Construction And Utilization Of Split-ubiquitin Membrane Yeast Two-hybrid System

It is revealed that approximately one-third of the predicted proteins of a given organism are likely to be associated with membranes.These membrane-associated proteins perform a wide range of essential cellular functions,including channels and transporters which facilitate the exchange of molecules,cell survival,differentiation and adhesion,cell signaling.Because of their central positions in mediating signaling, membrane proteins are considerable diagnostic and pharmacological targets today.Interactions among proteins are essential for proper cellular functioning.Various protein interactions are involved in the regulation and execution of biochemical pathways within cells.The study of protein-protein interactions has been greatly facilitated by the invention of yeast two-hybrid technology which is the most powerful genetic method to explore the protein interactions.Since its description, various modifications of the YTH system have been described,which include one-hybrid,reverse two-hybrid,et al.Despite significant progress in development of the yeast two-hybrid system,the analysis of interactions between membrane proteins remained a significant challenge because of the hydrophobic nature of these proteins. In addition,the two-hybrid system has limitations since it requires the interacting proteins locating in the nucleus to detect the interaction.The invention of split-ubiquitin assay is a more useful technology for the in vivo detection of interactions between membrane proteins.The split-ubiquitin membrane yeast two-hybrid system takes advantage of the ability of the N- and C-terminal halves of ubiquitin,Nub and Cub,to reassemble into a split-ubiquitin in vivo spontaneously.Ubiquitin-specific proteases(UBPs) recognize such reconstituted split-ubiquitin and cleave off a protein that is linked to Cub.A point mutation in the Nub half(Ile13 to Glyl3) abolishes the ability the two halves to reconstitute the split-ubiquitin.However,if the two ubiquitin halves are linked to two proteins that interact with each other,the interaction brings the mutated Nub half and Cub half close and split-ubiquitin reconstitution is recovered.If an artificial transcription factor(TF) fused to the Cub,it can be cleaved off to enter the nucleus, leading to the activation of the yeast reporter genes.In this study,we have generated a set of split-ubiquitin membrane yeast two-hybrid system which contains a pBait vector,a pPrey vector and a positive control plasmid.The system has been verified by LASS2 and VPL,a pair of human membrane proteins interacting with each other.We have demonstrated the effectiveness of this approach using either BNIP3 or Wag31 as bait,and have identifiedACAA2,Acetyl-Coenzyme A acyltransferase 2,or XCL2,a member of C chemokines,as the binging partners of BNIP3 or Wag31,separately.We expect this system to be valuable for the identification of interacting partners of membrane proteins,and it may also prove useful for drug discovery purposes.