Sphingomyelin-enriched microdomains at the Golgi complex

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as det ergent-resistant membranes from total cell extracts (total-DRM). It is gene rally believed that this total-DRM represents microdomains of the plasma me mbrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipi ds, containing 25% of total Golgi phospholipids including 67% of Golgi-deri ved sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to tot al-DRM, GICs contain only 10 major proteins, present in nearly stoichiometr ic amounts, including the alpha- and beta -subunits of heterotrimeric G pro teins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morpholo gical data show a brefeldin A-sensitive and temperature-sensitive localizat ion to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cel ls, GICs can be isolated from a fused Golgi-endoplasmic reticulum. organell e. This indicates that GIC microdomains are not in a dynamic equilibrium wi th neighboring membrane proteins and lipids. After disruption of the microd omains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indi cates that several of the identified GIC proteins localize to the same micr odomains and that the microdomain scaffold is not required for protein inte ractions between these GIC proteins but instead might modulate their affini ty.