Outline of the split-ubiquitin membrane yeast two-hybrid system. (A) A membrane bait protein of interest X is fused to Cub followed by an artificial transcription factor (TF), while another membrane (or cytoplasmic) protein Y is fused to the NubG domain (Y-NubG). On interaction of the X and Y proteins, ubiquitin reconstitution occurs, leading to proteolytic cleavage by UBPs, and the subsequent release of the transcription factor. This factor activates reporter genes to result in HIS3+/lacZ+ yeast cells. (B) If X and Y do not interact, there is no ubiquitin reconstitution and thus no UBP-mediated cleavage, resulting in HIS-/lacZ- yeast cells.
