Sprenger Richard R, Fontijn Ruud D, van Marle Jan, Pannekoek Hans, Horrevoets Anton J G
Department of Medical Biochemistry, Academic Medical Center K1-114, Meibergdreef 15, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
Biochem J. 2006 Dec 15;400(3):401-10. doi: 10.1042/BJ20060355.
Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), 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 [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] 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 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with 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.
脂筏和小窝在生物化学上相似，是质膜（细胞膜）的特殊区域，可聚集特定蛋白质。然而，它们在形态上有所不同，这意味着其功能可能不同但互补。在通过质谱鉴定蛋白质之前，使用二维凝胶电泳来比较从人脐静脉内皮细胞（HUVEC）分离的耐去污剂膜（DRM）和从小窝免疫纯化的DRM组分中蛋白质的相对丰度。鉴定出了各种信号和转运蛋白，并通过额外的细胞表面生物素化表明大多数蛋白是暴露的，证明它们存在于质膜上。在静息内皮细胞中，免疫分离的小窝支架仅由少数驻留蛋白组成，这些蛋白与结构相关[小窝蛋白-1（CAV1）、波形蛋白]和转运（V-ATP酶），以及糖基磷脂酰肌醇（GPI）连接的、表面暴露蛋白CD59有关。通过对已知的[内皮型一氧化氮合酶（eNOS）和CAV1]、不太知名的[23 kDa突触体相关蛋白（SNAP-23）和脑酸溶性蛋白1（BASP1）]和新的[8号染色体开放阅读框2（C8ORF2）]蛋白进行免疫印迹和共聚焦显微镜进一步定量表征显示，这些蛋白具有不同的亚细胞分布，且没有一种蛋白仅存在于小窝或DRM中。然而，与筏蛋白SNAP-23不同，新蛋白C8ORF2的DRM相关部分（约占总蛋白的5%）与免疫分离的小窝相关。在增殖细胞中，CAV1在空间上与eNOS、SNAP-23和BASP1分离，从小窝和脂筏的分离在视觉上得到了证实。这些结果为先前提出的内皮细胞质膜特殊区域之间的转运和信号功能分离提供了直接证据。
