Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and their fusion with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids. Here we report, using a previously established cell-free system derived from a neuroendocrine cell line, the purification of cytosolic factors that stimulate the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. One such factor, referred to as CAST1, was identified as the alpha and beta isoforms of the mammalian phosphatidylinositol transfer protein (PtdIns-TP) (refs 3-5). The yeast PtdIns-TP, SEC14p (ref. 6), which has no sequence homology to mammalian PtdIns-TP (refs 7,8), was able to substitute for the mammalian PtdIns-TP in secretory vesicle formation. Our results suggest a highly conserved role for phosphoinositides in vesicle formation.

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5′-[gamma-thio]triphosphate-mediated phospholipase C beta activity.

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The charge isomers of bovine brain PI-TP␣ (i.e. PI-TP␣I containing a phosphatidylinositol (PI) molecule and PI-TP␣II containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the doublereciprocal plot, it was estimated that the V max values for PI-TP␣I and II were 2.0 and 6.0 nmol/min, respectively; the K m values for both charge isomers were about equal, i.e. 0.7 M. Phosphorylation of charge isomers of recombinant mouse PI-TP␣ confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32 P-labeled PI-TP␣s showed that serine was the major site of phosphorylation. Degradation of 32 P-labeled PI-TP␣ by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one 32 P-labeled peptide (amino acids 104 -190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TP␣. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TP␣ to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TP␣ expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TP␣(S166A) and Myc-tagged PI-TP␣(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TP␣ is linked to the degradation of PI.

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)␤ is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser 262 was identified as the major site of phosphorylation and Ser 165 as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP␤ and PI-TP␤(S262A) were identical, whereas PI-TP␤(S165A) was completely inactive. PKC-dependent phosphorylation of Ser 262 also had no effect on the transfer activity of PI-TP␤. To investigate the role of Ser 262 in the functioning of PI-TP␤, wtPI-TP␤ and PI-TP␤(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP␤ from its phosphorylated form. After Western blotting, wtPI-TP␤ was found to be 85% phosphorylated, whereas PI-TP␤(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP␤ was strongly reduced. Immunolocalization showed that wtPI-TP␤ was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP␤ was distributed throughout the cell similar to what was observed for PI-TP␤(S262A). In contrast to wtPI-TP␤ overexpressors, cells overexpressing PI-TP␤(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP␤ to express its effect on sphingomyelin metabolism.Eukaryotic phosphatidylinositol transfer proteins (PI-TPs) 1 belong to a family of highly conserved proteins that are able to transfer phospholipids between membranes or from membrane to enzyme (1). In mammalian tissues at least two isoforms, PI-TP␣ and PI-TP␤, are found. PI-TP␣ is able to transfer phosphatidylinositol (PI) and, to a lesser extent, phosphatidylcholine (PC) (2-6) and is mainly localized in the cytosol and in the nucleus (7). Similar to PI-TP␣, PI-TP␤ is able to transfer PI and PC but is also able to transfer sphingomyelin (SM) (8). PI-TP␤ is mainly associated with the Golgi apparatus (7). The primary sequences of PI-TP␣ and PI-TP␤ are very similar, with an identity of 77% and a similarity of 94% (9).To date, little is known about the exact cellular function of PI-TP␣ and PI-TP␤. In a cell-free system containing transGolgi membranes, both PI-TP␣ and PI-TP␤ stimulated the formation of constitutive secretory vesicles and immature granules (10). In permeabilized, cytosol-depleted HL60 cells, both isoforms restored GTP␥S-stimulated protein secretion and phospholipase C-mediated inositol lipid signaling (11,12). In these assays, PI-TP␣ and PI-TP␤ were found to function equally well despite their different biochemical properties and cellular localizations. On the other hand, NIH3T3 cells with increased expression of either PI-TP␣ or PI-TP␤ demonstrated remarkable differences in lipid metabolic pathways. Cells overexpressing PI-TP␣ (SPI␣ cells) showed a...