FIGURE 8. Effect of the T455A mutation on the synthesis of phospholipids.

S. cerevisiae ura7Δ ura8Δ cells expressing the wild type (WT) and T455A mutant human CTP synthetase 1 enzymes were grown to the exponential phase of growth. Cells were incubated with ³²P_i (5 μCi/ml) (A) or with [methyl-¹⁴C]choline (0.5 μCi/ml) (B) for 30 min to label all phospholipids and to label CDP-choline pathway-derived phosphatidylcholine, respectively. Phospholipids were extracted and analyzed by two-dimensional thin-layer chromatography. The ³²P-labeled phospholipids were analyzed by phosphorimaging and quantified using ImageQuant software. The ¹⁴C-labeled phosphatidylcholine was determined by scintillation counting. Each data point represents the average of duplicate determinations from two independent experiments ± S.D.