Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.
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.
Thaminy
Safia
S
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, CH-8057 Zurich, Switzerland.
Auerbach
Daniel
D
Arnoldo
Anthony
A
Stagljar
Igor
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D025941:Protein Interaction Mapping; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020893:Receptor, ErbB-3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D046911:Macromolecular Substances; D008565:Membrane Proteins; D020710:RGS Proteins; D025801:Ubiquitin; C098799:RGS4 protein; D020893:Receptor, ErbB-3