title: Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.
abstract: Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein-coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, CH-8057 Zurich, Switzerland.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Iyer
authors.forename: Lukas
authors.initials: L
authors.lastname: Bürkle
authors.forename: Daniel
authors.initials: D
authors.lastname: Auerbach
authors.forename: Safia
authors.initials: S
authors.lastname: Thaminy
authors.forename: Martin
authors.initials: M
authors.lastname: Dinkel
authors.forename: Kim
authors.initials: K
authors.lastname: Engels
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D003124:Colorimetry / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D015723:Gene Library; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007202:Indicators and Reagents; D007532:Isoleucine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D012641:Selection, Genetic; D012697:Serine Endopeptidases / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*; D064570:Ubiquitin-Specific Proteases; D000099160:LexA Repressor Protein
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D010450:Endopeptidases; D017405:Herpes Simplex Virus Protein Vmw65; D007202:Indicators and Reagents; D007532:Isoleucine; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D025801:Ubiquitin; D064570:Ubiquitin-Specific Proteases; D000099160:LexA Repressor Protein