title: The lipid transfer activity of phosphatidylinositol transfer protein is sufficient to account for enhanced phospholipase C activity in turkey erythrocyte ghosts.
abstract: BACKGROUND
  The minor membrane phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) has been implicated in the control of a number of cellular processes. Efficient synthesis of this lipid from phosphatidylinositol has been proposed to require the presence of a phosphatidylinositol/phosphatidylcholine transfer protein (PITP), which transfers phosphatidylinositol and phosphatidylcholine between membranes, but the mechanism by which PITP exerts its effects is currently unknown. The simplest 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-factor which stimulates the activity of phosphoinositide kinases and phospholipase C (PLC) by presenting protein-bound lipid substrates to these enzymes. We have exploited turkey erythrocyte membranes as a model system in which it has proved possible to distinguish between the above hypotheses of PITP function.
  
  
  RESULTS
  In turkey erythrocyte ghosts, agonist-stimulated PIP2 hydrolysis is initially rapid, but it declines and reaches a plateau when approximately 15% of the phosphatidylinositol has been consumed. PITP did not affect the initial rate of PIP2 hydrolysis, but greatly prolonged the linear phase of PLC activity until at least 70% of phosphatidylinositol was consumed. PITP did not enhance the initial rate of phosphatidylinositol 4-kinase activity but did increase the unstimulated steady-state levels of both phosphatidylinositol 4-phosphate and PIP2 by a catalytic mechanism, because the amount of polyphosphoinositides synthesized greatly exceeded the molar amount of PITP in the assay. Furthermore, when polyphosphoinositide synthesis was allowed to proceed in the presence of exogenous PITP, after washing ghosts to remove PITP before activation of PLC, enhanced inositol phosphate production was observed, whether or not PITP was present in the subsequent PLC assay.
  
  
  CONCLUSIONS
  PITP acts by catalytically transferring 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 enzymes present in turkey erythrocyte ghosts.
authors.affiliation: Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, DD1 4HN, UK. racurrie@bad.dundee.ac.uk
authors.forename: R A
authors.initials: RA
authors.lastname: Currie
authors.forename: B M
authors.initials: BM
authors.lastname: MacLeod
authors.forename: C P
authors.initials: CP
authors.lastname: Downes
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000097:blood* / Q000502:physiology; D002417:Cattle; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008563:Membrane Lipids / Q000097:blood*; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000097:blood*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates / Q000097:blood; D049294:Phospholipid Transfer Proteins; D010727:Phosphoric Diester Hydrolases / Q000097:blood*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000097:blood; D015290:Second Messenger Systems / Q000502:physiology*; D014422:Turkeys / Q000097:blood
chemical_list: D002352:Carrier Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D049294:Phospholipid Transfer Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase