Auth Tanja, Schlüter Sharazad, Urschel Stephanie, Kussmann Petra, Sonntag Stephan, Höher Thorsten, Kreuzberg Maria M, Dobrowolski Radoslaw, Willecke Klaus
Institute of Genetics, Division of Molecular Genetics, University of Bonn, 53117 Bonn, Germany.
Exp Cell Res. 2009 Apr 1;315(6):1053-62. doi: 10.1016/j.yexcr.2008.12.025. Epub 2009 Jan 13.
Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.
几乎在所有组织中，间隙连接介导细胞间的电通讯和代谢通讯，并被认为在细胞生长控制、分化和胚胎发育中发挥重要作用。间隙连接通讯和通道组装被认为受连接蛋白与包括激酶和磷酸酶在内的不同蛋白质相互作用的调节。在此，我们在酵母双杂交蛋白相互作用筛选中鉴定出肿瘤易感基因101（TSG101）蛋白与连接蛋白45的羧基末端尾巴结合。谷胱甘肽S-转移酶下拉实验和免疫沉淀表明，在下拉分析中，不仅连接蛋白45，而且连接蛋白30.2、-36和-43的羧基末端区域都与TSG101蛋白相关，并且在HM1胚胎干细胞的裂解物中，连接蛋白31、-43和-45与内源性TSG101蛋白共沉淀。TSG101已被证明参与细胞周期控制、转录调控和内吞蛋白的周转。因此，我们决定研究这种相互作用的功能作用。在HM1胚胎干细胞中，小干扰RNA介导的TSG101敲低导致连接蛋白43和-45水平升高、这些连接蛋白的半衰期延长以及显微注射的荧光素黄转移增加。我们的结果表明，TSG101通过与连接蛋白相互作用参与连接蛋白的降解。
