Detecting interactions between membrane proteins in Vivo using chimeras

Abstract

Publisher Summary This chapter describes a method that can detect interactions of membrane proteins with other soluble or membrane proteins in vivo . The method employs the split-ubiquitin technique. This technique is based on the ability of the N- and C-terminal fragments of ubiquitin (Nub and Cub) to assemble into quasi-native ubiquitin (splitubiquitin). Ubiquitin-specific proteases (UBPs) do not recognize Nub or Cub alone, but recognizes the reconstituted split-ubiquitin. The UBPs cleave a reporter protein attached to the C terminus of Cub from the assembled split-ubiquitin. Analogous to the two-hybrid system, the libration of the reporter serves as a readout indicating the reconstruction of ubiquitin. The system is designed in such a way that productive association of Nub and Cub is prevented unless the two ubiquitin halves are linked to proteins that interact in vivo . Knowledge of the precise interactions of a given protein is crucial for the understanding of its functions. While identification of protein interactions initially occurred almost solely by the use of biochemical methods, recently yeast two-hybrid systems have been employed as powerful genetic tools to find proteins that interact specifically with a protein of interest.