We would like to thank Dawn Edmonds for a critical reading of this manuscript. The Stagljar lab is supported by funds from the Canadian Foundation for Innovation (CFI), the Canadian Institute for Health Research (CIHR), the Heart and Stroke Foundation, the Canadian Cancer Society, and Novartis.

1. Background Information
Protein-protein interactions (PPIs) are the fundamental building blocks involved in governing all cellular processes. Consequently, it is essential that all interactions are tightly regulated in order to maintain cellular homeostasis, as a shift in this biological equilibrium commonly plays a role in disease and cancer cell transformation. Membrane associated proteins are amongst the most biologically important class of proteins as they can initiate complex signaling c...

<ol>
	<li>Stagljar, I., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+membrane+protein+interactions+using+yeast-based+technologies.">Analysis of membrane protein interactions using yeast-based technologies.</a> Trends Biochem Sci. 27 (11), 559-563 (2002).</li><li>Iyer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+the+split-ubiquitin+membrane+yeast+two-hybrid+system+to+identify+protein-protein+interactions+of+integral+membrane+proteins.">Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.</a> Sci STKE. 275, pl3-pl3 (2005).</li><li>Paumi, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+protein-protein+interactions+for+the+yeast+ABC+transporter+Ycf1p+by+integrated+split-ubiquitin+membrane+yeast+two-hybrid+analysis.">Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.</a> Mol Cell. 26 (1), 15-25 (2007).</li><li>Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+system+based+on+split-ubiquitin+for+the+analysis+of+interactions+between+membrane+proteins+in+vivo.">A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo.</a> Proc Natl Acad Sci U S A. 95 (9), 5187-5192 (1998).</li><li>Gisler, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein-protein+interactions+between+the+mammalian+integral+membrane+transporters+and+PDZ-interacting+partners+using+a+modified+split-ubiquitin+membrane+yeast+two-hybrid+system.">Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.</a> Mol Cell Proteomics. 7 (7), 1362-1377 (2008).</li><li>Scheper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+N-glycosylation+and+protein+translocation+across+the+endoplasmic+reticulum+membrane+by+Sss1+protein.">Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.</a> J Biol Chem. 278 (39), 37998-38003 (2003).</li><li>Thaminy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+ErbB3-interacting+factors+using+the+split-ubiquitin+membrane+yeast+two-hybrid+system.">Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.</a> Genome Res. 13 (7), 1744-1753 (2003).</li><li>Johnsson, N., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+ubiquitin+as+a+sensor+of+protein+interactions+in+vivo.">Split ubiquitin as a sensor of protein interactions in vivo.</a> Proc Natl Acad Sci U S A. 91 (22), 10340-10344 (1994).</li><li>Kelleher, D. J., Gilmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Saccharomyces+cerevisiae+oligosaccharyltransferase+is+a+protein+complex+composed+of+Wbp1p,+Swp1p,+and+four+additional+polypeptides.">The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides.</a> J Biol Chem. 269 (17), 12908-12917 (1994).</li><li>Chevallier, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+transcriptional+control+of+a+eucaryotic+permease+gene.">Cloning and transcriptional control of a eucaryotic permease gene.</a> Mol Cell Biol. 2 (8), 977-984 (1982).</li></ol>

Igor Stagljar is a co-founder of DualSystems Biotech, Switzerland.

MYTH is the first high throughput system that allows the identification of interactions between full-length membrane proteins and cytosolic or membrane-bound partners. It has been used to study membrane protein from a range of organisms [3-7]. There are, however, specific details that may need to be scrutinized to ensure the protein-of-interest is amenable to study with MYTH.
Many membrane-bound proteins are directed to the plasma membrane via a signal sequence that is subsequently cleaved to...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Polyethenlene Glycol (PEG3350)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>PEG335</td>
<td>&nbsp;</td>
<tr>
<td>Lithium Acetate Bihydrate</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LIA001</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-galactopyranoside)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>XGA001</td>
<td>&nbsp;</td>
<tr>
<td>N`,N-dimethyl formamide</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>DMF 451</td>
<td>&nbsp;</td>
<tr>
<td>3-amino-1,2,4-triazole (3-AT)</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ATT124</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>SPD307</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate monobasic</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP329-500</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA80601-120</td>
<td>&nbsp;</td>
<tr>
<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>GLU501</td>
<td>&nbsp;</td>
<tr>
<td>LB Broth LENOX</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LBL405</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Nitrogen Base</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>YNB406</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>YEX401</td>
<td>&nbsp;</td>
<tr>
<td>Peptone</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>211677</td>
<td>&nbsp;</td>
<tr>
<td>Bio-Tryptone</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>TRP402</td>
<td>&nbsp;</td>
<tr>
<td>Adenine Sulphate</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ADS201</td>
<td>&nbsp;</td>
<tr>
<td>L-Uracil</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>URA241</td>
<td>&nbsp;</td>
<tr>
<td>L-Threonine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>THR002</td>
<td>&nbsp;</td>
<tr>
<td>L-Histidine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>HIS200</td>
<td>&nbsp;</td>
<tr>
<td>L-Methionine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>MET222</td>
<td>&nbsp;</td>
<tr>
<td>L-Valine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>VAL201</td>
<td>&nbsp;</td>
<tr>
<td>L-Phenylalanine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>PHA302</td>
<td>&nbsp;</td>
<tr>
<td>L-Isoleucine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ISO910</td>
<td>&nbsp;</td>
<tr>
<td>L-Tyrosine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>TYR333</td>
<td>&nbsp;</td>
<tr>
<td>L-Leucine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LEU222</td>
<td>&nbsp;</td>
<tr>
<td>L-Arginine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>ARG006</td>
<td>&nbsp;</td>
<tr>
<td>L-Tryptophane</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP395-100</td>
<td>&nbsp;</td>
<tr>
<td>L-Lysine</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>LYS101</td>
<td>&nbsp;</td>
<tr>
<td>L-Alanine</td>
<td>&nbsp;</td>
<td>FisherBiotech</td>
<td>BP369-100</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>AGR001</td>
<td>&nbsp;</td>
<tr>
<td>Soda Lime Galss Beads</td>
<td>&nbsp;</td>
<td>BioSpec Product</td>
<td>11079105</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Bioshop</td>
<td>SLD002</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Watch this Scientific Journal Video about Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions at JoVE.com

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

split-ubiquitin-based-membrane-yeast-two-hybrid-myth-system-powerful

Research

JoVE Journal

Biology

30.8K Views.  University of Toronto. MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.