Isolation of a somatic-cell mutant defective in phosphatidylserine biosynthesis

Mutant clones of Chinese hamster ovary (CHO) cells defective in the base-exchange reaction of phospholipids with choline were isolated by using an in situ enzymatic assay for the reaction in cell colonies immobilized on polyester cloth. The specific activities of the choline-exchange reaction in extracts of one of the mutants (designated 64) grown at 33°C and 40°C were 13% and 6% of those in parental (CHO-K1) cells, respectively. The choline-exchange activity in the mutant was moe thermolabile in cell extracts than that in the parent, suggesting that a mutation in the structural gene for the choline-exchange enzyme might have been induced in this mutant. In culture medium supplemented with lipoprotein-deficient serum, mutant 64 grew almost normally at 33°C but divided only twice at 40°C and then stopped growing. Labeling of intact cells with [³²P]P(i) showed that mutant 64 was also strikingly defective in the biosynthesis of phosphatidylserine at 40°C but was normal at 33°C. Most temperature-resistant revertants of mutant 64 exhibited nearly normal ability to synthesize phosphatidylserine at 40°C and also showed choline-exchange activity similar to that in parental cells. The addition of phosphatidylserine to medium supplemented with newborn calf serum, in which mutant 64 grew more slowly than parental cells at 40°C, restored the growth rate of the mutant to the parental level. Our findings suggest that the choline-exchange enzyme functions as the major route for the formation of phosphatidylserine and that the temperature-sensitive growth of mutant 64 is due to a defect in phosphatidylserine biosynthesis at 40°C.