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PIGF is a protein involved in the ethanolamine phosphate (EtNP) transfer steps of glycosylphosphatidylinositol (GPI) biosynthesis. PIGF forms a heterodimer with either PIGG or PIGO, two enzymes that transfer an EtNP to the second or third mannoses of GPI respectively. Heterodimer formation is essential for stable and regulated expression of PIGO and PIGG, but the functional significance of PIGF remains obscure. In the present study, we show that PIGF binds to PIGO and PIGG through distinct molecular domains. Strikingly, C-terminal half of PIGF was sufficient for its binding to PIGO and PIGG and yet this truncation mutant could not complement the PIGF defective mutant cells, suggesting that heterodimer formation is not sufficient for PIGF function. Furthermore, we identified a highly conserved motif in PIGF and demonstrated that the motif is not involved in binding to PIGO or PIGG, but critical for its function. Finally, we identified a PIGF homologue from Trypanosoma brucei and showed that it binds specifically to the T. brucei PIGO homologue. These data together support the notion that PIGF plays a critical and evolutionary conserved role in the ethanolamine-phosphate transfer-step, which cannot be explained by its previously ascribed binding/stabilizing function. Potential roles of PIGF in GPI biosynthesis are discussed.

The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ethanolaminephosphotransferase, respectively. Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids. A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/Ept1p enzymes to gain insight into their mechanism(s) of activation. Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, and Mn2+ and Co2+. This allowed the use of chimaeric enzymes to determine the mechanism of cation activation. Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves. Cpt1p and Ept1p also displayed distinct phospholipid activation profiles. Phospholipid activation required a phosphate and/or glycero-phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle. Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation. Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S. cerevisiae. Hence CPT displays product activation. Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition.

Essential-fatty acid deficiency produces a 52% increase in the rate of phosphatidyl-ethanolamine synthesis in rat liver as calculated from results obtained in vivo [Trewhella & Collins (1973) Biochem. Biophys. Acta 296, 34--50]. This flux change was used to test the possible regulatory roles of ethanolamine kinase and of phosphoethanolamine cytidylyltransferase, which are rate-limiting enzymes of the cytidine pathway for the synthesis of phosphatidylethanolamine [Infante (1977) Biochem. J. 167, 847--849]. The results show that essential-fatty acid deficiency produces 50% and 53% increases respectively in the specific activity of these enzymes, accounting for the increased rate of phosphatidylethanolamine synthesis produced by this dietary insufficiency. This evidence leads to the conclusion that ethanolamine kinase and phosphoethanolamine cytidylyl-transferase have co-ordinated regulatory roles in the flux control of the cytidine pathway, and its sphinganine 1-phosphate lyase branch reaction, for the synthesis of phosphatidylethanolamine.