Phosphatidylglycerophosphate Synthase Activity Research Articles

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.

Isolation of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylglycerophosphate (PGP) Synthase, Expression of Which Corrects the Mitochondrial Abnormalities of a PGP Synthase-defective Mutant of CHO-K1 Cells

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.

Regulation of Phosphatidylglycerophosphate Synthase Levels inSaccharomyces cerevisiae

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 beta-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.

The PEL1 Gene (Renamed PGS1) Encodes the Phosphatidylglycero-phosphate Synthase ofSaccharomyces cerevisiae

Phosphatidylglycerophosphate (PG-P) synthase catalyzes the synthesis of PG-P from CDP-diacylglycerol and sn-glycerol 3-phosphate and functions as the committed and rate-limiting step in the biosynthesis of cardiolipin (CL). In eukaryotic cells, CL is found predominantly in the inner mitochondrial membrane and is generally thought to be an essential component of many mitochondrial functions. We have determined that the PEL1 gene (now renamed PGS1), previously proposed to encode a second phosphatidylserine synthase of yeast (Janitor, M., Jarosch, E., Schweyen, R. J., and Subik, J. (1995) Yeast 13, 1223-1231), in fact encodes a PG-P synthase of Saccharomyces cerevisiae. Overexpression of the PGS1 gene product under the inducible GAL1 promoter resulted in a 14-fold increase in in vitro PG-P synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast did not lead to a loss of viability but did result in a dependence on a fermentable carbon source for growth, a temperature sensitivity for growth, and a petite lethal phenotype. The pgs1 null mutant exhibited no detectable in vitro PG-P synthase activity and no detectable CL or phosphatidylglycerol (PG); significant CL synthase activity was still present. The growth arrest phenotype and lack of PG-P synthase activity of a pgsA null allele of Escherichia coli was corrected by an N-terminal truncated derivative of the yeast PG-P synthase. These results unequivocally demonstrate that the PGS1 gene encodes the major PG-P synthase of yeast and that neither PG nor CL are absolutely essential for cell viability but may be important for normal mitochondrial function.

A somatic cell mutant defective in phosphatidylglycerophosphate synthase, with impaired phosphatidylglycerol and cardiolipin biosynthesis.

Phosphatidylglycerophosphate (PGP) synthase catalyzes a reaction involved in the synthesis of phosphatidylglycerol (PG), which serves as a metabolic precursor for cardiolipin (CL), found primarily in the mitochondrial membranes of eukaryotic cells. We isolated a Chinese hamster ovary cell mutant (designated PGS-S) with a specific lesion in PGP synthase by using an in situ enzymatic assay for the enzyme. This mutant was obtained by introducing a second mutation into mutant PGS-P that had been generated by first-step mutagenesis. The PGP synthase activities in cell extracts of mutant PGS-S grown at 33 and 40 degrees C were 14 and 1% of those in the wild type cells, respectively; in addition, PGP synthase in cell extracts of mutant PGS-S exhibited higher sensitivity to heat than that of the wild type. Mutant PGS-S also showed a temperature-dependent defect in the synthesis of PG and CL in vivo, together with temperature sensitivity for cell growth. A temperature-resistant revertant of mutant PGS-S simultaneously restored PGP synthase activity and the ability to synthesize PG and CL in vivo to nearly the same levels as those of mutant PGS-P. These results constitute genetic evidence that PGP synthase is responsible for PG synthesis and is essential for cell growth.

Regulation of Phospholipid Synthesis in Response to Nutritional Environments in Mitochondria ofSaccharomyces cerevisiae

Two phospholipid-biosynthetic enzymes of mitochondria, phosphatidylglycerophosphate synthase (PGPS) and phosphatidylserine decarboxylase (PSD), have been assayed in Saccharomyces cerevisiae under various culture conditions. D-Glucose, but not D-fructose, D-galactose, or nonfermentable lactate, repressed PGPS activity in wild-type cells, as well as in a rho0 mutant that lacked the mitochondrial DNA. On the contrary, PSD activity was enhanced specifically by culturing with D-glucose. myo-Inositol not only repressed PGPS and PSD but also altered divergently the activities of several mitochondrial enzymes. Novel mutants defective in PSD activity were isolated and shown to display the pet phenotypes, suggesting the presence of regulatory linkage between respiratory functions and PSD in mitochondria. These results indicate that D-glucose and myo-inositol play special roles by widely regulating the cellular functions to adapt to nutritional environments.

Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides

Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 microM for sn-glycerol-3-phosphate and 60 microM for CDP-diacylglycerol; the apparent Km of PS synthase for L-serine was near 165 microM. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10-50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase was distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromato-focusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C.

HOSPHATIDYLGLYCEROPHOSPHATE SYNTHASE FROM GERMINATING SOYBEANS

Phosphatidylglycerophosphate synthase (CDP-diacylglycerol: sn-glycerol-3-phosphate phosphatidyltransferase, EC 2.7.8.5) activity was identified from the crude mitochondrial fraction of germinating soybeans. The mitochondrial enzyme exhibited pH optima at 7.0 and 9.5. Phosphatidylglycerophosphate was the predominate product at pH 9.5 while phosphatidylglycerol was the predominate product at pH 7.0. At pH 9.5, maximum phosphatidylglycerophosp-hate synthase activity was dependent on manganese (0.5 mM), magnesium (10 mM), or cobalt (20 mM) ions and Triton X-100 (0.5 mM). The apparent Km values for CDP-diacylglycerol and glycerol-3-phos-phate were 0.1 mM and 0.17 mM, respectively. Activity was completely stable to temperatures up to 70°C and the energy of activation was 3.16 Kcallmole. Thioreactive agents slightly inhibited activity.

Detection of phospholipid biosynthetic enzyme activities in Saccharomyces cerevisiae by colony autoradiography

The colony autoradiography method ( C. R. H. Raetz (1975) Proc. Nat. Acad. Sci. USA 72, 2274–2278) was modified for the detection of CDP-diacylglycerol synthase, phosphatidylinositol synthase, phosphatidylserine synthase, and phosphatidylglycerophosphate synthase activities of Saccharomyces cerevisiae colonies on filter paper replica prints. Colonies were replica printed onto filter paper, permeabilized by air drying, and assayed for enzyme activities with labeled substrates. Autoradiograms of replica prints, following enzyme assays, showed dark halos indicating the enzymatic synthesis of labeled phospholipid products. The method was also used to detect a cho 1 mutant defective in phosphatidylserine synthase and a strain that overproduces phosphatidylserine synthase. The method should become a valuable tool in isolating yeast strains defective in phospholipid biosynthetic enzyme activities and strains with overproduced enzyme activities.

Phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol): sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) activity was characterized from the mitochondrial fraction of Saccharomyces cerevisiae. The pH optimum for the reaction was 7.0. Maximum activity was dependent on manganese (0.1 mM), magnesium (0.3 mM), or cobalt (1 mM) ions and the nonionic detergent Triton X-100 (1 mM). The apparent Km values for CDP-diacylglycerol and glycerol-3-phosphate were 33 and 27 microM, respectively. Optimal activity was at 30 degrees C with an energy of activation of 5.4 kcal/mol (1 cal = 4.1868 J). Phosphatidylglycerophosphate synthase activity was thermally labile above 40 degrees C. p-Chloromecuriphenylsulfonic acid, N-ethylmaleimide, and mercurous ions inhibited activity. Phosphatidylglycerophosphate synthase activity was partially solubilized from the mitochondrial fraction with 1% Triton X-100.