TAILIEUCHUNG - Báo cáo khoa học: Construction of a novel detection system for protein–protein interactions using yeast G-protein signaling

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 csubunit (Gc) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-termi-nus, and that this localization of Gcis required for signal transduction. | ễFEBS Journal Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling Nobuo Fukuda1 Jun Ishii2 Tsutomu Tanaka2 Hideki Fukuda2 and Akihiko Kondo1 1 Department of ChemicalScience and Engineering Graduate Schoolof Engineering Kobe University Japan 2 Organization of Advanced Science and Technology Kobe University Japan Keywords G-protein signaling membrane localization of Gc subunit protein-protein interaction yeast two-hybrid system Correspondence A. Kondo Department of ChemicalScience and Engineering Graduate School of Engineering Kobe University 1-1 Rokkodaicho Nada-ku Kobe 657-8501 Japan Fax Tel 81 78 803 6196 E-mail akondo@ Received 18 December 2008 revised 18 February 2009 accepted 3 March 2009 doi 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 c subunit Gc is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-termi-nus and that this localization of Gc is required for signal transduction. In our system mutated Gc Gccyto lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when Gccyto was expressed in the cytoplasm of yeast cells from which the endogenous Gc 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 Gccyto. The Fc-Gccyto fusion protein migrated towards the membrane via the ZZ-Fc interaction and signal transduction was therefore restored. .

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