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

Summary

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.

Abstract

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 (C_ub) and the N-terminal half of the ubiquitin moiety (N_ub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to C_ub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the N_ub 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.

Protocol

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 cascades, and mediate both the import and export of various molecules, including drugs, which has been of recent significance in the field of health ca....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethenlene Glycol (PEG3350)	BioShop Canada	PEG335
Lithium Acetate Bihydrate	BioShop Canada	LIA001
X-Gal (5-Bromo-4-Chloro-3-Indolyl-b-D-galactopyranoside)	BioShop Canada	XGA001
N`,N-dimethyl formamide	BioShop Canada	DMF 451
3-amino-1,2,4-triazole (3-AT)	BioShop Canada	ATT124
Sodium phosphate dibasic	BioShop Canada	SPD307
Sodium phosphate monobasic	Fisher Scientific	BP329-500
Salmon Sperm DNA	VWR international	CA80601-120
D-Glucose	BioShop Canada	GLU501
LB Broth LENOX	BioShop Canada	LBL405
Yeast Nitrogen Base	BioShop Canada	YNB406
Yeast Extract	BioShop Canada	YEX401
Peptone	BD Biosciences	211677
Bio-Tryptone	BioShop Canada	TRP402
Adenine Sulphate	BioShop Canada	ADS201
L-Uracil	BioShop Canada	URA241
L-Threonine	BioShop Canada	THR002
L-Histidine	BioShop Canada	HIS200
L-Methionine	BioShop Canada	MET222
L-Valine	BioShop Canada	VAL201
L-Phenylalanine	BioShop Canada	PHA302
L-Isoleucine	BioShop Canada	ISO910
L-Tyrosine	BioShop Canada	TYR333
L-Leucine	BioShop Canada	LEU222
L-Arginine	BioShop Canada	ARG006
L-Tryptophane	Fisher Scientific	BP395-100
L-Lysine	BioShop Canada	LYS101
L-Alanine	Fisher Scientific	BP369-100
Agar	BioShop Canada	AGR001
Soda Lime Galss Beads	Biospec Products	11079105
Sodium Chloride	BioShop Canada	SLD002