Genetic and biochemical studies on biosynthesis and physiological roles of phosphatidylserine in mammalian cells

Abstract

The base-exchange enzymes which catalyze exchange of free serine, choline, and ethanolamine with polar head groups of phospholipids are known to occur in eukaryotic cells. To understand the physiological roles of the base-exchange reactions, a rapid autoradiographic in situ enzyme assay has been developed for detecting the base-exchange activities in Chinese hamster ovary (CHO) cell colonies immobilized on filter paper or polyester cloth and obtained several mutants strikingly defective in the choline-exchange activity. CHO cell mutants that required exogenously added phosphatidylserine for cell growth were also isolated by using the replica technique with polyester cloth. Biochemical characterization of these mutants has revealed the following results. 1) There are at least two kinds of serine-exhange enzyme in CHO-K1 cells ; one of the enzymes (serine-exchange enzyme I) can catalyze the base-exchange reaction of phospholipids with serine, choline and ethanolamine, and the other enzyme (serine-exchange enzyme II) does not use choline as a substrate. 2) Phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions : phosphatidylcholine→phosphatidylserine→phosphatidylethanolamine→phsophatidylserine. The three reactions are catalyzed by serine-exchange I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively. 3) Serine-exchange enzyme I is essential for the growth of CHO cells. In addition, we have found that exogenous phosphatidylserine can be efficiently incorporated into CHO cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.