The structural gene (CHOI) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevmae from a bank of yeast genomic DNA on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome. The DNA sequence on an integrative plasmid was shown to recombine into the CHOi locus, confwrming its genetic identity. The chol yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase. Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6-to 7-fold as compared with the wild-type strain. The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate. The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.The characterization of mutants altered in the synthesis of phospholipids has given considerable insight into the regulation of phospholipid metabolism in Escherichia coli and Saccharomyces cerevisiae (1, 2). Using yeast phospholipid mutants and the transformation techniques now available, it is possible to clone by complementation a number of the structural and regulatory genes involved in yeast lipid metabolism. These cloned genes can then be used as probes to study the synthesis, regulation, and assembly of the membrane-associated enzymes of phospholipid synthesis.The chol mutants of S. cerevisiae require either ethanolamine or choline for growth and are deficient in the synthesis of the phospholipid, phosphatidylserine (PtdSer) (3-5). PtdSer is the normal precursor for both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). However, under conditions of ethanolamine or choline supplementation, chol mutants are able to use an alternative pathway described by Kennedy and Weiss (6) for the synthesis of PtdEtn and PtdCho (3-5). All chow mutants have very reduced levels of the enzyme phosphatidylserine synthase (CDPdiacylglycerol:L-serine 0-phosphatidyltransferase, EC 2.7.8.8). Thus, the CHOJ locus has the characteristics expected of the structural gene for this enzyme (4). Yeast PtdSer synthase is a regulated integral membrane protein (7,8) MATERIALS AND METHODS Strains. The haploid S. cerevsiae strain VAL2C (Mata leu2-3 leu2-112 ade6 chol) was derived from a cross of strain DC5 (Mata leu2-3 leu2-112 his3 cani-11, provided by J. Hicks) to strain VAL12 (MATa ade6 ural chol). Strain VAL12 was isoklted as an ethanolamine auxotroph from wild-type strain SHID SC (MATa ade6 ural). Strain 399 (UAA), lysl-1 (UAA), met8-1 (UAG), trpl-l (UAG), leu2 (UGA)] was obtained from G. Fink and wild-type strain S288C was used for the enzyme preparations. The E. coli strain used wa...
The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyri mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis. The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.Phosphatidylserine (PS) synthase plays a major role in the overall regulation of phospholipid biosynthesis in Saccharomyces cerevisiae (16,17,21). PS synthase is regulated by phosphorylation via cyclic AMP (cAMP)-dependent protein kinase (13). The phosphorylation of the PS synthase 23,000-Mr subunit results in a 60 to 70% reduction in PS synthase activity (13). Immunoprecipitation of the phosphorylated and dephosphorylated forms of the PS synthase 23,000-Mr subunit from a bcyl mutant (which has high cAMP-dependent protein kinase activity) and a cyri mutant (which has low cAMP-dependent protein kinase activity) with corresponding reduced and elevated levels of PS synthase activity, respectively, confirmed that the enzyme is regulated by phosphorylation in vivo (13). In the present study we showed that the levels of cAMP and thus of cAMP-dependent protein kinase activity regulated the rates of phospholipid biosynthesis in vivo.Our studies were facilitated by the use of strain AM18-5C (MATot ade6 ade8 ampi cami cam2 cam3 cyrl-2), a cyri mutant defective in adenylate cyclase (19 (50 ,uCi) was then added to the cultures, which were incubated for 20 min. All incubations were at 30°C. The labeled cells were harvested by centrifugation and washed with distilled water. Phospholipids were extracted (9) and analyzed by two-dimensional paper chromatography (1, 14). The amount of label in the individual phospholipids was quantitated by scintillation spectroscopy (16). The percentages of 32p; incorporation into each phospholipid represented the relative rates of synthesis during the 20-min pulse (11). Although phosphatidylethanolamine and phosphatidylcholine are major membrane phospholipids (2, 7), they were not heavily labeled during the pulse since they are derived from PS (11). In wild-type cells, the synthesis of phosphatidylinositol (PI) accounted for 44% of the label and the synthesis of PS accounted for 33% of the label (Fig. 1). In contrast, when mutant cyri cells were incubated in the absence of cAMP, the percentage of 32p; found in PS increased to 47% whereas the amount of label found in PI decreased to 32% (Fig. 1). The addition of increasing concentrations of cAMP to the incubation medium of cyri cells resulted in an increase in the rate of PI synthesis at the expense of PS synthesis (Fig. 1). The rates of PI and PS syntheses in cyri cells supplemented with 1 mM cAMP were essentially identical to those in wild-type cells incubated in the absence of cAMP (Fig. 1). The percentages of label recovered in phosphati...
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