Progress 04/01/87 to 06/30/10
Outputs
OUTPUTS: This project has been funded by NIH grant GM28140 and supports NJ10141. The annual reports and publications have been listed under NJ10141. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The work carried out under this project has provided fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This project is funded by NIH grant GM28140 and supports NJ10141. The annual report and publications are listed under NJ10141. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project is funded by NIH grant GM28140 and supports NJ10141. The annual report and publications are listed under NJ10141. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The annual report and publications are listed under NJ10141.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The progress report and publications are listed under NJ10141.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The progress report and publications are listed under NJ10141.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The progress report and publications are listed under NJ10141
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The progress report and publications are listed under NJ10141
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
We showed that the LSB6 gene encodes a membrane-associated phosphatidylinositol 4-kinase. The enzyme, which was not essential for cell growth, could partially suppress the lethal phenotype of a mutant defective in the STT4-encoded phosphatidylinositol 4-kinase. The LSB6-encoded enzyme was localized to the plasma membrane and vacuolar membrane, and possessed enzymological properties similar to that of the membrane-associated 55-kDa phosphatidylinositol 4-kinase enzyme.
Impacts
The work carried out under this project provides fundamental information on the synthesis of membrane phospholipids and its impact on the growth and metabolism of yeast. This information will be applicable to plants and animals, and be useful for development of strategies for increased survival of crops/animals in times of stress and disease.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
This project is funded by NIH grant GM28140 and supports NJ10141. The progress report and publications are listed under NJ10141
Impacts
Stated under NJ10108
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
This project is funded by NIH grant GM28140 and supports NJ10108. The progress report and publications are listed under NJ10108
Impacts
Stated under NJ10108
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
We will continue studies on the characterization of phospholipid synthesis enzymes in yeast. The phospatase sequence motif in the DPP1-encoded lipid phoaphatase was essential for activity. The mammalian homolog of the lipid phosphatase served as an ecto-enzyme. The yeast lipid phospatase enzymes utilized isoprenoid phosphates as substrates. The enzyme substrate DGPP caused an inflammatory response in mouse macrophages. The regulation of yeast choline kinase by protein kinase A phosphorylation was examined and the ethanolamine kinase gene of yeast was cloned and characterized. The protein kinase A target site in CTP synthetase was identified and a dUTP-dependent CTP synthetase activity was characterized.
Impacts
Our studies on characterization of phospholipid synthesis in yeast advance the understanding of regulation of membrane phospholipids in eukaryotic organisms. The work is applicable to higher eukaryotes including mammals and plants. The may impact on controlling thr growth and metabolism of normal cells.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
Amino acid sequence information was obtained from pure yeast DGPP phosphatase and was used to identify the gene (DPP1)encoding the enzyme. Yeast DPP1 was isolated and expressed in yeast and in insect cells. The expressed protein possessed DGPP phosphatase and PA phosphatase activities. The gene was characterized and its encoded protein contained a novel phoaphatase sequence motif. A dpp1 null mutant was constructed and it lacked DGPP phosphatase and PA phosphatase activities. The LPP1 gene was identified based on its sequence homology to DPP1. LPP1 was cloned and expressed in yeast and in insect cells. An lpp1 null mutant was constructed and it had reduced DGPP phosphatase and PA phosphatase activities. Mutants defective in DPP1 and LPP1 accumulated DGPP and PA, and had reduced PI levels. The nucleotide-dependent tetramerization of yeast CTP synthetase was examined in detail. Enzyme tetramerization was dependent on UTP and ATP hydrolysis. Phosphorylation by protein
kinase A and by protein kinase C was required for the nucleotide-dependent tetramerization. A CTP synthetase mutant (E161K) was constructed and shown to be defective in CTP synthetase inhibition by CTP. Mutant cells exibited elevated levels of CTP and a misregulation of phospholipid synthesis. The yeast CKI-encoded choline kinase was expressed in insect cells and purified to homogeneity. Pure choline kinase was characterized with respect to its enzymological and kinetic properties.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/96 to 12/30/96
Outputs
The regulation of yeast PA phosphatase by phospholipids was examined. Negativelycharged phospholipids activated activity whereas zwitterionic phospholipids were slightly ingibitory. We discovered, purified and characterized a novel enzyme called DGPP phosphatase. Regulation of phospholipid metabolism in yeast was studied.
Impacts
(N/A)
Publications
- Wu, W.I., Y. Liu, B. Riedel, J.B. Wissing, A.S. Fischl and G,M. CARMAN. 1996. Purification and characterization of diacylglycerol prophosphate phosphatase from Saccharomyces cerevisiae. J. Biology Chem. 271: 1868-1876.
- Wu, W.I. and G.M. CARMAN. 1996. Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids. Biochemistry. 35: 3790-3796.
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Progress 01/01/95 to 12/30/95
Outputs
During the past project period the laboratory studied the regulation of profilinlocalization in yeast. We found that the metabolism of phosphatidylinositol regulated the translocation of profilin between the plasma membrane and the cytosol. We also examined the regulation of overalll lipid biosynthesis in yeast by the toxin fumonisin B1. This toxin inhibited sphingolipid biosynthesis resulting in accumulation of sphingoid bases. The sphingoid bases in turn inhibited phospholipid and neutral lipid synthesis. Findally, an examination of lipid signaling enzymes and surface dilution kinetics was performed.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Over the past project period we examined the regulation of phosphatidate phosphatase activity by nucleotides. We found that ATP and CTP were potent inhibitors of the enzyme. The mechanism of nucleotide inhibition did not involve an interaction of nucleotides with the enzyme or its substrate phosphatidate. Instead, the mechanism of inhyibtion was the chelation of the enzyme's cofactor magnesium ions. This mechanism of inhibition was shown to be relevant in vivo.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
This project is supported by an NIH grant. The regulation of yeast purified phosphatidate phosphatase activity by sphingoid bases was examined. The sphingoid bases sphingosine, phytosphingosine, and sphinganine inhibited activities in a dose-dependent manner. Detailed kinetic analyses of the enzyme showed that sphingoid bases wer parabolic competitive inhibitors. The physiological relevance of this inhibition was examined by analyzing free sphingoid bases in yeast cells.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
The regulation of the 45-kDa and 104-kDa forms of phosphatide phosphatase from yeast by phosphorylation was examined. The 45-k-Da form of the nezyme was phosphorylated by cAMP-dependent protein kinase which resulted in enzyme activation. The 104-kDa from of the enzyme was not regulated by phosphorylation. The phosphorylation of the 45-kDa enzyme in growing yeast cells was demonstrated. Methods for the purification of several phospholipid biosynthetic enzymes were published based on previous work from this project. GRADUATE STUDENTS= 0.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
A 45-kDa form of phosphatidate phosphatase was identified in yeast mitochondria.The enzyme was purified to near homogeneity and its enzymological and kinetic properties were characterized. The properties of the 45-kDa phosphatidate phosphatase were similar to the 104-kDa form of the enzyme previously purified from this laboratory. Using antibodies against the 45-kDa and 104-kDa forms of phosphatidate phosphatase, we found that the enzymes were regulated differentially by inositol. Inositol induced the 45-kDa enzyme but had no effect on the 104-kDa enzyme. Both enzymes were induced as yeast cells entered stationary phase of growth. The regulation of phosphatidylethanolamine methyltransferase.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
A kinetic analysis of purified yeast phosphatidate phosphatase was conducted using Trition X-100/phosphatidate mixed micelles. The enzyme followed surface dilution kinetics where activity was dependent on the bulk and surface concentrations of phosphatidate. Physical data indicated that the enzyme bound to the surface of the micelle prior to its binding to substrate and catalysis. The effect of cAMP on the regulation of phospholipid synthesis in yeast was examined. The addition of cAMP to yeast cells resulted in an increase in phosphatidylinositol at the expense of phosphatidylserine. This effect was the result of a down regulation of phosphatidylserine synthase by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol was not due to the phosphorylation of phosphatidylinositol synthase.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Membrane associated phosphatidate phosphatase was purified 9833 fold from yeast.the purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE-53, 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. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH7. The Km value for phosphatidate was 50 uM and the V max was 30 mmol/min/mg. The turnover number for the enzyme was 2.7 x 10 3 min -1 at pH 7 and 30C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Thiophosphatidate was chemically synthesized and tested for its effects on pure phosphatidat phosphatase and pure CDP diacylglycerol synthase. Thiophosphatidate was a potent inhibitor of phosphatidate
phosphatase in a competitive manner and exhibited an apparent Ki of 60 uM. In contrast, pure CDP diacylglycerol synthase was able to convert thiophosphatidate to CDP diacylglycerol. Calculation of the specificity constant (V max/Km) demonstrated that phosphatidate was the preferred substrate.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
A S. cerevisiae mutant (cdgl) was isolated on the basis of an inositol excretionphenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol activity in mutant cdgl was due to a reduced level of the CDP-diacylglycerol synthase Mr = 56,000 subunit rather than an alteration in the enzymological properties of the enzyme. The regulation of phosphatidate phosphatase activity was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of inositol to the growth medium of wild-type cells resulted in a 2-fold increase in phosphatidate phosphatase activity. The phosphatidate phosphatase acitivity associated with the mitochondrial, microsomal, and cytosolic fractions of the cell was regulated by inositol in the same manner. Cells supplemented
with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared to unsupplemented cells. Enzyme activity was not regulated in inositol biosynthesis regulatory mutants suggesting that regulation by inositol is coupled to the regulation of inositol biosynthesis.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
PS synthase was purified and reconstituted into phospholipid vesicles containingphosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), and PS by gel filtration of detergent-phospholipid mixed micelles. The average diameter of the vesicles was 90nm and the enzyme was reconstituted with its active site facing outward. Enzyme activity was modulated by the PI to PS ratio of the vesicles. Changes in the PC to PE ratio did not effect reconstituted PS synthase activity. The modulation of activity observed in vesicles was enough to account for some of the flucturations in the PS content in vivo.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
The addition of ethanolamine or choline to inositol-containing medium resulted in a repression of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae. This activity was not repressed in the regulatory mutant opil. A method was developed to detect the phospholipid biosynthetic enzymes phosphatidylinositol synthase and phosphatidylserine synthase activities after SDS-polyacrylamide gel electrophoresis and electroblotting. Phosphatidylinositol synthase activity was reconstituted into phospholipid vesicles by passing a octylglucoside-phospholipid-Triton X-100 enzyme mixed micelle over a Sephadex G-50 column. The vesicles had an average diameter of 40 nm and 90% of the enzyme active site faced outward.
Impacts
(N/A)
Publications
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Progress 04/01/84 to 12/30/84
Outputs
The CHO 1 gene product of Saccharomyces cerevisiae was purified from the overproducing strain VAL2C (YEpCHO 1). The enzyme was identical with respect to enzymological properties and physical properties to that from wild-type yeast. Genetic and biochemical analysis of mutant strains of yeast indicate that the biosynthesis of phosphatidylcholine is coordinately regulated to inositol synthesis.
Impacts
(N/A)
Publications
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