Mapping of Eps15 domains involved in its targeting to clathrin-coated pits
Mapping of Eps15 domains involved in its targeting to clathrin-coated pits
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DOI：
10.1074/jbc.275.5.3288
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发表时间：
2000-02-04
影响因子：
4.8
通讯作者：
Dautry-Varsat, A
中科院分区：
文献类型：
--
作者：
Benmerah, A;Poupon, V;Dautry-Varsat, A
网格蛋白包被凹坑 (CCP) 的形成是由于胞质外壳蛋白（主要是质膜网格蛋白相关蛋白复合物 (AP-2) 和网格蛋白）靶向和组装到质膜的细胞内面而发生的。在本研究中，通过跟踪与绿色荧光蛋白融合的 Eps15 突变体的细胞内定位，分析了 AP-a 结合蛋白 Eps15 靶向 CCP 的机制。我们之前的结果表明 N 端 Eps15 同源 (EH) 结构域是 CCP 靶向所必需的。然而，我们现在表明，EH 结构域不足以靶向 CCP，同样，中央卷曲螺旋和 C 端 AP-2 结合结构域都无法将绿色荧光蛋白定位到 CCP，因此，Eps15 靶向 CCP 可能是 EM 结构域和蛋白质的另一个结构域之间协作的结果。缺乏位于 CCP 的卷曲螺旋结构域的 Eps15 突变体表明 Eps15 二聚化不是严格要求的。相比之下，缺乏所有AP-2结合位点的Eps15突变体显示质膜染色显着减少，表明AP-2结合位点与EH结构域一起在将Eps15靶向CCPs中发挥重要作用。最后，通过免疫荧光和流式细胞术测试了Eps15突变体对网格蛋白依赖性内吞作用的影响，获得的结果表明： 仅在能够干扰 CCP 组装的突变体中观察到转铁蛋白摄取的抑制。
Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-a-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15 homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs, Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs, Thus, targeting of Eps15 to CCPs likely results from the collaboration between EM domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs, Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry, The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly.