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.
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
D B
DB
Ostrander
D J
DJ
O'Brien
J A
JA
Gorman
G M
GM
Carman
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