Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism

Abstract

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (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 biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPalpha cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPalpha were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPalpha protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (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 amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.

Structure–function relationships of phosphatidylinositol transfer proteins: involvement of phosphorylation sites

“…Microinjection of fluorescently labeled PI-TPa and PI-TPb into living cells as well as indirect immunofluorescence studies have shown that PI-TPa is localized in the nucleus and cytosol whereas PITPb is associated with the Golgi system [8]. In intact cells, decreased expression of PI-TPa was shown to decrease the rate of proliferation and a lower rate of synthesis was observed for PC, lysoPC, SM, Choline and Glycerophosphocholine [9]. However, knock out of PI-TPa in mouse embryonic stem cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism.…”

Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

“…A role for PITP␣ in regulating metabolism of choline phospholipids in mammalian cells has also been proposed. Antisense approaches suggest that modest reductions in PITP␣ levels result in significant alterations in PtdCho and SM metabolism in one breast cancer cell line (Monaco et al, 1998).…”

Phosphatidylinositol transfer protein α regulates growth and apoptosis of NIH3T3 cells

“…In mammalian tissues, at least two isoforms are identified: PI-TP ␣ , which is localized in the nucleus and cytosol, and PI-TP ␤ , which is associated with the Golgi system (3). Possible cellular functions of these proteins have been obtained from experiments with permeabilized cells (4), reconstituted Golgi membrane systems (5), and cells in which the expression of the proteins has been altered (6)(7)(8). In permeabilized, cytosol-depleted cells, both isoforms restored guanosine 5 Ј -[ ␥Ϫ thio]triphosphate-stimulated protein secretion as well as phospholipase C-mediated inositol lipid signaling (9)(10)(11).…”