title: mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases.
abstract: Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.
authors.affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5.
authors.forename: Leslie
authors.initials: L
authors.lastname: Mitchell
authors.forename: Sylvain
authors.initials: S
authors.lastname: Huard
authors.forename: Michael
authors.initials: M
authors.lastname: Cotrut
authors.forename: Roghayeh
authors.initials: R
authors.lastname: Pourhanifeh-Lemeri
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Steunou
authors.forename: Akil
authors.initials: A
authors.lastname: Hamza
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Ning
authors.forename: Amrita
authors.initials: A
authors.lastname: Basu
authors.forename: Jacques
authors.initials: J
authors.lastname: Côté
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Figeys
authors.forename: Kristin
authors.initials: K
authors.lastname: Baetz
mesh_terms: D000107:Acetylation; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D051548:Histone Acetyltransferases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D051548:Histone Acetyltransferases; C504740:NuA4 protein, S cerevisiae; D008239:Lysine