EVIDENCE THAT MAMMALIAN PHOSPHATIDYLINOSITOL TRANSFER PROTEIN REGULATES PHOSPHATIDYLCHOLINE METABOLISM

Phosphatidylinositol transfer proteins (PITPs) and their yeast counter part (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns ) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the bio chemical consequences of lowering the cellular content of this protein . WRK-1 rat mammary tumour cells were transfected with a plasmid conta ining a full-length rat PITP alpha cDNA inserted in the antisense orie ntation and the resultant cell clones were analysed; Three clones expr essing antisense mRNA for PITP alpha: were compared with three clones transfected with the expression vector lacking the insert. The three a ntisense clones had an average of 25 % less PITP alpha protein than co ntrol clones. Two of the three antisense clones also exhibited a decre ased rate of growth. All three antisense crones exhibited a significan t decrease in the incorporation of labelled precursors into PtdCho dur ing a 90-min incubation period. Under the same conditions, however, th ere was no change in precursor incorporation into PtdIns. Further expe rimentation indicated that the decrease in precursor incorporation see n in antisense clones was not due to an increased rate of turnover. Wh en choline metabolism was analysed more extensively in one control (2- 5) and one antisense (4-B) clone using equilibrium-labelling condition s (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amou nt of PtdCho was lower in antisense-transfected clones (this was confi rmed by mass measurements); (2) a similar decrease was seen in cellula r sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an avera ge two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, de creased; and (5) cellular CDPcholine was not significantly altered.