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Construction of a novel detection system for protein–protein interactions using yeast G‐protein signaling
Author(s) -
Fukuda Nobuo,
Ishii Jun,
Tanaka Tsutomu,
Fukuda Hideki,
Kondo Akihiko
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.06991.x
Subject(s) - signal transduction , two hybrid screening , fusion protein , vesicle associated membrane protein 8 , biology , protein subunit , g protein , cytoplasm , protein–protein interaction , membrane protein , microbiology and biotechnology , protein g , protein a , protein a/g , yeast , biochemistry , gene , membrane , antibody , genetics , recombinant dna
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 γ subunit (Gγ) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C‐terminus, and that this localization of Gγ is required for signal transduction. In our system, mutated Gγ (Gγ cyto ) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G‐protein signal was observed when Gγ cyto was expressed in the cytoplasm of yeast cells from which the endogenous Gγ 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 Gγ cyto . The Fc–Gγ 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 × 10 3 to 6.8 × 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.