Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system
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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DOI：
10.1074/mcp.m800079-mcp200
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发表时间：
2008-07-01
影响因子：
7
通讯作者：
Stagljar, Igor
中科院分区：
文献类型：
--
作者：
Gisler, Serge M.;Kittanakom, Saranya;Stagljar, Igor
PDZ结合基序存在于许多完整的膜蛋白的C末端，它们通过与含有PDZ的蛋白质结合来介导特定的蛋白质之间的相互作用。传统的酵母双杂交筛选已被用来探索这些可溶性C末端的蛋白质-蛋白质相互作用。然而，到目前为止，还没有活体技术来研究全长完整膜蛋白与其同源PDZ相互作用伙伴之间的相互作用。我们以前开发了一种裂解泛素膜酵母双杂交系统来测试这种完整的膜蛋白之间的相互作用，方法是使用基于膜插入诱饵C端转录因子切割的转录产物。在这里，我们修改了Myth，通过将转录因子部分从给定的完整膜蛋白的C末端重定向到N末端，从而释放了它们的天然C末端，从而允许检测C-末端PDZ结构域的相互作用。我们成功地将这个“神话2.0”系统应用于五种不同的哺乳动物全长肾脏转运蛋白，并确定了新的含有PDZ结构域的磷酸(Napi-IIa)和硫酸盐(NaS1)转运蛋白，否则将无法检测到它们。此外，本实验还对NaS1蛋白上的PDZ结合域进行了定位。我们发现，PDZK1在NaS1上的PDZ结合域位于其C末端的上游，而两个相互作用的蛋白NHERF-1和NHERF-2结合在离NaS1的N端更近的位置。此外，当与NaS1共表达时，NHERF-1和NHERF-2增加了非洲爪哇卵母细胞对硫酸盐的功能性摄取。最后，我们使用Myth 2.0证明了Napi-IIa转运蛋白通过脂双层内的蛋白质-蛋白质相互作用进行同源二聚。总之，我们的研究建立了Myth 2.0系统，为膜蛋白复合体的相互作用蛋白质组学研究提供了一种新的工具。
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.