS enzyme activity increased as cells enter the stationary phase (14). Consistent with enzyme activity maximal PGS1 mRNA levels were observed as cells entered the stationary phase (Fig. 2B) in contrast to the UAS INO -containing gene INO1, which was fully derepressed in the logarithmic phase and decreased as cells entered the stationary phase (Fig. 2B).
PGS1 mRNA Abundance Is Not Altered by Inositol and Choline-As discussed above, PGPS enzyme activity is decreased in the presence of inositol. To determine whether the effect of inositol is because of transcriptional regulation, the effect of inositol on the steady state level of PGS1 mRNA was examined in cells grown to the early stationary growth phase. PGS1 mRNA abundance was not altered in cells growing in the presence of exogenous inositol or choline (Fig. 3A). In contrast, expression of INO1, which is known to be repressed by inositol, was reduced more than 10-fold (Fig. 3B). (INO1 expression was measured in the logarithmic phase, which is the time of maximal expression of this gene (see Fig. 2).) We showed previously that PGPS activity was decreased within minutes after inositol supplementation (13). We looked at PGS1 expression as a function of time after addition of inositol. PGS1 mRNA levels did not decrease after the addition of inositol, as seen in Fig. 4A, although PGPS enzyme activity was significantly decreased within 60 min following supplementation with inositol (Fig.  4C). In contrast, expression of INO1 was fully repressed within 30 min following the addition of inositol (Fig. 4B).
Effect of Valproate-induced Inositol Depletion on PGS1 Expression-VPA is an 8-carbon branched fatty acid used in the treatment of bipolar disorder. VPA causes a decrease in intracellular inositol mass leading to a dramatic increase in expression of INO1 in S. cerevisiae (30). To examine the effect of inositol depletion on PGS1 expression PGS1 mRNA abundance was measured in cells grown in the presence of VPA. As shown in Fig. 5A, VPA did not lead to an increase in PGS1 mRNA levels under conditions that led to more than 10-fold increase in INO1 mRNA (Fig. 5B). Interestingly, the rate of synthesis of PG was increased up to 3-fold in the presence of VPA. 2 Consistent with this observation, cells grown in the presence of VPA exhibited a 2-fold increase in PGPS activity (Fig. 5C). Therefore, inositol depletion by VPA led to an increase in PGPS  (10). The ino2 and ino4 mutants require exogenous inositol for growth because these mutants fail to express the INO1 gene product required for de novo inositol synthesis (31). In contrast, wild-type levels of PGPS activity were found in ino2 and ino4 mutants suggesting that INO2 and INO4 are not required for derepression of PGPS (13). To address this possibility, PGS1 expression was characterized in ino2 and ino4 mutants. PGS1 expression was observed at derepressed levels in both mutants in all conditions (Fig. 6A) in contrast to the INO1 gene, which was barely detectable in these mutants (Fig. 6B). Interestingly, PGS1 expression was increased 2-3-fold in the ino4 mutant, which suggests a possible negative regulatory role of the INO4 gene product in transcription of PGS1.
The Steady State Level of PGS1 mRNA Is Not Altered in the opi1 Mutant-Disruption of the negative regulatory gene OPI1 results in constitutive overexpression of UAS INO -containing genes (10). The opi1 mutant excretes inositol (32) because of overexpression of the INO1 gene even in inositol-free medium. PGPS activity, however, was reduced in the opi1 mutant (13) suggesting that OPI1 does not cause repression of PGS1 expression. PGS1 mRNA abundance was measured in the opi1 mutant. As seen in Fig. 7A, wild-type levels of PGS1 were observed in the opi1 mutant in the absence of inositol. In contrast, INO1 levels were more than 20-fold higher in the opi1 mutant than in the wild type in inositol-free medium (Fig. 7B). These findings and those in Fig. 6 demonstrate that transcrip-tional control of PGS1 is not mediated by the INO2-INO4-OPI1 regulatory circuit.
Effect of Mitochondrial Development on PGS1 mRNA Abundance-PGPS activity is regulated by factors affecting mitochondrial development, such as growth phase, carbon source, and mitochondrial genome (14). Only 30 -70% of wild-type levels of PGPS activity are present in mutants lacking mitochondrial DNA (rho 0 mutants) compared with isogenic rho ϩ strains. To determine whether decreased PGPS activity in rho 0 mutants can be explained by decreased PGS1 mRNA, mRNA abundance was compared in rho 0 and rho ϩ cells that were isogenic with respect to nuclear DNA. In synthetic glucose medium, PGS1 mRNA was fully derepressed in the rho 0 mutant (Fig. 8B). Therefore, the decrease in PGPS activity in the rho 0 mutant is not because of decreased PGS1 expression.
PGPS activity is increased 2-3-fold in non-fermentable versus fermentable medium (14). PGS1 mRNA levels were measured in wild-type cells grown in the presence of glucose or glycerol/ethanol as the sole carbon source (Fig. 9B). In the mid-logarithmic growth phase, PGS1 mRNA levels were about 2-fold higher in glycerol/ethanol. However, glucose-grown cells exhibited an increase in PGS1 as cells entered the stationary phase, at which point PGS1 mRNA abundance was higher in glucose than in glycerol/ethanol. DISCUSSION In this report, we used Northern analysis to look directly at the mRNA abundance of the PGS1 gene in response to regulatory stimuli and concluded the following points. Regulation of PGPS activity by inositol does not occur at the level of mRNA abundance and is not mediated by the INO2-INO4-OPI1 genetic regulatory circuit. Therefore, cross-pathway control of general and mitochondria-specific phospholipid pathways oc- curs by different regulatory mechanisms. In glucose, PGS1 expression increases during the stationary growth phase. In the logarithmic phase, the increase in PGPS in glycerol/ethanol versus glucose is accompanied by an increase in PGS1 mRNA. However, the decrease in PGPS activity in the absence of a mitochondrial genome is not accompanied by a decrease in PGS1 mRNA. Therefore, PGS1 expression is controlled by the mitochondrial development factors including growth phase and carbon source. Loss of mitochondrial genome, which does lead to decreased PGPS activity, is unaccompanied by altered PGS1 expression.
Our finding that regulation of PGPS by inositol is not via transcriptional control of PGS1 by INO2-INO4-OPI1 is consistent with the results of a previous study in which PGPS enzyme activity was measured in response to inositol supplementation (13). Specifically, that PGS1 expression is fully derepressed in ino2 and ino4 null mutants (Fig. 6) is in accord with the finding that PGPS enzyme activity was not altered in these mutants. Similarly, mutation in the OPI1 gene did not lead to increased activity of PGPS (13) nor did it affect regulation of PGS1 (Fig.  7). Furthermore, inositol depletion induced by the drug VPA stimulated synthesis of PG without an accompanying increase in PGS1 mRNA abundance (Fig. 5). These results are also predicted by the lack of a functional UAS INO element in the promoter region of PGS1 (16). Finally, the increased expression of PGS1 in the stationary phase (Fig. 2) differs from the characteristic increase in expression of UAS INO -containing genes during the logarithmic phase of growth. Our findings are not in agreement with results obtained from a study in which PGS1 promoter sequences fused to a reporter construct on an exogenous plasmid appeared to be regulated by inositol (15). In that study, activity of the reporter gene was correlated with expression of native PGS1 mRNA even though it is clear that promoter-driven reporter constructs do not necessarily reflect the regulatory control of a gene in its natural chromosomal context (33). Regulatory sequences may not be present in the construct, and the effects of histone modification and chromatin structure are not necessarily observed with an extrachromosomal reporter. Therefore, it is unlikely that the reporter construct reflected regulation of native PGS1.
PGPS activity responds to mitochondrial development factors such as loss of the mitochondrial genome, carbon source, and growth phase. Loss of the mitochondrial genome did not affect PGS1 expression (Fig. 8), whereas carbon source and growth phase did (Figs. 2 and 9). In this regard, regulation of PGS1 is in concert with expression of the CRD1 gene encoding CL synthase, expression of which increases 7-10-fold in the early stationary phase (34). Therefore, in contrast to the other phospholipids, synthesis of the mitochondrial lipid CL increases during the stationary phase during growth in glucose, a time in which mitochondrial development increases to respond to the switch from fermentative to respiratory growth. In fact, the relative CL composition doubles as cells enter the stationary phase (35,36). CL may play an important role in cell survival during the stationary growth phase, as both pgs1 and crd1 null mutants lose viability during prolonged liquid culture at elevated temperature (19). 3 How PGPS activity is regulated by factors that do not effect PGS1 expression is not known. PGPS activity drops within 20 min following addition of exogenous inositol to the growth medium (13). The rapidity of this response could be attributed 3  to degradation or inactivation of the protein in the presence of inositol. In a cardiac cell line, stimulation of PGPS activity by n-acetylsphingosine is attenuated when cells are exposed to phosphatase inhibitors. In addition, 8-(4-chlorophenylthio)-cAMP(CPT-cAMP), a non-hydrolyzable cAMP analogue that activates cAMP-dependent protein kinase, stimulates PGPS activity (37). These findings suggest that phosphorylation may be involved in the regulation of mammalian PGPS. At least one enzyme in general phospholipid synthesis in S. cerevisiae, PS synthase, is regulated by phosphorylation. This enzyme, which catalyzes the committed step of PC synthesis, is inactivated by cAMP-dependent protein kinase-catalyzed phosphorylation (38). Phosphorylation of PGPS in S. cerevisiae has not been studied. Experiments to characterize the mechanism underlying PGPS regulation in response to inositol are in progress.
In addition to the regulation of enzyme activity, decreased synthesis of PG by inositol may also be because of a shift in the pool of CDP-DAG in response to inositol. When yeast cells are grown in the presence of exogenous inositol, the partitioning of CDP-DAG shifts toward the synthesis of phosphatidylinositol at the expense of PS (39). Like PS, the rate of synthesis of PG and CL in S. cerevisiae is significantly decreased in the presence of inositol. 4 In summary, although both the mitochondrial and general phospholipid pathways are subject to cross-pathway control by inositol and choline, we have shown here that regulation of the mitochondrial pathway does not involve the same genetic regulatory circuit as the general pathway enzymes. Whereas PGPS activity is decreased in the presence of inositol, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.