Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

Lipid rafts and caveolae are biochemically similar, specialized domains of the plasma membrane (PM) which cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. 2-D gel electrophoresis preceding identification of proteins by mass spectrometry was used to compare the relative abundance of proteins in detergent resistant membranes (DRM), isolated from human umbilical-vein endothelial cells (HUVEC), and caveolae, immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure (caveolin-1 [CAV1], vimentin) and transport (V-ATPase), as well as the glycosylphosphatidylinositol (GPI)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known (endothelial nitric oxide synthase [eNOS], CAV1), less known (synaptosomal-associated protein 23 [SNAP-23], brain acid soluble protein 1 [BASP1]) and novel (chromosome 8 open reading frame 2 [C8ORF2]) proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (~5% of total protein) associated with immunoseparated caveolae, in contrast to the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane. ; r.r.sprenger@amc.uva.nl (Horrevoets, Anton JG) ; Medical Biochemistry, Academic Medical Center - NETHERLANDS (Horrevoets, Anton JG) ...