Journal
FEBS JOURNAL
Volume 276, Issue 9, Pages 2636-2644Publisher
WILEY-BLACKWELL PUBLISHING, INC
DOI: 10.1111/j.1742-4658.2009.06991.x
Keywords
G-protein signaling; membrane localization of G gamma subunit; protein-protein interaction; yeast two-hybrid system
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Funding
- Japanese Society for the Promotion of Science
- Global COE Program 'Global Center for Education and Research in Integrative Membrane Biology'
- Ministry of Education, Culture, Sports, Science and Technology of Japan
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In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein gamma subunit (G gamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of G gamma is required for signal transduction. In our system, mutated G gamma (G gamma(cyto)) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when G gamma(cyto) was expressed in the cytoplasm of yeast cells from which the endogenous G gamma gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to G gamma(cyto). The Fc-G gamma(cyto) fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore restored. This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling. Finally, several Z variants displaying affinity constants ranging from 8.0 x 10(3) to 6.8 x 10(8) m(-1) were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity. In conclusion, our system appears to be a reliable and versatile technique for detection of protein-protein interactions, and may prove useful in future protein interaction studies.
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