COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos.
COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos.
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DOI：
10.1083/jcb.138.4.891
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发表时间：
1997-08-25
期刊：
影响因子：
--
通讯作者：
Goodenough DA
中科院分区：
文献类型：
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作者：
Chen Y;Merzdorf C;Paul DL;Goodenough DA
闭塞蛋白是目前已知的唯一一种定位于紧密连接的膜-膜相互作用点上的完整膜蛋白。我们使用非洲爪蟾胚胎作为检测系统来检验：(a)胚胎中突变体occludin的表达是否会破坏紧密连接的屏障功能，以及(b) occludin结构内是否存在靶向连接相互作用位点所需的信号。从一系列cooh末端截断的occludin突变体转录的mrna被微注射到8细胞胚胎的前背卵裂球中。注射后8 h，在与内源性occludin和occludens-1共定位的紧密连接处检测到全长和5个cooh末端截断的蛋白，表明外源性occludin正确靶向紧密连接。重要的是，我们的数据显示，含有四种cooh末端截断的occludin蛋白的紧密连接是渗漏的；顶端细胞间的细胞间隙被磺基琥珀酰氨基-6-（生物胺）己酸酯（nhs - lc -生物素）渗透。相比之下，胚胎注射编码全长、最小截短或可溶性COOH末端的mrna后，nhs - lc -生物素示踪剂仍然无法渗透。通过与全长封闭蛋白mRNA共注射，可以挽救突变封闭蛋白引起的渗漏。用洗剂溶解胚胎膜的免疫沉淀分析显示，外源occludin在体内与内源occludin结合，表明occludin在紧密连接组装过程中发生了寡聚。我们的数据表明，咬合蛋白的COOH末端对于紧密结屏障功能的正确组装是必需的。我们还首次提供了证据，证明闭塞蛋白在紧密连接组装的正常过程中形成低聚物。我们的数据表明，突变型封闭蛋白通过其与全长内源分子寡聚的能力靶向紧密连接。
Occludin is the only known integral membrane protein localized at the points of membrane– membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero–dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.