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PMID: 12840049 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.

Genome research ·Vol. 13 ·No. 7 ·2003-07-00 ·Pages 1744-53

Thaminy S, Auerbach D, Arnoldo A, Stagljar I

Abstract

Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Brain Cell Line Cloning, Molecular DNA, Complementary/genetics Gene Library Genetic Vectors/biosynthesis,genetics Humans Kidney/chemistry,embryology,metabolism Macromolecular Substances Membrane Proteins/biosynthesis,genetics,metabolism Molecular Sequence Data Peptide Mapping Protein Interaction Mapping RGS Proteins/chemistry,genetics,metabolism Rats Receptor, ErbB-3/biosynthesis,chemistry,genetics,metabolism Saccharomyces cerevisiae/chemistry,metabolism Two-Hybrid System Techniques Ubiquitin/biosynthesis,genetics,metabolism
Chemicals
DNA, Complementary Macromolecular Substances Membrane Proteins RGS Proteins Ubiquitin RGS4 protein Receptor, ErbB-3
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Thaminy Safia
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, CH-8057 Zurich, Switzerland.
Auerbach Daniel
Arnoldo Anthony
Stagljar Igor
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Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1088-9051
DOI
10.1101/gr.1276503
Published
2003-07-00
Pages
1744-53
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC403748
Subset
IM
External Links
PubMed source DOI: 10.1101/gr.1276503 PMC Full Text View journal details
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