Genetic and Biochemical Study of Phosphatidylserine biosynthesis and its regulation in animal cells

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Background : We previously isolated a CHO cell mutant (designated as PSA-3) which requires phosphatidylserine (PS) for cell growth and showed that mutant PSA-3 is strikingly defective in PS biosynthesis. On characterization of mutant PSA-3, it was shown that there are at least two PS synthases (designated as PSS I and II,respectively) in CHO cells and that the mutant PSA-3 is defective in one of the synthases, PSS I.Furthermore, we isolated two different CHO cDNAs (designated as pssA and pssC,respectively) which are capable of conferring PS-prototrophy on mutant PSA-3.Research Results : 1) Using antibodies raised against synthetic pssA peptides, we showed that the pssA gene encodes PSS I.2) The pssC gene was shown to encode a mitochondrial phosphatidylserine decarboxylase that matures through three steps of post-translational processing. 3) A CHO cDNA (designated as pssB) similar in sequence to the pssA cDNA was isolated and shown to encode PSS II.PSS II deduced from the cDNA sequence is a 55kD protein containing nine potential membrane-spanning domains. 4) A CHO cell mutant (designated as #16) defective in both PSS I and II was isolated from mutant PSA-3. The characterization of mutant #16 showed that PSS II catalyzes the conversion of phosphatidylethanolamine to PS.5) We found that PSS I carries a domain which regulates own catalytic activity, and identified an amino acid residues involved in this regulation. 6) In order to purify PSS I and II,we developed methods for overexpression of PSS I and II in insect cells.