Abstract
Transport by discrete vesicular carriers is well established at least in part because of recent discoveries identifying key protein mediators of vesicle formation, docking, and fusion. A general mechanism sensitive to N-ethylmaleimide (NEM) is required for the transport of a divergent group of vesicular carriers in all eukaryotes. Many endothelia have an abundant population of non-coated plasmalemmal vesicles or caveolae, which have been reported with considerable controversy to function in transport. We recently have shown that like other vesicular transport systems, caveolae-mediated endocytosis and transcytosis are inhibited by NEM (Schnitzer, J. E., Allard, J., and Oh, P. (1995) Am. J. Physiol. 268, H48-H55). Here, we continue this work by utilizing our recently developed method for purifying endothelial caveolae from rat lung tissue (Schnitzer, J. E., Oh, P., Jacobson, B. S., and Dvorak, A. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1759-1763) to show that these caveolae contain key proteins known to mediate different aspects of vesicle formation, docking, and/or fusion including the vSNARE VAMP-2, monomeric and trimeric GTPases, annexins II and VI, and the NEM-sensitive fusion factor NSF along with its attachment protein SNAP. Like neuronal VAMPs, this endothelial VAMP is sensitive to cleavage by botulinum B and tetanus neurotoxins. Caveolae in endothelium are indeed like other carrier vesicles and contain similar NEM-sensitive molecular machinery for transport.
Highlights
In the last few years, studies have shown that specific proteins on the endothelial cell surface facilitate the select transendothelial transport of insulin and blood carrier proteins such as albumin (5) and transferrin
By removing cholesterol from plasma membranes, filipin selectively disassembles caveolae in lung endothelium, which produces a significant reduction in the scavenger endocytosis of modified albumins, transcytosis of insulin and native albumin, and even transcapillary permeability of albumin in the rat lung in situ (7)
We recently developed a method for selectively purifying endothelial caveolae from rat lung tissue (31)
Summary
Vol 270, No 24, Issue of June 16, pp. 14399-14404, 1995 Printed in U.S.A. Endothelial Caveolae Have the Molecular Transport Machinery for Vesicle Budding, Docking, and Fusion Including VAMP, NSF, SNAP, Annexins, and GTPases*. By removing cholesterol from plasma membranes, filipin selectively disassembles caveolae in lung endothelium, which produces a significant reduction in the scavenger endocytosis of modified albumins, transcytosis of insulin and native albumin, and even transcapillary permeability of albumin in the rat lung in situ (7) Such transport by caveolae is sensitive to alkylation with N-ethylmaleimide (NEM),l suggesting by analogy with other vesicular carrier systems a dependence on membrane fusion and specific NEM-sensitive factors (8). In the SNARE model, NEM inhibits transport by modifying a specific ATPase called NSF (NEM-sensitive fusion protein), which interacts with soluble NSF attachment proteins called SNAPs (26-28) Together, they form a functional SNARE fusion complex by direct association with complemen-. The studies presented here show that these purified endothelial caveolae do have the typical molecular machinery necessary for vesicle formation, docking, and fusion as found in many other vesicular carriers
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