CLONING OF A CHINESE-HAMSTER OVARY (CHO) CDNA-ENCODING PHOSPHATIDYLSERINE SYNTHASE (PSS) II, OVEREXPRESSION OF WHICH SUPPRESSES THE PHOSPHATIDYLSERINE BIOSYNTHETIC DEFECT OF A PSS I-LACKING MUTANT OF CHO-K1 CELLS

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preex isting phospholipid, We previously showed the presence of two differen t enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synth ase (PSS) I, which also catalyzes the exchange of the base moiety of p hospholipid(s) for ethanolamine and choline, In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the ser ine and ethanolamine base exchange activities, In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exc hange activity, The expression of the pssB gene in Sf9 insect cells al so results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-aux otrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy, T he PtdSer content of the resultant transformant grown without exogenou s PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant, These results sugges ted that the pssB cDNA encoded the second PtdSer synthase PSS II, whic h catalyzed the serine and ethanol amine base exchange, but not the ch oline base exchange.