Properties and intracellular localization of phosphatidylinositol transfer protein in Swiss mouse 3T3 cells

Abstract

In most mammalian cells the endoplasmic reticulum has been identified as the main site of phosphatidylinositol (PI) biosynthesis [ 1, 21. Since the receptor-mediated breakdown of phosphatidylinositol 4,s-bisphosphate (PIP,) and the conversion of PI into PIP, occurs in the plasma membrane [3, 41, the question by what mechanism the level of PI in the plasma membrane is maintained occurs. In a mechanism similar to that proposed for transport of cholesterol [5, 61, it is conceivable that phosphoinositides including PI are transported from the endoplasmic reticulum to the plasma membrane by intracellular vesicle flow [7]. In another proposed mechanism, transfer of PI to the plasma membrane occurs directly by a monomolecular insertion reaction involving the phosphatidylinositol transfer protein (PI-TP) [2,8,9].Recently the interest in the physiological role of PI-TP has strongly increased with the observation that PI-TP in yeast is identical to the SEC14 protein [ 101 and hence involved in secretory processes at the level of the late Golgi compartment [ 11, 121. Immunolocalization studies showed that Abbreviations used: I'C, phosphatidylcholine; 1' 1, phosphatidylinositol; PIPL, phosphatidylinositol 4,s-bisphosphate; 1'1-TI'. phosphatidylinositol-transfer protein; I'MA, phorbol 12-myristate 13-acetate. *To whom correspondence should be addressed. PI-TP in yeast is associated with Golgi structures[ 131. Furthermore deletion of the gene encoding PI-TP was shown to be lethal for the organism 1141. The requirement for PI-TP can be bypassed by a mutation in the CDP-choline pathway for phosphatidylcholine (PC) biosynthesis [ 13, 151. Since PI-TP has the ability to transfer both PI and, to a lesser extent, PC [9] this observation has been interpreted to indicate that PI-TP has the capacity to control the relative PI/PC composition of the yeast Golgi membranes, which is proposed to be critical to the secretory competence of these membranes. PropertiesThe first PI-TP to be purified was that from bovine brain [ 16, 171. This protein has a molecular mass of 33 kDa and consists of two isoforms with isoelectric points of 5.3 and 5.6, closely resembling rat and human 191. This resemblance is reflected in the crossreactivity of the anti-rat PI-TP antibody with PI-TP in bovine and human brain cytosol [ 191. In fact PI-TP appears to be strongly conserved, since anti-bovine PI-TP antibody reacted with a 35-36 kDa protein in the membranefree cytosol from mammals, birds, reptiles, amphibians and insects [20]. The sequence of rat PI-TP as deduced from cDNA analysis is composed of 271 amino acid residues and the N-terminal peptide Volume 21

Phosphatidylinositol Transfer Proteins: Emerging Roles in Cell Proliferation, Cell Death and Survival

“…Activation of protein kinase C by phorbol esters affected the cellular localization of the PI-TPs, increasing the association with the Golgi membranes. Both proteins could be phosphorylated in vitro but PI-TPb was phosphorylated to a much higher level than PI-TPa (15,16,32). Actually, it was shown that in vivo 85% of PI-TPb is already phosphorylated on the unique phosphorylation site Ser 262 and that this phosphorylation is essential for the association with the Golgi membranes.…”