Ljubljana/splitubiquitinassay

Split Ubiquitin Assay

Yeast two-hybrid system is widely used to detect the interactions between proteins, e.g. to determine the interactome. For integral membrane proteins most systems don't work and one of the few succesfull techniques is the split ubiquitin system. This assay, first described by Johnsson and Varshavsky (Johnsson and Varshavsky, 1994) is usually used for studying yeast membrane protein interactions. Ubiquitin can be expressed as N-terminal (Nub) and C-terminal (Cub) half, which retain affinity for each other, and assemble into functional split ubiquitin. The assembling takes place only if both parts are brought together, thus applying an additional step, which can be controlled during the experiment, thus preventing spontaneous activation of the system. Another protein (e.g. reporter or effector) can be linked onto the Cub half without of major influence on ubiquitin assembly. The formed complex Nub-Cub-reporter is recognized by the ubiquitin specific protease and the reporter protein is cleaved off. Usually a transcription factor is used instead of the reporter protein, which then induces the promoter and starts transcription of a reporter gene. The system was developed to work in yeast cells, but it hasn't been shown before to work in mammalian membrane proteins.