By use of indirect immunofluorescence it was shown that the phosphatidylinositol transfer protein (PI-TP) in 3T3 mouse fibroblast cells is associated with the Golgi system. This was concluded from double-labeling experiments with TRITC-labeled Ricin which binds to sugar residues that are specifically processed in the Golgi system. Independent evidence for this association was provided by the fact that dissociation of the Golgi system by brefeldin A was reflected in an extensive redistribution of PI-TP labeling. In addition, PI-TP is localized in the cytoplasm and in the nucleus. In exponentially growing cells an enhanced labeling of PI-TP was observed in the cytosol and in the cytosol and in the Golgi system in comparison with quiescent cells. By Western blot analysis and by transfer activity assays, it was confirmed that the concentration of PI-TP was increased in exponentially growing cells. These results strongly suggest that PI-TP fulfills a role in the functioning of the Golgi complex.

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 protein (PI‐TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI‐TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI‐carrying form of PI‐TP (pl 5.5) and of a novel, more acidic form of PI‐TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI‐transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI‐TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA‐induced differentiation. Immunoprecipitation of PI‐TP from [32P]Pi labeled cells demonstrated that the level of phosphorylation of PI‐TP is high in undifferentiated P19 EC cells and low after 5 days of RA‐induced differentiation. These results strongly suggest that changes in the levels of PI‐TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA‐induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI‐TP is an essential cytosolic factor in stimulating phospholipase C activity.