THE LIPID TRANSFER ACTIVITY OF PHOSPHATIDYLINOSITOL TRANSFER PROTEIN IS SUFFICIENT TO ACCOUNT FOR ENHANCED PHOSPHOLIPASE-C ACTIVITY IN TURKEY ERYTHROCYTE-GHOSTS

Background: The minor membrane phospholipid phosphatidylinositol 4,5-b isphosphate (PIP2) has been implicated in the control of a number of c ellular processes. Efficient synthesis of this lipid from phosphatidyl inositol has been proposed to require the presence of a phosphatidylin ositol/phosphatidylcholine transfer protein (PITP), which transfers ph osphatidylinositol and phosphatidylcholine between membranes, but the mechanism by which PITP exerts its effects is currently unknown. The s implest hypothesis is that PITP replenishes agonist-sensitive pools of inositol lipids by transferring phosphatidylinositol from its site of synthesis to sites of consumption, Recent cellular studies, however, led to the proposal that PITP may play a more active role as a co-fact or which stimulates the activity of phosphoinositide kinases and phosp holipase C (PLC) by presenting protein-bound lipid substrates to these enzymes. We have exploited turkey erythrocyte membranes as a model sy stem in which it has proved possible to distinguish between the above hypotheses of PITP function. Results: In turkey erythrocyte ghosts, ag onist-stimulated PIP2 hydrolysis is initially rapid, but it declines a nd reaches a plateau when similar to 15% of the phosphatidylinositol h as been consumed. PITP did not affect the initial rate of PIP2 hydroly sis, but greatly prolonged the linear phase of PLC activity until at l east 70% of phosphatidylinositol was consumed. PITP did not enhance th e initial rate of phosphatidylinositol 4-kinase activity but did incre ase the unstimulated steady-state levels of both phosphatidylinositol 4-phosphate and PIP2 by a catalytic mechanism, because the amount of p olyphosphoinositides synthesized greatly exceeded the molar amount of PITP in the assay. Furthermore, when polyphosphoinositide synthesis wa s allowed to proceed in the presence of exogenous PITP, after washing ghosts to remove PITP before activation of PLC, enhanced inositol phos phate production was observed, whether or not PITP was present in the subsequent PLC assay. Conclusions: PITP acts by catalytically transfer ring phosphatidylinositol down a chemical gradient which is created as a result of the depletion of phosphatidylinositol at its site of use by the concerted actions of the phosphoinositide kinases and PLC. PITP is therefore not a co-factor for the phosphoinositide-metabolizing en zymes present in turkey erythrocyte ghosts.