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Abstract
Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.
Highlights
Phosphoinositides and their cleavage products play a critical role as second messengers in signal transduction at the cell surface and as regulators that modulate the function of proteins involved in intracellular signal transduction [1– 4]
Recent studies have shown that targeted deletion of the murine phospholipase C (PLC)1-␥1 resulted in embryonic lethality [5] and that the vibrator mutation in mice caused neural degeneration via reduced expression of the soluble type phosphatidylinositol transfer protein ␣ (PITP␣) [6]
The deletion mutant, the ⌬-(258 –1242) (Fig. 1A), which lacks the acidic and whole membrane domains of PITPnm, was detected as a 33-kDa protein present in the cytosolic fraction under the same experimental conditions (Fig. 1E). These results indicate that PITPnm is an integral membrane protein with its carboxyl terminus anchored in the lipid bilayer
Summary
Construction of Expression Vectors—To construct an epitope tagged PITPnm, an XhoI site was introduced into the 5Ј and 3Ј ends of the full-length mouse PITPnm cDNA and ligated into the mammalian expression vector pcDNA 3.1 Myc-His (Invitrogen Corp., arlsbad, CA). The XhoI-BglII fragment (2.7 kilobases) was excised from full-length PITPnm cDNA and ligated into the pcDNA Myc-His (designated ⌬-(707–1242)). The lipid-coated plates were blocked with 5% skim milk (w/v) in PBS for 1 h at room temperature, and various amounts of recombinant proteins were added to each well and incubated at 4 °C overnight. The supernatants were divided and incubated for 2 h with a monoclonal anti-FLAG-M2 antibody (Eastman Kodak Co.), anti-c-Myc antibody, or control anti-mouse IgG (Zymed Laboratories Inc.) antibodies bound to protein G-Sepharose beads. The sections were mounted on nickel grids, blocked with PBS containing 1% bovine serum albumin for 40 min, and incubated for 4 h at 37 °C with an appropriate dilution of affinity-purified anti-PITPnm primary antibody. After staining with uranyl acetate and lead citrate, the sections were observed in a JEM-1200 EX electron microscope (JEOL, Tokyo, Japan) operated at 100 kV
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