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首页> 外文期刊>Molecular & cellular proteomics: MCP >Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.
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Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.

机译：使用改良的分裂泛素膜酵母双杂交系统监测哺乳动物整合膜转运蛋白与PDZ相互作用伙伴之间的蛋白质相互作用。

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摘要

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.
机译：在许多整合膜蛋白的C末端尾巴中发现了PDZ结合基序，它们通过与含PDZ的蛋白质结合来介导特定的蛋白质-蛋白质相互作用。常规的酵母双杂交筛选已用于探测这些可溶性C末端的蛋白质-蛋白质相互作用。然而，迄今为止，还没有体内技术可用于研究全长整合膜蛋白与其同源PDZ相互作用伴侣之间的相互作用。我们以前开发了一种分裂泛素膜酵母双杂交（MYTH）系统，通过使用基于从插入膜的诱饵C末端切割转录因子的转录输出来测试这种整合膜蛋白之间的相互作用。在这里，我们将MYTH修改为允许通过将转录因子部分从C重定向到给定完整膜蛋白的N末端，从而释放其天然C末端，从而检测C端PDZ域相互作用。我们成功地将该“ MYTH 2.0”系统应用于五个不同的哺乳动物全长肾脏转运蛋白，并鉴定了磷酸盐（NaPi-IIa）和硫酸盐（NaS1）转运蛋白的新颖的含PDZ结构域的伙伴，而这些蛋白否则将无法检测到。此外，该测定法被应用于在NaS1蛋白上定位PDZ结合结构域。我们显示，NaS1上PDZK1的PDZ结合域位于其C末端的上游，而两个相互作用的蛋白质NHERF-1和NHERF-2则在更靠近NaS1 N末端的位置结合。此外，当与NaS1共表达时，NHERF-1和NHERF-2增加了非洲爪蟾卵母细胞功能性硫酸盐的摄取。最后，我们使用MYTH 2.0来证明NaPi-IIa转运蛋白通过脂质双层中的蛋白质-蛋白质相互作用同二聚。总而言之，我们的研究将MYTH 2.0系统建立为膜蛋白复合物相互作用蛋白质组学研究的新工具。

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