Morimoto Shinya, Nishimura Noriyuki, Terai Tomoya, Manabe Shinji, Yamamoto Yasuyo, Shinahara Wakako, Miyake Hidenori, Tashiro Seiki, Shimada Mitsuo, Sasaki Takuya
Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan.
J Biol Chem. 2005 Jan 21;280(3):2220-8. doi: 10.1074/jbc.M406906200. Epub 2004 Nov 4.
During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.
在上皮形态发生过程中，黏着连接（AJs）和紧密连接（TJs）会经历动态重组，而上皮极性会暂时丧失并重新建立。尽管ARF6介导的E-钙黏蛋白的内吞循环已被阐明，并与AJs的快速重塑有关，但TJs动态重排的分子基础仍不清楚。闭合蛋白和紧密连接蛋白是构成TJ链的整合膜蛋白，被认为负责建立和维持上皮极性。在这里，我们研究了闭合蛋白和紧密连接蛋白在细胞表面的进出细胞内转运。利用细胞表面生物素化和免疫荧光技术，我们发现，在成纤维细胞系幼仓鼠肾细胞和上皮细胞系MTD-1A细胞中，一部分闭合蛋白持续被内吞并循环回到细胞表面。生化内吞和循环分析表明，Rab13显性活性突变体（Rab13 Q67L）抑制了MTD-1A细胞中闭合蛋白的内吞后循环，但不影响转铁蛋白受体和多聚免疫球蛋白受体的循环。双重免疫标记显示，在MTD-1A细胞中，一部分内吞的闭合蛋白与Rab13共定位。这些结果表明，Rab13在成纤维细胞和上皮细胞中特异性地介导闭合蛋白持续内吞循环回到细胞表面。
