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Y2H Membrane Pairwise Interaction Kit

Y2H Membrane Pairwise Interaction Kit
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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: Split-Ubiquitin System

MoBiTec's Y2H Membrane Pairwise Interaction 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).

yeast two hybrid membran protein splitting ubiquitin

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).

yeast two hybrid membran protein system Ubiquitin NubG

Fig. 1e

Description

In our Y2H Membrane Pairwise Interaction Kit, 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).

yeast two hybrid ubiquitin Protein Membran interaction

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 Pairwise Interaction Kit, 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 Pairwise Interaction Kit, 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 Pairwise Interaction Kit 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 Pairwise Interaction Kit is not dependent on the localization of bait and prey in the nucleus. Using the Y2H Membrane Pairwise Interaction Kit 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 Pairwise Interaction Kit includes

Optimized bait and prey vectors,
The yeast reporter strain NMY51 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 Pairwise Interaction Kit screen.

In addition to the Y2H Membrane Pairwise Interaction 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 Pairwise Interaction Kit and conventional yeast two-hybrid systems.

Order Information

Y2H Membrane Pairwise Interaction Kit
	Introduction
	Order Information
	Accessory Products
	Downloads
	Literature
	Support
		FAQ
		Protein Prediction Tools
		Putative false positive interactors

ORDER INFORMATION, SHIPPING & STORAGE:
order#	description	amount
P01501DS	Y2H Membrane Pairwise Interaction Kit	1 kit
	Kit components: pBT3-C - Type I bait vector pBT3-SUC - Type I bait vector pBT3-STE - Type I bait vector pBT3-N - Type II bait vector pPR3-N - prey vector pPR3-C - prey vector pPR3-SUC - prey vector pPR3-STE - prey vector pTSU2-APP positive control bait pOst1-NubI control prey pNubG-Fe65 positive control prey NMY51 yeast reporter strain Y2H Trafo kit P01003DS HTX β-galactosidase assay kit Bait and prey vectors also include constructs with yeast leader and Type I signal sequence. *supplied as purified and lyophilized plasmid	5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* 5 µg* lyophilized culture 5 library scale transformations or 80 small scale transformations 4 x 96 assays
Vectors to be used with the Y2H Membrane Pairwise Interaction Kit:
P03230DS	pBT3-N , type II bait vector	5 µg
P03231DS	pBT3-C, type I bait vector	5 µg
P03232DS	pBT3-SUC, type I bait vector	5 µg
P03233DS	pBT3-STE, type I bait vector	5 µg
P03234DS	pPR3-N prey library vector (NubG-cDNA orientation)	5 µg
P03235DS	pPR3-C prey library vector (cDNA-NubG orientation)	5 µg
P03236DS	pPR3-SUC prey library vector (cDNA-NubG orientation)	5 µg
P03237DS	pPR3-STE prey library vector (cDNA-NubG orientation)	5 µg
shipped on dry ice; store vectors and primers at -20°C yeast strain at -70°C

Accessory Products

Y2H Membrane Pairwise Interaction Kit
	Introduction
	Order Information
	Accessory Products
	Downloads
	Literature
	Support
		FAQ
		Protein Prediction Tools
		Putative false positive interactors

ORDER INFORMATION, SHIPPING & STORAGE:
order#	description	amount
P01002DS	HTX beta-galactosidase assay kit	Kit
P01003DS	Y2H Yeast transformation kit	Kit
P01013DS	TOTAL protein extraction kit	Kit
P06001DS	Single-stranded carrier DNA	1
P09001DS	Media package	1 Pack
P06004DS	Mouse anti-LexA antibody	1
P06005DS	Mouse anti-HA antibody	1

Downloads

Y2H Membrane Pairwise Interaction Kit
	Introduction
	Order Information
	Accessory Products
	Downloads
	Literature
	Support
		FAQ
		Protein Prediction Tools
		Putative false positive interactors

Download: Y2H Membrane Pairwise Interaction Kit Handbook (pdf, 1.327 KB, update 05.2010 )

Please download vector maps and sequences:

Vector Order#	Description	*Yeast /E. coli* Marker**	Map (pdf)	Sequence (txt)
pBT3-N #P03230DS	Type II bait vector (N-terminal fusion). cDNAs are expressed as fusions to the C-terminus of the LexA-VP16-Cub reporter cassette (e.g. LexA-VP16-Cub-BAIT). Suitable for cloning type II transmembrane proteins as baits.	LEU2/Kan	pBT3-N (758 KB)	pBT3-N.txt (8 KB)
pBT3-C #P03231DS	Type I bait vector (C-terminal fusion). cDNAs are expressed as fusions to the N-terminus of Cub-LexA-VP16. Expression is driven by the weak CYC1 promoter.	LEU2/Kan	pBT3-C (877 KB)	pBT3-C.txt (8 KB)
pBT3-SUC #P03232DS	Type I bait vector (C-terminal fusion). Contains a signal sequence derived from the invertase (SUC2) gene, followed by a multiple cloning site and Cub-LexA-VP16. Use pCCW-SUC if your bait is a type I mammalian transmembrane protein.	LEU2/Kan	pBT3-SUC (745 KB)	pBT3-SUC.txt (8 KB)
pBT3-STE #P03233DS	Type I bait vector (C-terminal fusion). Contains the N-terminal 15 amino acids from the yeast Ste2 protein, followed by a multiple cloning site and Cub-LexA-VP16. Addition of the Ste2 sequence may enhance expression of certain baits in yeast. Expression is driven by the weak CYC1 promoter.	LEU2/Kan	pBT3-STE (744 KB)	pBT3-STE.txt (8 KB)
pPR3-N #P03234DS	Library vector (NubG-cDNA orientation). cDNAs or cDNA libraries are expressed as fusions to the C-terminus of NubG. Expression is driven by the weak CYC1 promoter.	TRP1/Amp	pPR3-N (746 KB)	pPR3-N.txt (7 KB)
pPR3-C #P03235DS	Library vector (cDNA-NubG orientation). cDNAs or cDNA libraries are expressed as fusions to the N-terminus of NubG. Expression is driven by the strong ADH1 promoter.	TRP1/Amp	pPR3-C (746 KB)	pPR3-C.txt (7 KB)
pPR3-SUC #P03236DS	Library vector (cDNA-NubG orientation). Contains a signal sequence derived from the invertase (SUC2) gene, followed by a multiple cloning site and NubG. Use pPR3-SUC to clone type I mammalian transmembrane proteins as fusions to the SUC2 leader sequence. Expression is driven by the strong ADH1 promoter.	TRP1/Amp	pPR3-SUCE (746 KB)	pPR3-SUC.txt (7 KB)
pPR3-STE #P03237DS	Library vector (cDNA-NubG orientation). Contains the N-terminal 15 amino acids from the yeast Ste2 protein, followed by a multiple cloning site and NubG. Addition of the Ste2 sequence may enhance expression of certain preys in yeast. Expression is driven by the strong ADH1 promoter.	TRP1/Amp	pPR3-STE (746 KB)	pPR3-STE.txt (7 KB)
pTSU2-APP	Control bait expressing a fusion of the yeast resident membrane protein App to Cub-LexA-VP16. Expression is driven by the TEF1 promoter.	LEU2/Kan	pCCW-Alg5 (741 KB)	pCCW-Alg5.txt (9 KB)
pOst1-NubI	Control prey expressing a fusion of the yeast resident ER protein Ost1 to the wild type Nub portion of yeast ubiquitin. Expression is driven by the strong ADH1 promoter.	TRP1/Amp	pAI-Alg5 (738 KB)	pAI-Alg5.txt (8 KB)
pNubG-Fe65	Control prey expressing the cytosolic protein Fe65 fused to NubG. Expression is driven by the strong ADH1 promoter.	TRP1/Amp	pDL2-Alg5 (739 KB)	pDL2-Alg5.txt (8 KB)

Y2H Membrane Pairwise Interaction Kit
	Introduction
	Order Information
	Accessory Products
	Downloads
	Literature
	Support
		FAQ
		Protein Prediction Tools
		Putative false positive interactors

Literature

Reviews about the Y2H Membrane Pairwise Interaction Kit

Suter, B., Auerbach, D., and Stagljar, I. (2006): Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond.
Biotechniques, May 2006; 40(5): 625-44. Pubmed
Thaminy S, Miller J, Stagljar I. (2004):The split-ubiquitin membrane-based yeast two-hybrid system.
Methods Mol Biol. 2004;261:297-312. Pubmed
Auerbach, D., Fetchko, M, and Stagljar, I. (2003): Proteomic approaches for generating comprehensive protein interaction maps
Targets (Drug Discovery Today) 2, 85-92 (2003) Download (PDF 427 kb)
Auerbach, D., Thaminy, S., Hottiger, M., and Stagljar, I. (2002): The post-genomic era of interactive proteomics: Facts and perspectives
Proteomics 2, 611-623 (2002) Download (PDF 283 kb)
Auerbach, D., Galeuchet-Schenk, B., Hottiger, M.O., and Stagljar, I. (2002): Genetic approaches to the identification of interactions between membrane proteins in yeast
J Recept Signal Transduct Res 22, 473-483 (2002) Download (PDF 137 kb)
Stagljar, I. and Fields, S.
Analysis of membrane protein interactions using yeast-based technologies
Trends Biochem Sci 27, 559-563 (2002) Download (PDF 6,9 MB)

Original publications using the Y2H Membrane Pairwise Interaction Kit

Zheng, J. et al.(2006): Analysis of the oligomeric structure of the motor protein prestin.
J Biol Chem. 2006 May 8; [Epub ahead of print] Pubmed Free
Matsuda, S. et al.(2005): The Familial Dementia BRI2 Gene Binds the Alzheimer Gene Amyloid- Precursor Protein and Inhibits Amyloid- Production
J Biol Chem. 2005 Aug 12;280(32):28912-6. Epub 2005 Jun 27. Pubmed Free
Miller, J. P. et al. (2005): Large-scale identification of yeast integral membrane protein interactions
PNAS, Aug 2005; 102: 12123 - 12128. Pubmed free
Mo, C. et al.(2005): Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex
J Lipid Res. 2005 Sep;46(9):1991-8. Epub 2005 Jul 1.Pubmed Free
Pasch, J.C. et al. (2005): The yeast split-ubiquitin system to study chloroplast membrane protein interactions
Appl Microbiol Biotechnol. 2005 Dec;69(4):440-7. Epub 2005 Jun 30. Pubmed
Yan, A. et al.(2005): Studies of yeast oligosaccharyl transferase subunits using the split-ubiquitin system: topological features and in vivo interactions
Proc Natl Acad Sci U S A. 2005 May 17;102(20):7121-6. Epub 2005 May 10. Pubmed Free
Chavan, M.M. et al. (2005): Subunits of the translocon interact with components of the oligosaccharyl transferase complex.
J Biol Chem. 2005 Jun 17;280(24):22917-24. Epub 2005 Apr 14. Pubmed Free
Mo, C. et al. (2004): The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p).
Biochim Biophys Acta. 2004 Nov 8;1686(1-2):30-6. Pubmed
Vitale, R. et al. (2004): Use of the Split-Ubiquitin Two-Hybrid System to Identify Proteins Interacting With the Alzheimer Proteins APP and LRP.
Biol. Bull., Oct 2004; 207: 167. Highwire Free
Wang, B. et al. (2004): The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.
Mol Cell Biol. 2004 Apr;24(7):2767-78. Pubmed Free
Pandey, S. et al. (2004): The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling.
Plant Cell. 16(6):1616-32 (2004) Highwire Free
Obrdlik et al. (2004): K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions.
Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12242-7. Epub 2004 Aug 6. Pubmed Free
Thaminy, S. et al. (2003): Identification of Novel ErbB3-interacting Factors Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System
Genome Res. 2003 Jul;13(7):1744-53. Pubmed Free
Reinders, A. et al.(2002): Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays
Structure. 2002 Jun;10(6):763-72. Pubmed
Stagljar et al. (1998): A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5187-92. Pubmed Free