Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae.
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.
Ostrander
D B
DB
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
O'Brien
D J
DJ
Gorman
J A
JA
Carman
G M
GM
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000494:pharmacology; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000096:biosynthesis*; D011743:Pyrimidines / Q000506:poisoning; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*
D000970:Antineoplastic Agents; D010743:Phospholipids; D011743:Pyrimidines; D005973:Glutamine; D003562:Cytidine; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D008239:Lysine