Hlubek Andrea, Schink Kay O, Mahlert Michael, Sandrock Björn, Bölker Michael
Philipps-University Marburg, Department of Biology, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany.
Mol Microbiol. 2008 May;68(3):615-23. doi: 10.1111/j.1365-2958.2008.06177.x.
The highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis, Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41-56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo. Expression of Rac1(W56F) in Delta cdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1(W56F) is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo.
高度保守的GTP结合蛋白Cdc42和Rac1调节胞质分裂、细胞极性的建立和囊泡运输。在二态真菌玉米黑粉菌中，Rac1是细胞极性和出芽所必需的，而Cdc42是胞质分裂期间细胞分离所必需的。在缺乏Dbl家族鸟嘌呤核苷酸交换因子（GEF）Don1的突变体中也观察到相同的细胞分离缺陷。我们生成了一系列由Cdc42和Rac1的不同部分组成的嵌合GTP结合蛋白。体内互补分析表明，包含氨基酸41-56的短区域决定了信号特异性。值得注意的是，在这个特异性结构域内第56位的单个氨基酸替换足以在体内赋予Rac1 Cdc42功能。在Δcdc42突变细胞中表达Rac1（W56F）导致细胞分离缺陷的互补。体外GDP/GTP交换分析表明，Dbl家族GEF Don1对Cdc42具有高度特异性，不能激活Rac1。然而，如果将Rac1（W56F）用作底物，Don1能够刺激GDP/GTP交换。这些数据共同表明，GEF Don1的激活是体内Cdc42特异性信号传导的重要决定因素。
