Using the Split-Ubiquitin Yeast Two-Hybrid System to Test Protein–Protein Interactions of Transmembrane Proteins

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1242)

Abstract

Proteins are responsible for many biological processes within living organisms. Many proteins have the ability to specifically interact with other proteins in order to function properly. The identification of protein–protein interactions (PPIs) can provide useful information about the function of a protein of interest. Historically, the properties of transmembrane proteins have caused difficulty in analyzing PPIs among transmembrane proteins. The development of an assay that is capable of analyzing PPIs involving transmembrane proteins, the split-ubiquitin yeast two-hybrid (SU-Y2H) assay, has provided a method to probe pairwise PPIs between two proteins of interest or to screen a single protein of interest for interaction partners. The following protocol explains how to use the SU-Y2H assay, which is compatible with the use of transmembrane proteins, to investigate PPIs between two proteins of interest and also briefly describes how to adjust the system to be used as a high-throughput screen for interaction partners of a particular protein of interest.

Key words

Split-ubiquitin Yeast two-hybrid Protein–protein interaction Transmembrane protein Yeast 

Abbreviations

3-AT

3-Amino-1,2,4-triazole

ADE2

Phosphoribosylaminoimidazole carboxylase gene in the adenine biosynthesis pathway

ADHp

Promoter of yeast alcohol dehydrogenase 1

AmpR

Ampicillin resistance gene

ccdb

Gene encoding cytotoxic gene

CmR

Chloramphenicol resistance gene

Cub

C-terminal half of ubiquitin

ddH2O

Double distilled water

DMF

Dimethylformamide

DMSO

Dimethyl sulfoxide

H or His

Histidine

HIS3

Imidazoleglycerol-phosphate dehydratase gene in the histidine biosynthesis pathway

L or Leu

Leucine

LacZ

β-Galactosidase gene

LEU2

Beta-isopropylmalate dehydrogenase gene in the leucine biosynthesis pathway

Met

Methionine

Met25p

Methionine repressive promoter

NaCl

Sodium chloride

Nub

N-terminal half of ubiquitin

NubG

Mutated N-terminal half of ubiquitin with reduced Cub binding affinity

PEG

Polyethylene glycol

PLV

ProteinA-LexA-VP16 reporter module

PPI

Protein–protein interaction

SD

Synthetic dropout

SpecR

Spectinomycin resistance gene

ssDNA

Salmon sperm DNA

SU-Y2H

Split-ubiquitin yeast two-hybrid

TE

Tris–HCl and EDTA buffer

TOPO

Topoisomerase

TRP

Tryptophan

TRP1

Phosphoribosylanthranilate isomerase gene of the tryptophan biosynthesis pathway

USP

Ubiquitin-specific protease

W

Tryptophan

X-Gal

5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside

Y2H

Yeast two-hybrid

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry & Molecular BiologyPennsylvania State UniversityUniversity ParkUSA

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