Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins
| Autor: | Kavitha Ravee Iyer, Kim Engels, Lukas Bürkle, Igor Stagljar, Safia Thaminy, Daniel Auerbach, Martin Dinkel |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
DNA
Complementary Two-hybrid screening Recombinant Fusion Proteins Saccharomyces cerevisiae Polymerase Chain Reaction Bacterial Proteins Endopeptidases Protein Interaction Mapping Escherichia coli Animals Humans Cloning Molecular Isoleucine Selection Genetic Integral membrane protein Gene Library FERM domain biology Membrane transport protein Ubiquitin Peripheral membrane protein Serine Endopeptidases Membrane Proteins Herpes Simplex Virus Protein Vmw65 General Medicine Membrane transport Transport protein Cell biology Protein Structure Tertiary Membrane protein Biochemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed Colorimetry Indicators and Reagents Ubiquitin-Specific Proteases Hydrophobic and Hydrophilic Interactions Plasmids Protein Binding |
| Zdroj: | Science's STKE : signal transduction knowledge environment. 2005(275) |
| ISSN: | 1525-8882 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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