Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions.
The characterization of protein-protein interactions provides the foundation for further studies concerning protein complex function and regulation. Since the advent of the yeast two-hybrid assay, many additional genetic systems based upon the principle of protein fragment complementation have been designed. One such system, the split-ubiquitin membrane yeast two-hybrid system (MbYTH), is able to analyze the interaction status between two integral membrane proteins. This ability of the MbYTH system augments genetic analysis of protein interactions by covering for the inherent limitation of the yeast two-hybrid system when studying membrane protein interactions. Herein, we provide a description of the MbYTH method and detailed protocols in order to monitor protein interactions and discover novel interacting partners using the MbYTH system.
Fetchko
Michael
M
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Stagljar
Igor
I
D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D006639:Histidine; D001616:beta-Galactosidase