One- and Two-Hybrid Systems

Easy detection and characterization of proteins interacting with membrane proteins

MoBiTec has extended its two-hybrid product line and is now offering a very innovative yeast two-hybrid system allowing a versatile screening for proteins interacting with membrane-integrated or membrane-associated proteins. This unique technology enables fast and reliable detection of membrane protein binding partners and the identification of their specific binding sites. Up to now, this was not possible using the currently available two-hybrid kits.

Applications

• Identify novel interaction partners of your membrane protein of interest by library screening
  
• Confirm the dimerization of known membrane proteins
  
• Define the critical amino acids of an interacting domain
  
• Generate comprehensive maps of membrane protein interactions

Advantages

• Efficient in vivo screening for specific binding partners of full-length integral membrane and membrane-associated proteins
  
• Posttranslational modifications such as glycosylation and disulfide bond formation are preserved
  
• Protein-protein interactions are identified in their natural compartment - the membrane

Background

MoBiTec's new Y2H Membrane Protein Kit exploits the fact that fusions of ubiquitin and a target protein are recognized and cleaved by ubiquitin-specific proteases (UBPs) after a double glycine motif (Gly-Gly-X) located at the C-terminus of ubiquitin. Unlike many other proteases, UBPs do not recognize a specific polypeptide sequence but instead the folded ubiquitin (Fig. 1a).

Fig 1a

Ubiquitin can be expressed in yeast as an N-terminal (Nub) as well as a C-terminal moiety (Cub) which retain their binding affinity for each other and are able to reassemble spontaneously to form the so-called split-ubiquitin (Fig. 1b).

Fig 1b

If the Nub and Cub moieties are co-expressed within a single cell, a reporter protein (the TF reporter, see below) that is fused to the C-terminus of ubiquitin will be cut off upon re-assembly of the Nub and Cub moieties into split-ubiquitin (Fig. 1c).

Fig 1c

A point mutation in the N-terminal moiety of ubiquitin (NubG) completely destroys the affinity of the moieties for each other. Thus, the separate NubG and Cub moieties are not recognized by ubiquitin-specific proteases (UBPs) and there is no cleavage of the reporter protein (Fig. 1d).

Fig 1d

The bait protein (e.g. the known membrane protein) is fused to the Cub domain that is on his part linked to a reporter protein (TF reporter). On the other hand, a prey protein (e.g. a membrane or a cytosolic protein from a library) is fused to a mutated NubG domain. Interaction of bait and prey brings the NubG and Cub domains close enough together to reconstitute split-ubiquitin, resulting in the release of the reporter protein (red) by the action of the UBPs (Fig 1e).

Fig. 1e

Description

In our Y2H Membrane Protein System, the reporter protein is a fusion of the LexA DNA-binding domain and the Herpes simplex VP16 transactivator (TF reporter complex; Fig. 2). This reporter is fused to the Cub moiety (Cub-TF) which in turn is fused to an integral membrane protein (depicted in red). A transmembrane prey protein (depicted in green) is fused to the NubG moiety. The only requirement is that both Cub as well as NubG are located on the cytoplasmic side of the membrane. Co-expression of Bait-Cub-TF with a non-interacting Prey-NubG does not result in the formation of split-ubiquitin that can be cleaved by UBPs. Consequently, the TF reporter cannot activate lacZ and His3 transcription and cells are His3- and lacZ- - thus not able to grow as blue colonies on medium lacking histidine but containing the β-galactosidase substrate X-Gal (Fig. 2).

Fig 2

If bait and prey interact, Cub and NubG are brought into close proximity so that they can reassemble to split-ubiqutin resulting in cleavage and release of the TF reporter complex. The TF reporter is now free to enter the nucleus, where it can bind to and activate the reporter genes resulting in histidin-prototroph cells that are able to grow on histidin-lacking medium and turn blue in a β-galactosidase assay.


Baits

In the Y2H Membrane Protein System, a given transmembrane bait can either be a transmembrane (TM) protein Type I (C-terminus in the cytosol) or Type II (C-terminus outside the cell or in the lumen of ER, Golgi etc.). 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 is directed to the cytosol, thus generating a Bait-Cub-TF fusion. If the bait to be studied is a Type II TM protein, the TF-Cub portion has to be fused to the N-terminus (TF-Cub-Bait). In both cases, an interaction between an integral membrane protein and a prey protein will result in UBP cleavage after the last amino acid of the Cub domain, thus releasing either TF (in the case of the Type I TM bait) or TF-Cub (in the case of Type II TM bait) (Fig. 3).

Fig. 3

Preys

In the Y2H Membrane Protein System, a given prey protein (or a cDNA library) can be studied in either Y-NubG or NubG-Y orientation (Y: cDNA or genomic DNA insert). 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 that 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 (Fig. 4). On the other hand, cytosolic prey proteins may also be fused with the NubG domain and studied.

Fig. 4

Y2H Membrane Protein System is ideal if you intend to screen for interaction partners of integral membrane proteins or membrane-associated proteins. Unlike the yeast two-hybrid system, the Y2H Membrane Protein System is not dependent on the localization of bait and prey in the nucleus. Using the Y2H Membrane Protein System you can screen full-length, integral membrane proteins, as well as membrane-associated proteins, against a cDNA library to identify and characterize novel interaction partners of your protein of interest.

The Y2H Membrane Protein Kit includes

• optimized bait and prey vectors,
  
• the yeast reporter strain NMY32 with two growth and one color marker enabling increased stringency of selection resulting in fewer false positives,
  
• a set of control plasmids for functional assay and detection by Western blot,
  
• and a complete step-by-step manual guiding you on how to perform a Y2H Membrane Protein screen.
  

In addition to the Y2H Membrane Protein Kit we also offer the respective bait and prey vectors separately as well as vectors for conventional yeast two-hybrid studies, general yeast expression vectors, yeast strains and cDNA libraries for use with our Y2H Membrane Protein System and conventional yeast two-hybrid systems.