Yeast one-hybrid gγ recruitment system for identification of protein lipidation motifs.

Nobuo Fukuda,Motomichi Doi,Shinya Honda,Howard Riezman

doi:10.1371/journal.pone.0070100

Nobuo Fukuda, Motomichi Doi + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0070100

Copy DOI

Abstract

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.

Highlights

A wide range of proteins are modified by covalent attachment of fatty acids and/or isoprenoid groups, modifications that play major roles in regulating protein structure and function [1]
Because recruitment of lipidated proteins to the plasma membrane influences complex signaling pathways that regulate specific physiological functions, there is a great interest in gaining a better understanding of protein trafficking via lipid modification
After removal of the N-terminal methionine residue by methionine aminopeptidase, myristate is attached to the Nterminal glycine of protein substrates with the consensus motif Met1-Gly2-Xaa3-Xaa4-Xaa5-Ser/Thr6-Xaa7-Xaa8 [3] in a reaction catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) [4]

Summary

Introduction

A wide range of proteins are modified by covalent attachment of fatty acids and/or isoprenoid groups, modifications that play major roles in regulating protein structure and function [1]. After removal of the N-terminal methionine residue by methionine aminopeptidase, myristate is attached to the Nterminal glycine of protein substrates with the consensus motif Met1-Gly2-Xaa3-Xaa4-Xaa5-Ser/Thr6-Xaa7-Xaa (where Xaa indicates any amino acid residue) [3] in a reaction catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) [4]. Further refinement of this consensus might be possible, as it has been shown that not all amino acid residues are allowable at the positions indicated by Xaa [4,5]. Protein acyltransferases (PATs) have a common DHHC Cys-rich domain [6], the consensus motif for palmitoylation of proteins is still unclear, due at least in part to the diversity of substrates that can be recognized by multiple PATs, including 7 DHHC proteins in yeast and 23 in humans [7]

Objectives

Methods

Results

Conclusion