Ward R E, Lamb R S, Fehon R G
Developmental, Cell, and Molecular Biology Group, Department of Zoology, Duke University, Durham, North Carolina 27708-1000, USA.
J Cell Biol. 1998 Mar 23;140(6):1463-73. doi: 10.1083/jcb.140.6.1463.
The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.
蛋白质4.1超家族由多种细胞质蛋白组成，其中许多蛋白已被证明可通过与特定跨膜蛋白结合而与质膜结合。果蝇中的coracle是一种蛋白质4.1同源物，在胚胎发育过程中是必需的，并且定位于上皮细胞中分隔连接的细胞质面。通过体外诱变，我们证明coracle的氨基末端383个氨基酸定义了一个功能域，该功能域对于转基因胚胎中分隔连接的正确定位既必要又充分。该结构域内的基因突变会破坏coracle亚细胞定位并严重影响其遗传功能，表明正确的亚细胞定位对于coracle功能至关重要。此外，coracle和跨膜蛋白神经纤毛蛋白在分隔连接处的定位显示出相互依赖的关系，这表明coracle和神经纤毛蛋白在分隔连接的细胞质面相互作用。与此观点一致，免疫沉淀和体外结合研究表明，coracle的氨基末端383个氨基酸与神经纤毛蛋白的细胞质结构域直接相互作用。这些结果共同表明，coracle在分隔连接处提供了重要的膜组织功能，并且这些功能由所有蛋白质4.1超家族成员中保守的氨基末端结构域执行。
