Investigation of membrane protein interactions using the split-ubiquitin membrane yeast two-hybrid system.
Proteins are generally organized into molecular complexes, in which multiple interaction partners collaborate to carry out cellular processes. Thus, techniques to map protein-protein interactions have become pivotal for biological studies of as yet uncharacterized proteins. Investigation of interaction partners of membrane proteins is of special interest, as they play a major role in cellular processes and are often directly linked to human diseases. Owing to their hydrophobic nature, however, it has proven difficult to study their interaction partners. To circumvent this problem, a yeast-based genetic technology for the in vivo detection of membrane protein interactions, the split-ubiquitin membrane yeast two-hybrid (MYTH) system, has been developed. MYTH allows for detection of both stable and transient interactions and can be applied to large- and small-scale screens. It uses the split-ubiquitin approach, in which the reconstitution of two ubiquitin halves is mediated by a specific protein-protein interaction. Briefly, the bait membrane protein is fused to the C-terminal half of ubiquitin and an artificial transcription factor. The mutated N-terminal moiety of ubiquitin is fused to the prey protein. Upon interaction of bait and prey proteins, ubiquitin is reconstituted and further recognized by ubiquitin-specific proteases, which subsequently cleave off the transcription factor, thus resulting in reporter gene activation. To date, MYTH has been successfully applied to study interactions of membrane proteins from various organisms and has only recently been adapted for the identification of interaction partners of mammalian receptor tyrosine kinases.
Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Julia
J
Petschnigg
Victoria
V
Wong
Jamie
J
Snider
Igor
I
Stagljar
D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017421:Sequence Analysis; D014169:Transformation, Bacterial; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D015003:Yeasts / Q000166:cytology / Q000235:genetics
D008565:Membrane Proteins; D025801:Ubiquitin