title: Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites.
abstract: Upon permeabilization of Swiss mouse 3T3 fibroblasts, an isoform of phosphatidylinositol transfer protein (PI-TP) was preferentially retained, a major part of which was associated with the perinuclear Golgi system (K. J. de Vries, A. Momchilova-Pankova, G. T. Snoek, and K. W. A. Wirtz, Exp. Cell Res. 215, 109-113, 1994). In the present study, the intracellular localization of this isoform (PI-TP beta) and the regular form (PI-TP alpha) was investigated in fetal bovine heart endothelial cells by microinjection of fluorescently labeled analogs followed by confocal laser scanning microscopy. The PI-TP alpha and PI-TP beta used were purified from bovine brain cytosol and covalently labeled with sulfoindocyanine dyes. By this novel method it was found that PI-TP beta was preferentially associated with perinuclear membrane structures whereas PI-TP alpha was predominantly present in the nucleus and in the cytoplasm. This intracellular localization was confirmed by indirect immunofluorescence indicating that the fluorescently labeled PI-TP alpha and PI-TP beta were targeted to the same sites as their endogeneous counterparts.
authors.affiliation: Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
authors.forename: K J
authors.initials: KJ
authors.lastname: De Vries
authors.forename: J
authors.initials: J
authors.lastname: Westerman
authors.forename: P I
authors.initials: PI
authors.lastname: Bastiaens
authors.forename: T M
authors.initials: TM
authors.lastname: Jovin
authors.forename: K W
authors.initials: KW
authors.lastname: Wirtz
authors.forename: G T
authors.initials: GT
authors.lastname: Snoek
mesh_terms: D000818:Animals; D001011:Aorta; D001923:Brain Chemistry; D002232:Carbocyanines; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003593:Cytoplasm / Q000737:chemistry; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D008845:Microinjections; D018613:Microscopy, Confocal / Q000379:methods*; D009685:Nuclear Envelope / Q000737:chemistry*; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins
chemical_list: D002232:Carbocyanines; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5