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 preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(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 serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous 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 suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.
Kuge
O
O
Department of Biochemistry and Cell Biology, National Institute of Health, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan. kuge@nih.go.jp
Saito
K
K
Nishijima
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D008969:Molecular Sequence Data; D009154:Mutation; D019884:Nitrogenous Group Transferases / Q000235:genetics* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010718:Phosphatidylserines / Q000096:biosynthesis*; D014162:Transfection; D014166:Transferases / Q000172:deficiency*
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D014166:Transferases; D019884:Nitrogenous Group Transferases; C111493:Ptdss2 protein, mouse; C022370:phospholipid serine base exchange enzyme