Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association
Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association
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DOI：
10.1074/jbc.m513720200
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发表时间：
2006-08-18
影响因子：
4.8
通讯作者：
Prohaska, Rainer
中科院分区：
文献类型：
--
作者：
Umlauf, Ellen;Mairhofer, Mario;Prohaska, Rainer
胞质取向的单调整合膜蛋白stomatin形成高阶寡聚体，并与脂筏。为了表征参与寡聚化和耐洗涤剂膜（DRM）关联的结构域，我们表达了气孔蛋白的截短和点突变体，并通过超离心方法分析了它们的大小和浮力。一个小的C-末端区域的气孔蛋白，主要是疏水性的，丝氨酸-苏氨酸-异亮氨酸-缬氨酸-苯丙氨酸-脯氨酸-亮氨酸-脯氨酸-异亮氨酸（残基264-272），被证明是至关重要的寡聚化，而N-末端结构域（残基1-20）和最后12个C-末端氨基酸（残基276 - 287）是不必要的。在区域264-272中引入丙氨酸取代导致单体的出现。值得注意的是，发现这些残基中只有三个Ile-Val-Phe（残基266-268）对于DRM关联是不可缺少的。有趣的是，交换Pro-269和在一定程度上的残基270-272，这是必要的寡聚化，不影响DRM协会的气孔蛋白。这表明，低聚物的形成是不必要的气孔蛋白与DRM的协会。膜锚定结构域附近的内部缺失导致形成中等大小的低聚物，这表明大部分C-末端区域的构象相互依赖性。标记的、单体的、非DRM突变ST-（1-262）-绿色荧光蛋白和野生型气孔蛋白Stom GFP的光漂白分析后的荧光恢复显示出截短突变体在质膜中的显著更高的横向移动性，这表明相应C-末端区域也在体内的膜相互作用。
The cytoplasmically oriented monotopic integral membrane protein stomatin forms high-order oligomers and associates with lipid rafts. To characterize the domains that are involved in oligomerization and detergent-resistant membrane (DRM) association, we expressed truncation and point mutants of stomatin and analyzed their size and buoyancy by ultracentrifugation methods. A small C-terminal region of stomatin that is largely hydrophobic, Ser-Thr-Ile-Val-Phe-Pro-Leu-Pro-Ile (residues 264-272), proved to be crucial for oligomerization, whereas the N-terminal domain (residues 1-20) and the last 12 C-terminal amino acids (residues 276 287) were not essential. The introduction of alanine substitutions in the region 264-272 resulted in the appearance of monomers. Remarkably, only three of these residues, Ile-Val-Phe (residues 266-268), were found to be indispensable for the DRM association. Interestingly, the exchange of Pro-269 and to some extent the residues 270-272, which are essential for oligomerization, did not affect the DRM association of stomatin. This suggests that the formation of oligomers is not necessary for the association of stomatin with DRMs. Internal deletions near the membrane anchoring domain resulted in the formation of intermediate size oligomers suggesting a conformational interdependence of large parts of the C-terminal region. Fluorescence recovery after photobleaching analysis of the tagged, monomeric, non-DRM mutant ST-(1-262)-green fluorescent protein and wild type stomatin Stom GFP showed a significantly higher lateral mobility of the truncation mutant in the plasma membrane suggesting a membrane interaction of the respective C-terminal region also in vivo.