pCCW-Alg5膜蛋白酵母双杂交载体对照诱饵质粒-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pBT3-C, pBT3-C, pPR3-N, pPR3-C plasmids

BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

The yeast two-hybrid membrane protein system was developed to identify and characterize

protein-protein interactions between integral membrane proteins, membrane-associated

proteins and soluble proteins in their natural setting.

Unlike the yeast two-hybrid system, there is no need for your bait to be transported to the

nucleus and therefore, interactions between full-length integral membrane proteins can be

readily detected in situ at the cellular membrane.

The yeast two-hybrid membrane protein kit version 3 contains improved vectors which

help you express mammalian integral membrane proteins or membrane-associated proteins

in yeast for screening against cDNA libraries, an expanded manual and a trial size HTX β-

galactosidase assay kit for fast, quantitative determination of β-galactosidase levels in yeast.

Y2H membrane cDNA libraries

The yeast two-hybrid membrane protein system can be used to assay the interaction

between two known proteins or to find novel interaction partners of a protein of interest by

screening cDNA libraries.

How does the Y2H membrane protein system work?

The Y2H membrane protein system takes advantage of a classical protein complementation

assay, the split-ubiquitin system (Johnsson and Varshavsky, 1994), to detect the interaction

of two heterologously expressed fusion proteins in the yeast Saccharomyces cerevisiae. In

contrast to the most popular yeast based screening system, the yeast two-hybrid system

(Fields and Song, 1989), there is no requirement for the interacting proteins to be located in

the nucleus. Therefore, the Y2H membrane protein system allows investigation of proteinprotein

interactions involving full-length integral membrane proteins or membrane-associated

proteins in their natural setting at the cellular membrane.

Advantages of the Y2H membrane protein system are:

Full-length proteins can be assayed

Interactions are detected at the cellular membrane

Interactions involving integral membrane proteins, membrane-associated proteins and

soluble proteins can be detected

Post-translational modifications such as glycosylation are preserved

2 How to carry out a Y2H membrane screen

YEAST TWO HYBRID MEMBRANE

The split-ubiquitin mechanism Target

The Y2H membrane protein system is based on the reconstitution of ubiquitin, a small and

highly conserved protein which tags other proteins for degradation (Hershko, 2005; Mayer,

2000). When an intracellular protein is destined for degradation, a series of enzymatic

reactions covalently attaches a chain of ubiquitin molecules to this protein (Figure 1A). The

poly-ubiquitin tagged protein is then transported to the 26S proteasome, where it is degraded

(Figure 1B). To recycle the ubiquitin, the cell has evolved a mechanism to cleave the polyubiquitin

chain off the target protein. This process is mediated by so-called ubiquitin specific

proteases (UBPs). The UBPs specifically recognize intact, folded ubiquitin and cleave the

polypeptide chain after the last residues of ubiquitin (a Gly-Gly motif). Free monomeric ubiquitin

is thus released back into the cytosol, whereas the target protein is degraded.

The Yeast Two-Hybrid Membrane Protein System

The Y2H membrane (Stagljar et al., 1998; Thaminy et al., 2003) system takes advantage of

the split-ubiquitin mechanism to measure the interaction between an integral membrane

protein and its interaction partners (either integral membrane proteins or soluble proteins).

As the interaction is detected in situ at the membrane, the assay represents a more

physiological situation than a conventional yeast two-hybrid assay, where only subdomains

of integral membrane proteins can be assayed and the interaction takes place in the nucleus.

A first membrane protein of interest (the bait) is fused to the C-terminal half of ubiquitin (Cub)

and the artificial transcription factor LexA-VP16 (Figure 4A). A second protein of interest (the

prey) is fused to the mutated N-terminal half of ubiquitin (NubG, Figure 4B). If bait and prey

interact NubG and Cub are forced into close proximity, resulting in the reconstitution of splitubiquitin.

Split-ubiquitin is immediately recognized by UBPs which then cleave the polypeptide

chain between Cub and LexA-VP16. As a result, the artificial transcription factor is released

from the membrane and translocates to the nucleus where it binds to the LexA operators

situated upstream of a reporter gene via its LexA DNA binding domain. The VP16 transactivator

domain then recruits the RNA polymerase II complex to the transcriptional start of the reporter

gene, resulting in its transcriptional activation (Figure 4C). The reporter genes used in the

Y2H membrane protein system are two auxotrophic growth markers (HIS3 and ADE2), whose

activation enables the yeast to grow on defined minimal medium lacking histidine or adenine,

and lacZ, encoding the enzyme β-galactosidase. Thus, the interaction between two proteins

at the membrane of yeast is translated into a transcriptional readout, resulting in growth

of yeast on selective medium and color development in a β-galactosidase assay.

What are the applications of the Y2H membrane protein system?

The Y2H membrane protein kit is intended for the detection and identification of interactions

involving integral membrane proteins and membrane-associated proteins in yeast.

The kit allows you to:

Investigate the interaction between a membrane protein (integral or membraneassociated)

and a membrane protein or soluble protein

Map domains or amino acids which are critical for an interaction

Screen cDNA libraries using a membrane protein as a bait to find novel interacting

proteins

cDNA library screening

Together with the use of NubG-fused cDNA libraries, the Y2H membrane protein system is

capable of identifying novel interaction partners of a given integral membrane protein of

interest. Both cytosolic interaction partners and membrane proteins can be identified in a

screen. cDNA libraries are available in two orientations: NubG-x (expressing fusion proteins

with N-terminal NubG) and x-NubG (expressing fusion proteins with C-terminal NubG). Please

visit www.mobitec.com for premade Y2H membrane protein system cDNA libraries.

Published literature

Below, we have listed some publications citing the use of the Y2H membrane protein system

for investigating pairwise interactions or finding novel protein interactions by screening cDNA

libraries. An updated list of publications can be found at www.mobitec.com

Interactions between defined proteins

Miller, J.P. et al. (2005) Large-scale identification of yeast integral membrane protein

interactions. Proc. Natl. Acad. Sci. USA 102:12123-1218

Yan, A. et al. (2005) Studies of yeast oligosaccharyl transferase subunits using the splitubiquitin

system: topological features and in vivo interactions. Proc. Natl. Acad. Sci. USA

102:7121-7126

Pandey, S. and Assmann, S.M. (2004) The Arabidopsis putative G protein-coupled receptor

GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling.

Plant Cell. 16:1616-1632

Library screens

Matsuda, S. et al. (2005) The familial dementia BRI2 gene binds the Alzheimer’s gene APP

and inhibits Abeta production. J. Biol. Chem. 280:28912-28916

Vitale, R. and Buxbaum, J.D. (2004) Use of the split-ubiquitin two-hybrid system to identify

proteins interacting with the Alzheimer proteins APP and LRP. Biol. Bull. 207:167

Wang 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. 24:276727-78

Overview of a Y2H membrane screen

A Y2H membrane screen is divided into three major parts:

Bait construction and expression verification

Library transformation and selection of positive clones

Confirmation of interactors and sequence analysis