Phosphatidylserine synthase from yeast

Abstract

Whereas mammalian cells produce PS by a base exchange reaction from preexisting phospholipids, yeast cells synthesize PS from CDP-diacylglycerol and serine by the PS synthase reaction. Yeast PS synthase was purified to homogeneity and shown to have a molecular mass of 23 kDa. The activity is dependent on either Mg 2+ or Mn 2+ and Triton X-100. The enzyme specifically transfers the phosphatidyl group from CDP-diacylglycerol or dCDP-diacylglycerol to l-serine, but not to threonine, cysteine and ethanolamine. The PSS/ CHO1 gene encoding the enzyme was cloned by the complementation of the choline auxotrophic pss/ cho1 mutant. The deduced protein comprises 279 amino acids with a calculated molecular mass of 30 804. The primary translate undergoes proteolytic processing to the enzymatically more active 23-kDa enzyme. The deduced amino acid sequence contains several putative membrane-spanning regions and resembles that of the Bacillus subtilis enzyme, but not those of the E. coli and Haemophilus influenzae enzymes. The sequence also contains the local, conserved region found in enzymes catalyzing the transfer of the phosphoalcohol moiety from CDP-alcohol, such as PI synthase, cholinephosphotransferase and phosphatidylglycerolphosphate synthase. The activity of PS synthase is maximal in the exponential phase, but decreases when cells enter the stationary phase. The enzyme is phosphorylated at a single serine residue by cyclic AMP-dependent protein kinase with a 60–70% decrease in enzymatic activity, but the primary translation product is not phosphorylated. PS synthase is inhibited by CTP, probably due to the chelation of the divalent cations, Mg 2+ and Mn 2+, and also by sphingoid bases, such as sphinganine and phytosphingosine. Phosphatidate, phosphatidylcholine and phosphatidylinositol are stimulatory, whereas cardiolipin and diacylglycerol are inhibitory. The expression of yeast PS synthase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes. The upstream regulatory region of the PSS/ CHO1 gene responsible for the myo-inositol–choline regulation was identified. An octameric sequence, CATRTGAA (R=A or G), plays an important role in the conferral of the myo-inositol–choline transcriptional regulation.