title: Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.
abstract: Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065;
authors.forename: Avital
authors.initials: A
authors.lastname: Percher
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065;
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Ramakrishnan
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065;
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Thinon
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065;
authors.forename: Xiaoqiu
authors.initials: X
authors.lastname: Yuan
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065; Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State University, Columbus, OH 43210 yount.37@osu.edu hhang@rockefeller.edu.
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Yount
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065; yount.37@osu.edu hhang@rockefeller.edu.
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
mesh_terms: D000215:Acylation; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005227:Fatty Acids; D008565:Membrane Proteins; D003545:Cysteine
keywords: IFITM3; PEGylation; fatty-acylation; influenza virus; palmitoylation