Revised Manuscript Received September 27, 7995® abstract: Caveolae are clathrin-free cell-surface organelles implicated in transmembrane transport. A fibroblast caveolar membrane fraction was isolated by sucrose density gradient ultracentrifugation and its identity confirmed by protein markers (caveolin, annexin II). When 3H-labeled free cholesterol was selectively transferred to thecells from labeled low density lipoprotein to increase cell free cholesterol—15%, there was a 6-fold increase in label in the caveolar fraction above baseline levels. Subsequent incubation of these cells with unlabeled native plasma or plasma high density lipoprotein selectively unloaded caveolar free cholesterol into the medium. Okadaic acid, which decreased caveolar activity as measured by cholera toxin binding and uptake, decreased cholesterol efflux in parallel. Cholesterol newly synthesized from [3H] mevalonate was also preferentially incorporated into the caveolar fraction and selectively released by plasma into the medium. Together these data indicate that caveolae represent a major site of efflux of both newly synthesized and low density lipoprotein-derived free cholesterol in these cells.

Caveolae and clathrin-coated pits represent alternative cell-surface organelles specialized for binding and internalizing extracellular solutes (Hansen et al., 1991). Caveolae are clathrin-free invaginations (40—80 nM diameter) of many cell surfaces. The caveolar membranefraction is enriched in GPI-anchored proteins, src-family tyrosine kinases, and Ca2+-ATPase (Rothberg et al., 1990; Stefanova et al., 1991; Brown & Rose, 1992; Dupree et al., 1993). Annexins, GTPases implicated in vesicle-mediated exocytosis (Creutz, 1992), are also located in the caveolarfraction (Fiedler et al., 1995). Highly purified caveolae contain proteins involved in boththe budding and fusion of cytoplasmic vesicles (Schnitzer et al., 1995).