Split ubiquitin systems

This is a yeast two-hybrid system used to detect protein-protein interactions between non-nuclear or insoluble proteins. For further information, please refer to Obrdlik et al. 2004, PNAS, vol. 101, no. 33, pp. 12242-12247.

The ubiquitin protein is split into two halves – the N-terminal “Nub” (from N-ubiquitin, amino acids 1-34) and the C-terminal “Cub” (from C-ubiquitin, amino acids 35-76). NubG is a mutant with decreased affinity for Cub; functional ubiquitin is reconstituted only when NubG and Cub are in closed proximity to each other. The C terminus of Cub is fused to an artificial transcription factor, protein A-LexA-VP16 (PLV). The Cub-PLV and NubG peptides are fused to bait and prey proteins, respectively.

If bait and prey proteins interact, Cub-PLV and NubG are brought into close vicinity. Functional ubiquitin is thereby reconstituted and recognized by ubiquitin-specific proteases (USPs). The activity of USPs leads to the release of PLV and the activation of LexA-driven reporter genes in the nucleus.