Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling
Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling
复制标题
DOI：
10.1111/j.1742-4658.2009.06991.x
复制
发表时间：
2009-05-01
期刊：
影响因子：
5.4
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
--
作者：
Fukuda, Nobuo;Ishii, Jun;Kondo, Akihiko
在当前的研究中，我们报告了利用酵母 G 蛋白信号传导检测蛋白质-蛋白质相互作用的新系统的构建。众所周知，G 蛋白伽马亚基 (G伽马) 通过 C 末端的脂质修饰锚定在质膜的内叶上，并且 G 伽马的这种定位是信号转导所必需的。在我们的系统中，缺乏膜定位能力的突变Gγ（Gγ（细胞））是通过删除脂质修饰位点来遗传制备的。当内源Gγ基因已被删除的酵母细胞的细胞质中表达Gγ(细胞)时，观察到G蛋白信号完全消失。为了证明我们的系统的潜在用途，我们利用金黄色葡萄球菌 ZZ 结构域和人免疫球蛋白 G (IgG) 的 Fc 部分作为模型相互作用对。为了设计我们的蛋白质-蛋白质相互作用检测系统，我们改变了 ZZ 结构域，使其与质膜的内部小叶结合，然后将 Fc 部分与 G gamma（细胞）融合。 Fc-G γ（细胞）融合蛋白通过 ZZ-Fc 相互作用向膜迁移，信号转导因此恢复。通过评估 G 蛋白信号传导的生长抑制和转录，成功检测到该信号。最后，制备了几种亲和力常数范围为8.0 x 10(3)至6.8 x 10(8) m(-1)的Z变体，并证明我们的系统能够区分亲和力的细微差异。总之，我们的系统似乎是检测蛋白质-蛋白质相互作用的可靠且通用的技术，并且可能在未来的蛋白质相互作用研究中有用。
In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein gamma subunit (G gamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of G gamma is required for signal transduction. In our system, mutated G gamma (G gamma(cyto)) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when G gamma(cyto) was expressed in the cytoplasm of yeast cells from which the endogenous G gamma gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to G gamma(cyto). The Fc-G gamma(cyto) fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore restored. This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling. Finally, several Z variants displaying affinity constants ranging from 8.0 x 10(3) to 6.8 x 10(8) m(-1) were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity. In conclusion, our system appears to be a reliable and versatile technique for detection of protein-protein interactions, and may prove useful in future protein interaction studies.