Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.
Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.
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盘基网柄菌中耐洗涤剂质膜微区的鉴定：信号转导蛋白的富集。
DOI：
10.1091/mbc.8.5.855
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发表时间：
1997
影响因子：
3.3
通讯作者：
Devreotes,PN
中科院分区：
文献类型：
--
作者：
Xiao,Z;Devreotes,PN
与大多数其他细胞蛋白质不同，网柄藻的化学引诱物受体cAR 1对两性离子去污剂CHAPS的提取具有抗性。我们利用这一特性，通过在蔗糖密度梯度中浮选细胞的CHAPS裂解物来分离高度富集cAR 1的亚细胞级分。免疫金电子显微镜研究揭示了膜双层片的均匀制备。这种制剂，命名为CHAPS不溶性漂浮部分（CHIEF），还含有一组确定的20种其他蛋白质和一种不带电荷的脂质。细胞表面生物素化和包埋前免疫电镜均证实了该制剂的质膜来源。细胞表面磷酸二酯酶（PDE）和下游效应的cAR 1，腺苷酸环化酶（ACA），专门定位在这些结构中，而细胞粘附分子gp 80，大多数主要的细胞表面膜蛋白，细胞骨架成分，肌动蛋白结合的整合膜蛋白脑桥蛋白，和G-蛋白α-和β-亚基的缺席。总的来说，CHIFF占细胞外部暴露膜蛋白的约3-5%。所有这些结果表明，CHIFF是来自质膜的专门的微域。分离的方法类似于Caveolae。然而，我们无法检测到与cAR 1相关的细胞表面上的明显的小窝样结构，其显示出弥漫性染色特征。CHIFF的发现促进了cAR 1和相关信号蛋白的纯化以及受体介导过程（如G蛋白活化和脱敏）的生物化学表征。它也有重要的意义，为“流体马赛克”模型的质膜结构。
Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.