In the DUALmembrane system, the reporter protein is a fusion of the DNA-binding domain of the LexA protein and the Herpes simplex VP16 transactivator. The reporter is fused to the Cub moiety, which in turn is fused to an integral membrane protein (depicted in red, called Cub-TF). A transmembrane prey protein (depicted in green) is fused to the NubG moiety. The only requirement is that both Cub and NubG are located on the cytoplasmic face of the membrane. Co-expression of Bait-Cub-TF with a non-interacting Prey-NubG does not lead to the formation of split-ubiquitin nor cleavage by UBPs, resulting in cells that are His3- and lacZ- (left panel).

If bait and prey interact, Cub and NubG are brought into close proximity, where they will form split-ubiqutin, resulting in cleavage and liberation of the TF reporter. The reporter is now free to enter the nucleus, where it will bind and activate the reporter genes to result in cells that are His+ and that turn blue in a ß-galactosidase assay.

 

 

 

DUALmembrane baits

In the DUALmembrane system, a given transmembrane bait can either be a Type I (C-terminus in the cytosol) or Type II (C-terminus outside the cell or in the lumen of ER, Golgi etc.) transmembrane (TM) protein. In the case of a Type I TM protein, the Cub-TF portion is fused to its C-terminus so that the Cub-TF portion faces the cytosol, thus generating a Bait-Cub-TF fusion. If a bait to be studied in the DUALmembrane system is a Type II TM protein, the TF-Cub portion has to be fused to N-terminus (TF-Cub-Bait). In both cases, an interaction between an integral membrane protein and a prey protein will result in the cleavage after the last amino acid of the Cub domain, thus liberating either TF (in the case of the Type I TM bait) or TF-Cub (in the case of Type II TM bait).

DUALmembranes preys

In the Dual membrane system, a given prey protein (or a cDNA library) can be studied in either Y-NubG (where Y is cDNA or genomic DNA insert) or NubG-Y orientation. In the case of a Type I TM prey, the cDNA (or a library of cDNAs encoding potential interactors) is fused at its C-terminus with the NubG domain, whereas in the case of a Type II prey, it is fused at its N-terminus with the NubG domain. In this way, it is possible to identify both Type I (Y-NubG orientation) and Type II (NubG-Y orientation) transmembrane proteins that interact with a particular membrane bait protein.