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Papers by George Carman

Research paper thumbnail of PAH1-encoded Phosphatidate Phosphatase Plays a Role in the Growth Phase- and Inositol-mediated Regulation of Lipid Synthesis in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of... more In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of growth occurs at the expense of the storage lipid triacylglycerol. As exponential phase cells progress into the stationary phase, the synthesis of triacylglycerol occurs at the expense of phospholipids. Early work indicates a role of the phosphatidate phosphatase (PAP) in this metabolism; the enzyme produces the diacylglycerol needed for the synthesis of triacylglycerol and simultaneously controls the level of phosphatidate for the synthesis of phospholipids. Four genes (APP1, DPP1, LPP1, and PAH1) encode PAP activity in yeast, and it has been unclear which gene is responsible for the synthesis of triacylglycerol throughout growth. An analysis of lipid synthesis and composition, as well as PAP activity in various PAP mutant strains, showed the essential role of PAH1 in triacylglycerol synthesis throughout growth. Pah1p is a phosphorylated enzyme whose in vivo function is dependent on its dephosphorylation by the Nem1p-Spo7p protein phosphatase complex. nem1Δ mutant cells exhibited defects in triacylglycerol synthesis and lipid metabolism that mirrored those imparted by the pah1Δ mutation, substantiating the importance of Pah1p dephosphorylation throughout growth. An analysis of cells bearing PPAH1-lacZ and PPAH1-DPP1 reporter genes showed that PAH1 expression was induced throughout growth and that the induction in the stationary phase was stimulated by inositol supplementation. A mutant analysis indicated that the Ino2p/Ino4p/Opi1p regulatory circuit and transcription factors Gis1p and Rph1p mediated this regulation.

Research paper thumbnail of Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C: IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION

Journal of Biological Chemistry, 2007

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purif... more Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coliexpressed CTP synthetase 1 as a substrate, protein kinase C activity was time-and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1 R398A -encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7⌬ ura8⌬ mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser 462 and Thr 455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser 462 and Thr 455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser 462 stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr 455 inhibits activity.

Research paper thumbnail of Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Journal of Biological Chemistry, 2006

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously sho... more Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.

Research paper thumbnail of Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion

Journal of Biological Chemistry, 2006

Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via... more Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The beta-galactosidase activity driven by the P(EKI1)-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of P(EKI1)-lacZ reporter gene activity in the zrt1deltazrt2delta mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1delta mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.

Research paper thumbnail of The Saccharomyces cerevisiae Lipin Homolog Is a Mg2+-dependent Phosphatidate Phosphatase Enzyme

Journal of Biological Chemistry, 2006

In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene tha... more In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg 2؉ -dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G. M. (1989) J. Biol. Chem. 264, 8641-8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg 2؉ -dependent PA phosphatase activity, whereas the pah1⌬ mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg 2؉ -dependent PA phosphatase enzyme and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA and had reduced amounts of diacylglycerol and its derivative triacylglycerol. The PAH1-encoded Mg 2؉dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg 2؉ -dependent PA phosphatase enzyme.

Research paper thumbnail of Regulation of the Saccharomyces cerevisiae CKI1-encoded Choline Kinase by Zinc Depletion

Journal of Biological Chemistry, 2008

In the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed st... more In the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine via the CDP-choline branch of the Kennedy pathway. Analysis of a P(CKI1)-lacZ reporter gene revealed that CKI1 expression was regulated by intracellular levels of the essential mineral zinc. Zinc depletion resulted in a concentration-dependent induction of CKI1 expression. This regulation was mediated by the zinc-sensing and zinc-inducible transcriptional activator Zap1p. A purified Zap1p probe interacted with two putative UAS(ZRE) sequences (ZRE1 and ZRE2) in the CKI1 promoter. Mutations of ZRE1 and ZRE2 to a nonconsensus UAS(ZRE) attenuated the induction of CKI1 expression in response to zinc depletion. A UAS(INO) element in the CKI1 promoter was responsible for stimulating CKI1 expression, but this element was not involved with the regulation by zinc depletion. The induction of CKI1 expression in zinc-depleted cells translated into increased choline kinase activity in vitro and in vivo, and an increase in phosphatidylcholine synthesis via the Kennedy pathway.

Research paper thumbnail of Phosphatidic Acid Plays a Central Role in the Transcriptional Regulation of Glycerophospholipid Synthesis in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2007

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Research paper thumbnail of Regulation of Phospholipid Biosynthesis in the Yeast Saccharomyces cerevisiae

Journal of Biological Chemistry, 1996

... Footnotes. ???* This work was supported by United States Public Health Service Grants GM-2814... more ... Footnotes. ???* This work was supported by United States Public Health Service Grants GM-28140, GM-35655, and GM-50679 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. ??? 1 The abbreviations used are: PC. ...

Research paper thumbnail of Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae

Journal of Biological Chemistry

Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of ... more Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of membrane phospholipids. Results: The expression of diacylglycerol kinase is induced by the Reb1p transcription factor, and the resulting activity increase is essential for the enzyme function in phospholipid synthesis. Conclusion: The Reb1p-mediated transcriptional activation regulates the expression of diacylglycerol kinase activity. Significance: Diacylglycerol kinase is regulated at the level of transcription.

Research paper thumbnail of Casein Kinase II Phosphorylation of the Yeast Phospholipid Synthesis Transcription Factor Opi1p

Journal of Biological Chemistry, 2006

The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevi... more The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UAS INO -containing genes (e.g., INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose binding protein (MBP)-Opi1p as a substrate, casein kinase II activity was dose-and time-dependent, and dependent on the concentrations of MBP-Opi1p (K m = 25μg/ml) and ATP (K m = 7 μM). Of three mutations (S10A, S38A, and S239A) in putative phosphorylation sites, only the S10A mutation affected Opi1p phosphorylation. That Ser 10 was a specific target of casein kinase II was confirmed by loss of a phosphopeptide in the S10A mutant protein. The S10A mutation did not affect phosphorylation of Opi1p by either protein kinase A or by protein kinase C. Likewise, phosphorylation of Opi1p by casein kinase II was not affected by mutations in protein kinase A (S31A and S251A) and protein kinase C (S26A) phosphorylation sites. Expression of the OPI1 S10A allele in an opi1δ mutant attenuated (2-fold) the repressive effect of Opi1p on INO1 expression, and this effect was only observed when cells were grown in the absence of inositol. These data supported the conclusion that casein kinase II phosphorylation at Ser 10 played a role in stimulating the repression of INO1 when Opi1p was not in its most active state (i.e., in inositoldeprived cells).

Research paper thumbnail of The Yeast Anaerobic Response Element AR1b Regulates Aerobic Antifungal Drug-dependent Sterol Gene Expression

Journal of Biological Chemistry, 2013

Research paper thumbnail of Phosphatidate phosphatase plays role in zinc-mediated regulation of phospholipid synthesis in yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated w... more In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated with mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G.-S. (2007) Biochim. Biophys. Acta 1771, 322-330; Eide, D.J. (2009) J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme), and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme), but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Research paper thumbnail of Purification and Characterization of Diacylglycerol Acyltransferase from the Lipid Body Fraction of an Oleaginous Fungus

Journal of Biochemistry, 1997

Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that cataly... more Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.

Research paper thumbnail of A hypomorphic mutation in Lpin1 induces progressively improving neuropathy and lipodystrophy in the rat

The Journal of biological chemistry, Jan 29, 2011

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critica... more The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular an...

Research paper thumbnail of Regulation of the DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol and growth phase. Inhibition of DGPP phosphatase activity by CDP-diacylglyceron and activation of phosphatidylserine synthase activity by DGPP

The Journal of biological chemistry, 2000

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) p... more The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in op...

Research paper thumbnail of Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

Genetics, 2012

Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces... more Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2-Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UAS INO -containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UAS INO -containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways.

Research paper thumbnail of Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Genes & Development, 2008

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from... more Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg 2+ -dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis.

Research paper thumbnail of Lipin-1γ isoform is a novel lipid droplet-associated protein highly expressed in the brain

FEBS Letters, 2011

Lipin-1 proteins are phosphatidic acid phosphatases catalyzing the conversion from phosphatidic a... more Lipin-1 proteins are phosphatidic acid phosphatases catalyzing the conversion from phosphatidic acid to diacylglycerol. Two alternative splicing isoforms, lipin-1α and -1β, are localized at different subcellular compartments. A third splicing isoform, lipin-1γ was recently cloned and its subcellular localization is unknown. Here, we demonstrate that lipin-1γ is localized to lipid droplets, an association mediated by a hydrophobic, lipin-1γ-specific domain. Additional expression of lipin-1γ altered lipid droplet morphology without affecting the triacylglycerol level. In human tissues, lipin-1γ is the main lipin-1 isoform expressed in normal human brain, suggesting a specialized role in regulating brain lipid metabolism.

Research paper thumbnail of Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Journal of Biological Chemistry, 1989

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast ... more Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

Research paper thumbnail of PAH1-encoded phosphatidate phosphatase activity plays an essential role in protecting yeast against fatty acid-induced toxicity

Chemistry and Physics of Lipids, 2011

Research paper thumbnail of PAH1-encoded Phosphatidate Phosphatase Plays a Role in the Growth Phase- and Inositol-mediated Regulation of Lipid Synthesis in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of... more In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of growth occurs at the expense of the storage lipid triacylglycerol. As exponential phase cells progress into the stationary phase, the synthesis of triacylglycerol occurs at the expense of phospholipids. Early work indicates a role of the phosphatidate phosphatase (PAP) in this metabolism; the enzyme produces the diacylglycerol needed for the synthesis of triacylglycerol and simultaneously controls the level of phosphatidate for the synthesis of phospholipids. Four genes (APP1, DPP1, LPP1, and PAH1) encode PAP activity in yeast, and it has been unclear which gene is responsible for the synthesis of triacylglycerol throughout growth. An analysis of lipid synthesis and composition, as well as PAP activity in various PAP mutant strains, showed the essential role of PAH1 in triacylglycerol synthesis throughout growth. Pah1p is a phosphorylated enzyme whose in vivo function is dependent on its dephosphorylation by the Nem1p-Spo7p protein phosphatase complex. nem1Δ mutant cells exhibited defects in triacylglycerol synthesis and lipid metabolism that mirrored those imparted by the pah1Δ mutation, substantiating the importance of Pah1p dephosphorylation throughout growth. An analysis of cells bearing PPAH1-lacZ and PPAH1-DPP1 reporter genes showed that PAH1 expression was induced throughout growth and that the induction in the stationary phase was stimulated by inositol supplementation. A mutant analysis indicated that the Ino2p/Ino4p/Opi1p regulatory circuit and transcription factors Gis1p and Rph1p mediated this regulation.

Research paper thumbnail of Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C: IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION

Journal of Biological Chemistry, 2007

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purif... more Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coliexpressed CTP synthetase 1 as a substrate, protein kinase C activity was time-and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1 R398A -encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7⌬ ura8⌬ mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser 462 and Thr 455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser 462 and Thr 455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser 462 stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr 455 inhibits activity.

Research paper thumbnail of Control of Phospholipid Synthesis by Phosphorylation of the Yeast Lipin Pah1p/Smp2p Mg2+-dependent Phosphatidate Phosphatase

Journal of Biological Chemistry, 2006

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously sho... more Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.

Research paper thumbnail of Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion

Journal of Biological Chemistry, 2006

Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via... more Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The beta-galactosidase activity driven by the P(EKI1)-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of P(EKI1)-lacZ reporter gene activity in the zrt1deltazrt2delta mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1delta mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.

Research paper thumbnail of The Saccharomyces cerevisiae Lipin Homolog Is a Mg2+-dependent Phosphatidate Phosphatase Enzyme

Journal of Biological Chemistry, 2006

In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene tha... more In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg 2؉ -dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G. M. (1989) J. Biol. Chem. 264, 8641-8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg 2؉ -dependent PA phosphatase activity, whereas the pah1⌬ mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg 2؉ -dependent PA phosphatase enzyme and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA and had reduced amounts of diacylglycerol and its derivative triacylglycerol. The PAH1-encoded Mg 2؉dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg 2؉ -dependent PA phosphatase enzyme.

Research paper thumbnail of Regulation of the Saccharomyces cerevisiae CKI1-encoded Choline Kinase by Zinc Depletion

Journal of Biological Chemistry, 2008

In the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed st... more In the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine via the CDP-choline branch of the Kennedy pathway. Analysis of a P(CKI1)-lacZ reporter gene revealed that CKI1 expression was regulated by intracellular levels of the essential mineral zinc. Zinc depletion resulted in a concentration-dependent induction of CKI1 expression. This regulation was mediated by the zinc-sensing and zinc-inducible transcriptional activator Zap1p. A purified Zap1p probe interacted with two putative UAS(ZRE) sequences (ZRE1 and ZRE2) in the CKI1 promoter. Mutations of ZRE1 and ZRE2 to a nonconsensus UAS(ZRE) attenuated the induction of CKI1 expression in response to zinc depletion. A UAS(INO) element in the CKI1 promoter was responsible for stimulating CKI1 expression, but this element was not involved with the regulation by zinc depletion. The induction of CKI1 expression in zinc-depleted cells translated into increased choline kinase activity in vitro and in vivo, and an increase in phosphatidylcholine synthesis via the Kennedy pathway.

Research paper thumbnail of Phosphatidic Acid Plays a Central Role in the Transcriptional Regulation of Glycerophospholipid Synthesis in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2007

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Research paper thumbnail of Regulation of Phospholipid Biosynthesis in the Yeast Saccharomyces cerevisiae

Journal of Biological Chemistry, 1996

... Footnotes. ???* This work was supported by United States Public Health Service Grants GM-2814... more ... Footnotes. ???* This work was supported by United States Public Health Service Grants GM-28140, GM-35655, and GM-50679 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. ??? 1 The abbreviations used are: PC. ...

Research paper thumbnail of Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae

Journal of Biological Chemistry

Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of ... more Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of membrane phospholipids. Results: The expression of diacylglycerol kinase is induced by the Reb1p transcription factor, and the resulting activity increase is essential for the enzyme function in phospholipid synthesis. Conclusion: The Reb1p-mediated transcriptional activation regulates the expression of diacylglycerol kinase activity. Significance: Diacylglycerol kinase is regulated at the level of transcription.

Research paper thumbnail of Casein Kinase II Phosphorylation of the Yeast Phospholipid Synthesis Transcription Factor Opi1p

Journal of Biological Chemistry, 2006

The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevi... more The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UAS INO -containing genes (e.g., INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose binding protein (MBP)-Opi1p as a substrate, casein kinase II activity was dose-and time-dependent, and dependent on the concentrations of MBP-Opi1p (K m = 25μg/ml) and ATP (K m = 7 μM). Of three mutations (S10A, S38A, and S239A) in putative phosphorylation sites, only the S10A mutation affected Opi1p phosphorylation. That Ser 10 was a specific target of casein kinase II was confirmed by loss of a phosphopeptide in the S10A mutant protein. The S10A mutation did not affect phosphorylation of Opi1p by either protein kinase A or by protein kinase C. Likewise, phosphorylation of Opi1p by casein kinase II was not affected by mutations in protein kinase A (S31A and S251A) and protein kinase C (S26A) phosphorylation sites. Expression of the OPI1 S10A allele in an opi1δ mutant attenuated (2-fold) the repressive effect of Opi1p on INO1 expression, and this effect was only observed when cells were grown in the absence of inositol. These data supported the conclusion that casein kinase II phosphorylation at Ser 10 played a role in stimulating the repression of INO1 when Opi1p was not in its most active state (i.e., in inositoldeprived cells).

Research paper thumbnail of The Yeast Anaerobic Response Element AR1b Regulates Aerobic Antifungal Drug-dependent Sterol Gene Expression

Journal of Biological Chemistry, 2013

Research paper thumbnail of Phosphatidate phosphatase plays role in zinc-mediated regulation of phospholipid synthesis in yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated w... more In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated with mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G.-S. (2007) Biochim. Biophys. Acta 1771, 322-330; Eide, D.J. (2009) J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme), and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme), but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Research paper thumbnail of Purification and Characterization of Diacylglycerol Acyltransferase from the Lipid Body Fraction of an Oleaginous Fungus

Journal of Biochemistry, 1997

Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that cataly... more Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.

Research paper thumbnail of A hypomorphic mutation in Lpin1 induces progressively improving neuropathy and lipodystrophy in the rat

The Journal of biological chemistry, Jan 29, 2011

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critica... more The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular an...

Research paper thumbnail of Regulation of the DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol and growth phase. Inhibition of DGPP phosphatase activity by CDP-diacylglyceron and activation of phosphatidylserine synthase activity by DGPP

The Journal of biological chemistry, 2000

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) p... more The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in op...

Research paper thumbnail of Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

Genetics, 2012

Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces... more Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2-Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UAS INO -containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UAS INO -containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways.

Research paper thumbnail of Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Genes & Development, 2008

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from... more Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg 2+ -dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis.

Research paper thumbnail of Lipin-1γ isoform is a novel lipid droplet-associated protein highly expressed in the brain

FEBS Letters, 2011

Lipin-1 proteins are phosphatidic acid phosphatases catalyzing the conversion from phosphatidic a... more Lipin-1 proteins are phosphatidic acid phosphatases catalyzing the conversion from phosphatidic acid to diacylglycerol. Two alternative splicing isoforms, lipin-1α and -1β, are localized at different subcellular compartments. A third splicing isoform, lipin-1γ was recently cloned and its subcellular localization is unknown. Here, we demonstrate that lipin-1γ is localized to lipid droplets, an association mediated by a hydrophobic, lipin-1γ-specific domain. Additional expression of lipin-1γ altered lipid droplet morphology without affecting the triacylglycerol level. In human tissues, lipin-1γ is the main lipin-1 isoform expressed in normal human brain, suggesting a specialized role in regulating brain lipid metabolism.

Research paper thumbnail of Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Journal of Biological Chemistry, 1989

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast ... more Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

Research paper thumbnail of PAH1-encoded phosphatidate phosphatase activity plays an essential role in protecting yeast against fatty acid-induced toxicity

Chemistry and Physics of Lipids, 2011