Purification and characterization of smooth muscle cell caveolae.
Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center at Dallas 75235.
W J
WJ
Chang
Y S
YS
Ying
K G
KG
Rothberg
N M
NM
Hooper
A J
AJ
Turner
H A
HA
Gambliel
J
J
De Gunzburg
S M
SM
Mumby
A G
AG
Gilman
R G
RG
Anderson
D000818:Animals; D051242:Caveolin 1; D022461:Caveolins; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D002645:Chickens; D019204:GTP-Binding Proteins / Q000302:isolation & purification; D005895:Gizzard, Avian / Q000166:cytology; D017261:Glycosylphosphatidylinositols; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000737:chemistry*; D016253:Microscopy, Immunoelectron; D009130:Muscle, Smooth / Q000737:chemistry* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry / Q000648:ultrastructure
D051242:Caveolin 1; D022461:Caveolins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D019204:GTP-Binding Proteins