Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells

We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO-K1. The resultant transfectant, CDT-1, exhibited about 20-fold higher specific activity of the enzyme in the membrane fraction than CHO-K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross-reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT-1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42-kDa protein was eliminated from solubilized membrane proteins of CDT-1 cells. Both the enzyme activity and the 42-kDa protein of CHO-K1 cells were enriched in the mitochondria-associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.