Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki, Japan.
PLoS One. 2013 Jul 26;8(7):e70100. doi: 10.1371/journal.pone.0070100. Print 2013.
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.
脂肪酸和异戊二烯可以共价连接到各种蛋白质上。这些脂质修饰调节蛋白质的结构、定位和功能。在这里，我们描述了一种基于 Gγ 募集系统的酵母单杂交方法，该方法可用于鉴定影响脂质修饰以招募蛋白质到质膜的序列基序。我们的方法通过利用诱导二倍体形成的 G 蛋白信号的简单易行的生长选择测定法，促进了表达脂质修饰蛋白的酵母细胞的分离。在本研究中，我们选择了 Gα 亚基的 N 端序列作为模型案例，研究了双重脂质修饰，即豆蔻酰化和棕榈酰化，这种修饰从酵母到高等真核生物都广泛保守。我们的结果表明，两种脂质修饰都需要恢复 G 蛋白信号。尽管我们无法区分豆蔻酰化和棕榈酰化，但 N 端第 7 位和第 8 位起着一些关键作用。此外，我们使用基于文库的筛选测试了第 7 位和第 8 位特定氨基酸残基的偏好性。这种新方法将有助于探索蛋白质-脂质的相互作用，并确定相应的序列基序。
