Phosphatidylglycerophosphate Synthase Research Articles

Expression in yeast of an Escherichia coli gene encoding a phospholipid biosynthetic enzyme

Cardiolipin (CL) is a structurally unique phospholipid having important functional roles in both prokaryotic and eukaryotic cells. The genes encoding CL biosynthetic enzymes have been identified and extensively studied in Escherichia coli, and manipulation of CL biosynthesis in this organism has elucidated a great deal about CL function in prokaryotes. In contrast, little is known about CL biosynthesis or its regulation in eukaryotic cells. We sought to determine whether we could utilize E. coli genes to manipulate expression of CL biosynthetic enzymes and CL content in yeast. The E. coli pgsA gene encodes phosphatidylglycerophosphate synthase (PGPS), catalyzing the first step in the CL biosynthetic pathway. We constructed plasmids with pgsA under the control of the yeast CUP1 promoter. Extracts of Saccharomyces cerevisiae cells transformed with this plasmid contained high levels of E. coli PGPS activity. However, when compared to cells transformed with a control plasmid, pgsA-transformed cells did not exhibit differences in phospholipid composition. The most likely explanation is that the in vitro activity of the E. coli pgsA product is not indicative of its activity in vivo, due to mislocalization of the enzyme and/or inaccessibility of the enzyme to the substrates. To our knowledge, this is the first demonstration of expression of a bacterial phospholipid biosynthetic enzyme in yeast.

Biosynthesis of Phosphatidylglycerol in Isolated Mitochondria of Etiolated Mung Bean (Vigna radiata L.) Seedlings.

Phosphatidylglycerophosphate synthase (sn-glycerol-3-phosphate:CDP-diacylglycerol phosphatidyltransferase) and phosphatidylglycerophosphate phosphatase were characterized in mung bean (Vigna radiata L.) mitochondria. The synthase has a rather broad pH optimum between 7 and 9, whereas the phosphatase has one of about 7. Both enzymic activities are stimulated by Triton X-100 and require divalent cations but differ in their cation specificities. The synthase shows apparent Km values of 9 and 3 [mu]M for sn-glycerol-3-phosphate and CDP-diacylglycerol, respectively. Phosphatidylglycerophosphate, in contrast to lysophosphatidic and phosphatidic acid, is effectively dephosphorylated by the phosphatase, which exhibits an apparent Km value of 12 [mu]M for its substrate. Each enzyme shows higher activities with the dipalmitoyl species of its substrate than with the dioleoyl species. These substrate specificities of both enzymes are predominantly based on differences in apparent Vmax values.

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.

Mitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant deficient in cardiolipin.

In our preceding paper, we reported that a temperature-sensitive Chinese hamster ovary cell mutant, PGS-S, with thermolabile phosphatidylglycerophosphate synthase was defective in the biogenesis of both phosphatidylglycerol and cardiolipin (CL) at a nonpermissive temperature (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913). To investigate the biological role of cardiolipin, we examined the structure and function of mitochondria in mutant PGS-S cells, since CL is primarily found in the mitochondrial membranes of eukaryotic cells. Under conditions where the formation of CL was impaired, this mutant had both morphological and functional mitochondrial abnormalities, manifested by more stringent temperature sensitivity for cell growth in glucose-deficient medium and by reduced ATP production, increased glycolysis, and reduced oxygen consumption in intact cells. Rotenone-sensitive NADH oxidase activity in cell extracts was also reduced in the mutant cultivated at a nonpermissive temperature, showing a defect(s) in the respiratory electron transport chain of mitochondria. Of the respiratory chain complexes, rotenone-sensitive NADH-ubiquinone reductase (Complex I) was most severely impaired in the mutant, whereas its activity was restored in a revertant of the mutant that had regained the ability to synthesize CL. These results suggest that CL plays a critical role in mitochondrial functions, at least in the respiratory electron transport chain.

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.

37] Phosphatidylglycerophosphate synthase from Escherichia coli

The phosphatidylglycerophosphate synthase of Escherichia coli (E.coli) is tightly associated with the cytoplasmic membrane. A similar activity has been reported to be associated with mitochondrial membranes from Saccharomyces cerevisiae and higher eukaryotic cells. Only the enzyme from E. coli has been purified to homogeneity and extensively studied. The enzyme catalyzes the displacement of deoxycytidine monophosphate ((d) CMP) from deoxycytidine diphosphate ((d)CDP)diacylglycerol by sn -glycero-3-phosphate in the presence of a nonionic detergent and magnesium ion. The activity is usually determined by following the incorporation of radiolabeled glycerophosphate into chloroform-soluble material. Genetic studies have established that the phosphatidylglycerophosphate synthase is the only enzyme catalyzing the committed step to phosphatidylglycerol synthesis and that this gene product is essential to cell growth.

25] Phosphatidylglycerophosphate phosphatase from Escherichia coli

This chapter discusses the synthesis of phosphatidylglycerophosphate phosphatase from Escherichia coli (E. coli). The synthesis of phosphatidylglycero[ 32 P]phosphate substrate requires phosphatidylglycerophosphate synthase from E. coli . Phosphatidylglycerophosphate phosphatase is determined by a modification of the method Phosphatidylglycero[ 32 P]phosphate is dried out of chloroform and suspended with vortexing to a final concentration of 0.2 mM in a 2-fold concentrated assay mixture consisting of 100 mM Tris-HCl (pH 7.4), 0.24% triton X-100, 2 mM dithiothreitol, and 2 mM ethylenediaminetetraacetic acid. The phosphatidylglycerophosphate phosphatase activity is responsible for reaction is associated with the membrane fraction of E. coli and appears to be a composite of the activities of at least three gene products; therefore, many properties of this activity are based on assays performed on this composite of enzymes. The activities catalyzing reactions are localized to the cell membrane fraction but have different distributions between the two membranes of the cell envelope.

Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation.

Alteration of the Phospholipid Composition of Escherichia coli through Genetic Manipulation

In order to study the function of individual phospholipids, we have constructed a strain of Escherichia coli in which the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin can be regulated. In this strain (HDL1001) the normal expression of the phosphatidylglycerophosphate synthase does not occur due to the presence of the pgsA30 allele (Heacock, P. N., and Dowhan, W. (1987) J. Biol. Chem. 262, 13044-13049). A second chromosomal copy of the pgsA gene is fused to the lacOP region in single copy within the lac operon. Strain HDL1001 is absolutely dependent for growth on an inducer of the lac operon. In addition, the level of the pgsA gene product, the content of the two major acidic phospholipids, and the growth rate are dependent on the level of inducer in the growth medium. Cells remain viable in the absence of inducer as evidenced by a rapid return to normal growth after the readdition of inducer. The growth rate and phospholipid composition are affected only after the level of phosphatidylglycerophosphate synthase drops below about 15% of normal levels; both phosphatidic acid and (d)CDP-diacylglycerol also begin to increase to significant levels. At the point of cell arrest the level of the major acidic phospholipids is reduced by about 90% of wild type levels.

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.

Regulation of the balanced synthesis of membrane phospholipids. Experimental test of models for regulation in Escherichia coli.

In Escherichia coli, highly effective regulation controls the balanced synthesis of membrane phospholipids, important for optimal growth. Regulation is such that normally about 70% of a common pool of cytosine liponucleotide precursor is utilized by phosphatidylserine synthase and eventually converted to phosphatidylethanolamine, while about 30% is utilized by the competing enzyme phosphatidylglycerophosphate synthase and converted to phosphatidylglycerol (25%) plus cardiolipin (5%). Although the ratio of phosphatidylglycerol to cardiolipin may vary with conditions of growth, the sum of these two lipids remains relatively constant at about 30% of the total. Alternative models, postulating coordinate regulation of the two competing enzymes, or independent feedback regulation are proposed. These models were tested in experiments in which phosphatidylglycerol was continuously removed from growing cells treated with arbutin (4-hydroxyphenyl-O-beta-D-glucoside), causing its conversion to arbutinphosphoglycerol (Bohin, J.-P., and Kennedy, E.P. (1984) J. Biol. Chem. 259, 8388-8393.) The synthesis of phosphatidylglycerol was increased by a factor of 7 in cells treated with arbutin, with only small changes in phospholipid composition and with no significant change in the level of phosphatidylglycerophosphate synthase. The synthesis of phosphatidylethanolamine was not significantly increased, decisively eliminating the model that requires coordinate regulation of phosphatidylserine synthase and phosphatidylglycerophosphate synthase, and supporting the model of independent feedback inhibition, sensitive to very small changes in composition of cellular phospholipids.

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.

Structure and expression of the gene locus encoding the phosphatidylglycerophosphate synthase of Escherichia coli.

This paper presents definitive results which establishes a direct gene-protein product relationship between the pgsA gene and the phosphatidylglycerophosphate synthase of Escherichia coli. The predicted protein sequence derived from the determined DNA sequence of pgsA is in close agreement with the amino acid composition and partially determined amino acid sequence of the purified enzyme. The purified synthase has the same apparent molecular mass as the gene product made by a plasmid-directed transcription-translation system. The plasmid-borne copy of the pgsA gene is also capable of expressing enzymatically active synthase in vitro. The DNA sequence analysis has established the exact linear relationship between the uvrC, pgsA, and glyW loci and revealed that these three genes are transcribed in the same direction. The terminal coding regions of these three genes also share common sequences with transcriptional regulatory elements for the adjacent genes.

Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis.

Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature. The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele [coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)] and the cls- allele (responsible for a defective cardiolipin synthase). The newly acquired mutations caused better growth at higher temperatures. One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase [glycerophosphate phosphatidyltransferase (EC 2.7.8.5)]. Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid. In media with low osmotic pressures, these cells grew more slowly than the wild-type cells. They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2. Formation of cardiolipin and phosphoglycerol derivatives of membrane-derived oligosaccharides was reduced in a pgsA3 mutant. E. coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered.

Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides.

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with 32Pi and L-[3H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.

Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis.

A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP-diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity.

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.

Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli.

The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector. The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo. The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E. coli. The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism. The isolated hybrid plasmids have been mapped by restriction endonuclease analysis. The presence and location of other genetic markers have also been established. The above data, along with analysis of deletion derivatives of these plasmids and subcloning of appropriate restriction fragments, have established the position of the pgsA locus on the hybrid plasmids. From this data, the position of the pgsA locus has been determined to le between flaI and uvrC on the E. coli genetic map.