Endothelial Caveolae Have the Molecular Transport Machinery for Vesicle Budding, Docking, and Fusion Including VAMP, NSF, SNAP, Annexins, and GTPases

doi:10.1074/jbc.270.24.14399

Endothelial Caveolae Have the Molecular Transport Machinery for Vesicle Budding, Docking, and Fusion Including VAMP, NSF, SNAP, Annexins, and GTPases (*)

From the (1)Department of Pathology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215

^§ To whom correspondence should be addressed:
Dept. of Pathology, Harvard Medical School, Research North-Beth Israel, 99 Brookline Ave., Boston, MA 02215.
Tel.: 617-667-3577; Fax: 617-667-3591; E-mail: jschnitz{at}BIH.HARVARD.EDU.

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 noncoated 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL43278 and HL52766 (to J. E. S.) and was done during the tenure of an Established Investigatorship Award from the American Heart Association and Genentech (to J. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NEM

N-ethylmaleimide

NSF

NEM-sensitive fusion protein

BoNT

botulinum neurotoxin B

TeTx

tetanus toxin

PAGE

polyacrylamide gel electrophoresis

DTT

dithiothreitol

P

pellet

H

homogenate

V

vesicle

T

Triton X-100-soluble fraction

P-V

pellet containing resedimented silica-coated membranes stripped of the caveolae

VAMP

vesicle-associated membrane protein

SNAP

soluble NSF attachment protein

SNARE

SNAP receptor

tSNARE

target-associated SNAP receptor

vSNARE

vesicle-associated SNAP receptor.
↵²J. E. Schnitzer, D. McIntosh, A. M. Dvorak, J. Liu, and P. Oh, submitted for publication.
↵³J. E. Schnitzer and P. Oh, manuscript in preparation.