Greenwood Brianna L, Oshima Kana, Stuart David T
Dept of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Bio Protoc. 2025 Aug 20;15(16):e5418. doi: 10.21769/BioProtoc.5418.
Protein-protein interactions facilitate cellular functions through the creation of networks and multi-protein complexes. Mapping the interactions within and between protein networks and elucidating the composition of protein complexes provides critical insight into biological processes. Interactions among soluble cytoplasmic proteins have been extensively investigated through the application of immunoaffinity capture as well as conventional nuclear two-hybrid testing. The integrated membrane yeast two-hybrid provides a method to investigate protein-protein interactions between integral membrane proteins in their native membrane environment. This procedure makes use of the ability of the amino-terminal fragment of ubiquitin (Nub) and the carboxyl-terminal fragment of ubiquitin (Cub) to refold reconstituting functional ubiquitin, which can be recognized by a ubiquitin peptidase. Appending a fusion protein composed of Cub fused to LexA and VP16 (CLV) to a candidate "bait" protein and Nub to candidate "prey" proteins allows a test of their interaction. If the two proteins interact closely, the CLV fragment is cleaved and enters the nucleus to activate the expression of reporter genes, signaling the interaction. When the bait and prey proteins are tagged with CLV and NubG, respectively, at their genomic loci, they are only copies of the bait and prey in the cell and are expressed under the regulation of their native promoters. This avoids overexpression artifacts that can occur if the tagged proteins are expressed from plasmids while the untagged chromosomally encoded copies of the bait and prey continue to be expressed. Key features • Allows an in vivo interaction test with integral membrane proteins in the native membrane environment. • Allows integration of NubG tag at the amino or carboxyl-terminus of prey proteins. • Avoids overexpression artifacts that can be caused by expression of CLV-tagged bait and NubG-tagged prey proteins from plasmid-based systems. • Avoids competition from untagged chromosomally encoded bait and prey proteins, as occurs when CLV-tagged bait and NubG-tagged prey are expressed from plasmids.
蛋白质-蛋白质相互作用通过形成网络和多蛋白复合物来促进细胞功能。绘制蛋白质网络内部和之间的相互作用，并阐明蛋白质复合物的组成，可为生物过程提供关键见解。通过免疫亲和捕获以及传统的核双杂交测试，对可溶性细胞质蛋白之间的相互作用进行了广泛研究。整合膜酵母双杂交提供了一种方法，可在天然膜环境中研究整合膜蛋白之间的蛋白质-蛋白质相互作用。该方法利用泛素的氨基末端片段（Nub）和泛素的羧基末端片段（Cub）重新折叠形成功能性泛素的能力，这种功能性泛素可被泛素肽酶识别。将由与LexA和VP16融合的Cub组成的融合蛋白（CLV）附加到候选“诱饵”蛋白上，并将Nub附加到候选“猎物”蛋白上，可对它们的相互作用进行测试。如果这两种蛋白紧密相互作用，CLV片段就会被切割并进入细胞核以激活报告基因的表达，从而表明存在相互作用。当诱饵蛋白和猎物蛋白在其基因组位点分别用CLV和NubG标记时，它们只是细胞中诱饵和猎物的拷贝，并在其天然启动子的调控下表达。这避免了如果标记蛋白从质粒表达而未标记的染色体编码的诱饵和猎物拷贝继续表达时可能出现的过表达假象。关键特性 • 允许在天然膜环境中对整合膜蛋白进行体内相互作用测试。 • 允许在猎物蛋白的氨基或羧基末端整合NubG标签。 • 避免基于质粒系统中CLV标记的诱饵蛋白和NubG标记的猎物蛋白表达所导致的过表达假象。 • 避免未标记的染色体编码的诱饵和猎物蛋白的竞争，这在从质粒表达CLV标记的诱饵和NubG标记的猎物时会发生。
