Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells
Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells
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DOI：
10.1111/tra.12548
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发表时间：
2018-03-01
期刊：
影响因子：
4.5
通讯作者：
Watanabe, Reika
中科院分区：
文献类型：
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作者：
Castillon, Guillaume A.;Burriat-Couleru, Patricia;Watanabe, Reika
最近，在动物模型中的研究表明网格蛋白和AP 1在上皮组织中的顶端蛋白分选中的潜在作用。然而，这些蛋白在顶端蛋白运输中的确切功能仍不清楚。在这里，我们揭示了网格蛋白重链或AP 1亚基敲低（KD）后Madin-Darby犬肾（MDCK）细胞中内源性糖基磷脂酰肌醇锚定蛋白（GPI-AP）和可溶性分泌蛋白的错误定位。使用一种新的定向内吞和再循环试验，我们发现这些KD细胞不仅在生物合成途径中对GPI-AP的顶端分选受到影响，而且还影响其顶端再循环和基底至顶端的转胞吞途径。已知t-SNARE突触融合蛋白3在生物合成和内吞途径中负责选择性靶向各种顶端目的货物蛋白的顶端分布受到损害，这表明KD细胞中表型的分子解释。我们的研究结果表明，生物合成和内吞途径的重要性，建立和维持顶端定位的GPI-AP极化MDCK细胞。
Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells.