%0 Journal Article
%T Yeast One-Hybrid G¦Ã Recruitment System for Identification of Protein Lipidation Motifs
%A Nobuo Fukuda
%A Motomichi Doi
%A Shinya Honda
%J PLOS ONE
%D 2013
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0070100
%X Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the G¦Ã recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of G¦Á subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0070100