GENETIC-EVIDENCE THAT PHOSPHATIDYLSERINE SYNTHASE II CATALYZES THE CONVERSION OF PHOSPHATIDYLETHANOLAMINE TO PHOSPHATIDYLSERINE IN CHINESE-HAMSTER OVARY CELLS

Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE), The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS syn thase (PSS) I and II. A PSS I-lacking mutant of CHO-III cells, PSA-S, which exhibits similar to 2-fold lower serine base exchange activity t han CHO-K1, is defective in the conversion of phosphatidylcholine to P S but has the ability to convert PE to PS. The PSA-3 mutant requires e xogenous PS or PE for cell growth. In the present study, from PSA-3 mu tant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was similar to 10% that of PSA-3 mutant cells an d similar to 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an si milar to 80% reduction in the PSS II mRNA level relative to that in PS A-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutan t is defective in PSS II. Like the PSA-S mutant, the PSB-2 mutant grew well in medium supplemented with PS, However, in the medium supplemen ted with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant wa s defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS, A metabolic labeling experiment with exogenous [ P-32]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS, This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complem ented by the PSS II cDNA In addition, the cDNA of the other PS synthas e, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These resu lts indicated that PSS LI is critical for the growth and PS biosynthes is of PSA-3 mutant cells cultivated with exogenous PE and suggested th at most of the PS formation from PE in CHO-gl cells is catalyzed by PS S II.