Yeast one-hybrid gγ recruitment system for identification of protein lipidation motifs.
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 Gγ 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 Gα 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.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki, Japan.
Nobuo
N
Fukuda
Motomichi
M
Doi
Shinya
S
Honda
D000595:Amino Acid Sequence; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054878:Lipoylation; D019814:Myristic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D044385:GTP-Binding Protein alpha Subunits; D044388:GTP-Binding Protein gamma Subunits; D029701:Saccharomyces cerevisiae Proteins; D019814:Myristic Acid