Caveolae require intact VAMP for targeted transport in vascular endothelium.
Caveolae appear to function in vesicular trafficking of specific molecular cargo into and across vascular endothelial and other cells. They contain the molecular machinery for docking and fusion, similar to other vesicular trafficking systems, yet the mechanisms mediating ligand internalization and targeted intracellular transport by caveolae remain unclear. Using immunoelectron microscopy, we show that caveolae in the microvascular endothelium of rat lung express vesicle-associated membrane protein (VAMP)-2 (also called synaptobrevin) on their cytoplasmic surface. Immunofluorescence studies of cholera toxin B (CTB)-FITC internalization in toxin-treated cells demonstrate that intact VAMP-2 is necessary for the efficient trafficking of caveolar ligands. The CTB subunit binds preferentially to GM1 in caveolae, and N-ethylmaleimide treatment drastically inhibits the intracellular accumulation of CTB. The cleavage of caveolar VAMP-2 with VAMP-specific neurotoxins (botulinum D and F but not A) significantly inhibits CTB endocytosis and targeted intracellular accumulation in cultured endothelial cells. This impairment of caveolae-mediated trafficking provides evidence that caveolae require intact VAMP-2 for efficient targeted delivery via vesicle docking with target organelles.
McIntosh
D P
DP
Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Schnitzer
J E
JE
D000818:Animals; D001011:Aorta; D001692:Biological Transport / Q000187:drug effects; D001905:Botulinum Toxins / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000493:pharmacokinetics / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005033:Ethylmaleimide / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D050683:R-SNARE Proteins; D051381:Rats
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D002772:Cholera Toxin; D001905:Botulinum Toxins; D005033:Ethylmaleimide