Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells.
Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an approximately 2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.
Kuge
O
O
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan. kuge@nih.go.jp
Hasegawa
K
K
Saito
K
K
Nishijima
M
M
D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D005246:Feedback; D006801:Humans; D007700:Kinetics; D019884:Nitrogenous Group Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010718:Phosphatidylserines / Q000096:biosynthesis* / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014162:Transfection
D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D012694:Serine; D019884:Nitrogenous Group Transferases; C022370:phospholipid serine base exchange enzyme