Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites.
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.
De Vries
K J
KJ
Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
Westerman
J
J
Bastiaens
P I
PI
Jovin
T M
TM
Wirtz
K W
KW
Snoek
G T
GT
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
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