Kail Mark, Barnekow Angelika
Department of Experimental Tumor Biology, University of Muenster, Muenster, Germany.
Methods Mol Biol. 2008;440:111-25. doi: 10.1007/978-1-59745-178-9_9.
Much of our knowledge of the mechanisms governing vesicular transport has come from the combination of genetic and biochemical approaches that have identified Ypt/Rab guanosine 5'-triphosphatases (GTPases) as key components of transport processes in both yeast and mammalian cells. More recently, research has focused on establishing the complex protein-protein interactions necessary for the regulation of vesicular transport by a variety of methods, including the yeast two-hybrid interaction assay. A central component of the signaling pathway regulated by Ypt/Rab proteins is the GTPase cycle, in which the proteins cycle between an active guanosine 5'-triphosphate (GTP)-bound form and an inactive guanosine 5'-diphosphate (GDP)-bound form. Alterations in the conformation of the Ypt/Rab proteins when either GTP or GDP is bound specify the interaction of effector proteins and influence membrane binding. Our work has focused on identifying interacting partners for the GTPases Rab1 and Rab6 and their isoforms, which regulate transport steps between the endoplasmic reticulum and Golgi in mammalian cells. We have employed both active (GTP-bound) and inactive (GDP-bound) Rab1 and Rab6 mutants to identify potential new interacting proteins using the yeast two-hybrid system and have verified these interactions using alternative methods.
我们对囊泡运输调控机制的许多了解都来自于遗传学和生物化学方法的结合，这些方法已确定Ypt/Rab鸟苷5'-三磷酸酶（GTP酶）是酵母和哺乳动物细胞运输过程的关键组成部分。最近，研究集中在通过多种方法建立囊泡运输调控所需的复杂蛋白质-蛋白质相互作用，包括酵母双杂交相互作用分析。由Ypt/Rab蛋白调控的信号通路的一个核心组成部分是GTP酶循环，其中蛋白质在活性鸟苷5'-三磷酸（GTP）结合形式和非活性鸟苷5'-二磷酸（GDP）结合形式之间循环。当结合GTP或GDP时，Ypt/Rab蛋白构象的改变决定了效应蛋白的相互作用并影响膜结合。我们的工作集中在鉴定GTP酶Rab1和Rab6及其异构体的相互作用伙伴，它们在哺乳动物细胞中调节内质网和高尔基体之间的运输步骤。我们使用活性（GTP结合）和非活性（GDP结合）的Rab1和Rab6突变体，通过酵母双杂交系统鉴定潜在的新相互作用蛋白，并使用其他方法验证了这些相互作用。
