Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been ut ilized to assess lipid distribution in the caveolae bilayer, taking advanta ge of the ability of the glycolipid, endogenous or exogenously added, to co ncentrate within this membrane compartment and to crosslink neighboring mol ecules upon illumination. After insertion into A431 plasma membrane and pho toactivation, a membrane-enriched and a detergent-resistant fraction, enric hed in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, o nly radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction In caveolae and the presence of ganglioside within this compartment Among lipids, crosslin ked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detec ted in the detergent-resistant fraction. These results suggest the enrichme nt in sphingomyelin-along with ganglioside-within the outer leaflet, and th e preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.