Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Hepatic plasma membrane fluidity and dietary proteins.

Effect of protein deficient diet on hepatic plasma membrane fluidity has been studied in rats using (i) steady state fluorescence polarization and anisotropy, (ii) phospholipid and cholesterol contents, (iii) phospholipid fatty acid composition, (iv) turnover of phosphatidyl choline (PC), and (v) activities of membrane-bound enzymes as parameters and rats fed casein (20%) diet as standard group. A significant increase in steady state fluorescence and anisotropy values was registered in the deficient group, indicating increased resistance and hence decrease in fluidity of the plasma membrane. Supplementation of the diet with lysine and threonine improved these values, thereby suggesting the significance of diet for membrane fluidity. Simultaneous significant alterations in other parameters, viz. (i) decrease in PC, PE and free cholesterol and increase in esterified cholesterol contents, (ii) decrease in unsaturation of fatty acids of PC, (iii) decrease in incorporation of NaH2 32PO4, [CH3-14C]choline and [CH3-14C]methionine into plasma membrane PC, and (iv) decrease in activities of plasma membrane 5'-nucleotidase and phosphodiesterase along with increase of (Na(+)-K+)ATPase and adenyl cyclase, were observed in the deficient group which on supplementation with lysine and threonine showed improvement over alterations.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

Gibberellin modulation of phosphatidyl-choline turnover in wheat aleurone tissue

Phosphatidyl choline (PC) is synthesised in wheat (Triticum aestivum L. cv. Flanders) aleurone tissue during early germination when new endomembranes are being formed. Although gibberellic acid does not ostensibly affect PC levels, it inhibits the incorporation of choline and differentially and specifically modulates the turnover of the N-methyl and methylene carbons of the choline headgroup of PC. Gibberellic acid has no effect on turnover of the phosphate moiety of either PC or the other major phosphatides. The possible biological importance of the findings is discussed.Abbreviations ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - PA phosphatidic acid - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol - PI phosphatidyl inositol - t_1/2 half-life     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.     

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin B12 diet

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B(12) on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 microg vitamin B(12) per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B(12) ameliorates the second messenger-mediated injury.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Hepatic Subcellular Fractions Lipids in Rats Fed Millet (Sorghum vulgarie) at Different Protein Levels

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5% protein levels respectively for six weeks has been studied on rat liver mitochondrial, microsomal and supernatant fractions total lipids, cholesterol, triglycerides, total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. The results have been compared with rats fed casein at 10% level for the same period. The metabolism of liver subcellular fractions lipids of millet diet and casein diet fed rats has been studied by the incorporation of acetate-1-¹⁴C and . A significant increase in mitochondrial triglycerides of rats fed millet diet at 5 and 10% protein level, in microsomes of rats fed millet diet at 5, 10 and 15% protein levels and in supernatant fractions of rats fed millet diet at 5 and 15% protein levels was observed. A significant increase in total cholesterol in mitochondria and microsomes and a significant decrease in supernatant fraction of rats fed millet diet at 10% protein level was observed. A significant increase in mitochondrial total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine in rats fed millet diet at 10% protein level and a decrease in these in rats fed millet diet at 5 per cent protein level was observed. In microsomes total phospholipids were increased in rats millet diet at 10% protein level and phosphatidyl choline was increased in rats fed millet diet at 15% protein level. Total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine were significantly reduced in the supernatant fraction of rats fed millet at 10% protein level.

Incorporation of acetate-1-¹⁴C into nonsaponifiable fraction of mitochondria, microsomes and supernatant fractions of rats fed millet diet at 5 and 15 % protein levels was significantly greater, and in saponifiable fractions of the above subcellular fractions was greater in rats fed millet diet at 5 per cent protein level. The specific activity (counts/min/mg) of free cholesterol in mitochondria, microsomes and supernatant fractions of millet diet fed rats was significantly greater, whereas the specific activity of triglycerides was not significantly different from the controls. The acetate-1-¹⁴C specific activity of phosphatidyl choline and phosphatidyl ethanolamine was significantly greater in all the above subcellular fractions of millet diet fed rats (except of phosphatidyl choline in rats fed millet diet at 5 % protein level). The specific activities of phosphatidyl choline were significantly greater in mitochondria of rats fed millet diet at 5 % protein level and of phosphatidyl choline and phosphatidyl ethanolamine in microsomes and supernatant fractions of rats fed millet diet at 5 and 15% protein levels. The specific activities of phosphatidyl choline were significantly decreased in mitochondria and microsomes of rats fed millet diet at 10% protein level. The total acetate-1-¹⁴C activities (counts/min/g equivalent wet liver) of free and esterified cholesterol triglycerides, phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis from acetate-1-¹⁴C was either enhanced in millet diet fed rats or was comparable to the controls. The total activity of (counts/min/g equivalent wet liver) into phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis was decreased in microsomes of rats fed millet diet at 10% protein level, increased in rats fed millet diet at 5 and 15% protein levels.     

The effect of dietary protein on the excretion of alpha2u, the sex-dependent protein of the adult male rat.

Adult male rats were maintained on normal (20% casein), protein-free (0% casein), high protein (50% casein), decicient protein (20% zein), and a supplemented, deficient protein (20% zein plus L-lysine and L-tryptophan) diets. Rats on a protein-free diet excreted approximately 1 mg alpha2u/24 h compared with a normal of 10-15 mg/24 h. Depleted rats placed on a 20% casein diet showed a rapid restoration of the normal alpha2u excretion as well as total urinary proteins. Accumulation of alpha2u in the blood serum was measured in nep-rectomized rats. Rats on a 0% casein diet accumulated only 30% of the alpha2u compared to normals. On a 50% casein diet, rats excreted 30-50 mg alpha2u/24 h. However, the accumulation was normal in the serum of nephrectomized rats. A high protein diet did not stimulate alpha2u synthesis but probably increased the renal loss of all urinary proteins. The excretion of alpha2u on a zein diet was reduced to the same degree as with the protein-free diet. Supplementation with lysine and tryptophan restored the capacity to eliminate alpha21 to near normal levels. Accumulation of alpha2u in the serum of nephrectomized rats kept on the zein diets showed that the effect to suppress the synthesis of the ahpha2u. Supplementation restored the biosynthesis of alpha2u. We conclude that the effect of dietary protein on the excretion of urinary proteins in the adult male rat is caused in large part by an influence on the hepatic biosynthesis of alphay2u. The biosynthesis of this protein, which represents approximately 30% of the total urinary proteins, is dependent on an adequate supply of dietary protein.     

Al(3+)-mediated changes on membrane fluidity affects the activity of PI-PLC but not of PLC

We investigated whether Al(3+)-mediated changes in membrane fluidity can affect the activity of prokaryotic enzymes phospholipase C (PLC) and phospholipase C-phosphatidyl inositol specific (PI-PLC) in liposomes of phosphatidyl choline (PC), PC:phosphatidyl inositol (PI), or PC and polyphosphoinositides (PPI). Al(3+) (10-100 microM) promoted membrane rigidification, evaluated with the probes 1,6-diphenyl-1,3,5-hexatriene and Laurdan, and followed the order: PC:PPI>PC:PI>PC. Al(3+) (25 and 50 microM) did not affect PLC-mediated hydrolysis of PC, PI and PIP(2), but stimulated PIP hydrolysis (48.6%). PI-PLC did not affect PC, PI, and PIP concentrations, but caused a 67% decrease in PIP(2). Al(3+) significantly inhibited PIP(2) hydrolysis in a concentration-dependent (25-50 microM) manner. Results suggest that the inhibition of PIP(2) hydrolysis by Al(3+) could be partially due to a higher lipid packing induced by Al(3+) which could affect the interaction between the enzyme and its substrate.     

A high-beef diet alters protein kinase C isozyme expression in rat colonic mucosa

We recently reported that a red meat (beef) diet relative to a casein-based diet increases protein kinase C (PKC) activity in rat colonic mucosa. The purpose of this study was to further elucidate the effects of a high-beef diet on colonic intracellular signal transduction by analyzing steady-state protein levels of different PKC isozymes as well as activities of the three types of sphingomyelinases. Male Wistar rats (n = 12/group) were fed AIN93G-based diets either high in beef or casein for 4 weeks. Rats fed the beef diet had significantly (P < 0.05) higher cytosolic PKC alpha and lower membrane PKC delta protein levels than rats fed the casein diet. The beef-fed rats also had alterations in subfractions of PKC zeta/lambda so that they had a significantly (P = 0.001) lower level of membrane 70 & 75 kDa fraction and a higher (P = 0.001) level of cytosolic 40 & 43 kDa fraction than rats fed the casein diet. Because protein levels analyzed with a PKC zeta-specific antibody were similar, these differences in PKC zeta/lambda were probably due to changes in PKC lambda expression. PKC beta2 levels did not differ between the dietary groups. Diet had no significant effect on the activity of acid, neutral, or alkaline sphingomyelinase. This study demonstrated that consumption of a high-beef diet is capable of modulating PKC isozyme levels in rat colon, which might be one of the mechanisms whereby red meat affects colon carcinogenesis.     

Simultaneous imaging of phosphatidyl inositol metabolism and Ca2+ levels in PC12h cells

To elucidate the spatial and temporal relationships between phosphatidyl inositol metabolism and changes in intracellular calcium levels, we developed a simultaneous imaging system using green fluorescent protein fused with the pleckstrin homology domain, and the fluorescent calcium indicator, FuraRed. The redistribution of the fusion protein, which represents the phosphatidyl inositol metabolism process, was quantified by calculating the coefficient of variance of the fluorescence over the entire cytosolic region, excluding the nucleus. This calculation increased the reproducibility, compared to the normalized fluorescence changes in arbitrarily selected cytosolic regions used in conventional analysis. The application of this method to analyzing the response of PC12h cells to a number of pharmacological stimuli showed that the extent of the phosphatidyl inositol metabolism was related to the calcium level, but not induced by calcium alone.     

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.     

Effects of orally administered cytidine 5'-diphosphate choline on brain phospholipid content

Cytidine, as cytidine 5'-diphosphate choline (CDP-choline), is important for the synthesis of phosphatidylcholine in cell membranes. To investigate whether exogenous CDP-choline could affect brain phospholipid composition, we supplemented the diet of mice with this drug (500 mg/kg/day) for 27 months in 3-month-old mice and for 90, 42, and 3 days in 12-month-old mice, and measured their levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and the content of phosphatidylinositol plus phosphatidic acid in the cerebral cortex. After 27 months of treatment, PC and PE increased significantly by 19% (P < 0.05) and by 20% (P < 0.01), respectively. PS levels increased by 18% (not statistically significant). Similar elevations in PC and PE levels were obtained when older mice were treated for only 3 months (P < 0.05). No changes were observed with shorter treatment periods. These results suggest that chronic administration of CDP-choline can have effects on brain phospholipid composition that may underlie its reported utility in various neurologic disorders.     

In vitro phosphorylation of high molecular weight glutenin subunits from wheat endosperm

We have investigated the in vitro phosphorylation of high molecular weight glutenin subunits (HMW-GS), a group of non-soluble proteins present in wheat endosperm. Computer aided searches of potential biological sites in the known sequences of these proteins have evidenced the presence of sequence motifs specific for protein kinase C (PKC), calcium-dependent protein kinase from wheat, casein kinase II, tyrosine protein kinase and glycosylation. We have demonstrated that subunit 1Bx7 is a substrate of a partially purified PKC from rat brain. Further experiments have shown that this subunit is phosphorylated by an endogenous protein kinase activity found in wheat flour. These preliminary results are important for the possible implications on the structure-function relationships of these proteins and could probably suggest, for the first time, a potential physiological role in particular situations for some HMW-GS.     

Evaluation of dietary essentiality of vitamins for Penaeus monodon (Fabricius)

The effect of exclusion of individual water-soluble (thiamine, riboflavin, pyridoxine, cyanocobalamin, pantothenic acid, folic acid, niacin, biotin, choline, inositol, ascorbic acid) and fat-soluble vitamins (vit. A, D, K and E) in semi-purified diets on growth and survival of juvenile shrimp, P. monodon was studied in the laboratory for 8 weeks. Diets lacking riboflavin and vitamin K did not affect growth and survival of shrimp. However, deletion of inositol and choline resulted in poor growth. Maximum growth was observed in the control diet (C1) which was supplemented with all vitamins. Diet deficient in ascorbic acid, biotin, folic acid, niacin, thiamine and alpha-tocopherol resulted in poor appetite and poorer feed conversion efficiency. All treatments except the control (C1) resulted in histological changes in the digestive gland cells. Detachment or destruction of the epithelial cells was observed in all treatments lacking individual vitamins but more severely in the treatment without a vitamin supplement followed by inositol, choline and ascorbic acid deficient diets.     

Effects of tumor necrosis factor α (TNFα) on the phospholipid metabolism of Tetrahymena pyriformis

The effect of (0·05 ng ml⁻¹ and 0·1 ng ml⁻¹) TNFα on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP₂) as well, as sphingomyelin (SM) content was lower in TNFα-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [³H]-palmitic acid or ³²P into the phospholipids. To control the functional effects of TNFα in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNFα at a low level of evolution, what is realized by changes in the phospolipid metabolism participating in signalling pathways. © 1998 John Wiley & Sons, Ltd.     

Phosphoinositides of sheep platelets plasma membranes.

Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively).     

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.     

Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.