Identification and characterization of interacting partners of Rab GTPases by yeast two-hybrid analyses

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.