Mapping the local protein interactome of the NuA3 histone acetyltransferase

ArticleinProtein Science 18(9):1987-97 · September 2009with15 Reads
DOI: 10.1002/pro.212 · Source: PubMed
Protein-protein interactions modulate cellular functions ranging from the activity of enzymes to signal transduction cascades. A technology termed transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) is described for the identification of stable and transient protein-protein interactions in vivo. The procedure combines mild in vivo chemical cross-linking and non-stringent affinity purification to isolate low abundance chromatin-associated protein complexes. Using isotopic labeling and mass spectrometric readout, purified proteins are categorized with respect to the protein 'bait' as stable, transient, or contaminant. Here we characterize the local interactome of the chromatin-associated NuA3 histone lysine-acetyltransferase protein complex. We describe transient associations with the yFACT nucleosome assembly complex, RSC chromatin remodeling complex and a nucleosome assembly protein. These novel, physical associations with yFACT, RSC, and Nap1 provide insight into the mechanism of NuA3-associated transcription and chromatin regulation.
    • "Proteins were resolved by SDS-PAGE, visualized by colloidal Coomassie staining, excised, and treated in-gel with trypsin for protein identifi cation (Smart et al. 2009 ). Peptides were subjected to tandem mass spectrometric analysis by nanospray ionization with a coupled nanoLC 2D reverse-phase liquid chromatography system (Eksigent) and Thermo LTQ mass spectrometer (Smart et al. 2009 ). Two proteins were identifi ed by UniProt database searching with Mascot using the Loxodonta africana protein sequences ( "
    [Show abstract] [Hide abstract] ABSTRACT: The chemical analysis of urine tells much about the physiological status of mammals, and often reveals compounds that function as chemical signals to conspecifics. Such is the case with mature male African (Loxodonta africana) and Asian (Elephas maximus) elephants in which there is odoriferous drainage from the temporal gland and dribbling of urine during musth, a periodic state in which serum androgens are elevated, food intake typically decreases, and aggressiveness between male conspecifics increases. We have employed solid phase dynamic extraction (SPDE)/GC-MS to identify a series of alkan-2-ones, alkan-2-ols, and a few simple aromatic compounds that increase in abundance in musth elephant urine. The primary focus of this report is on the alkan-2-ones and their corresponding alkan-2-ols, specifically: (1) the probable biosynthesis of these compounds via a secondary pathway for fatty acid metabolism, (2) the proximate cause for their increased abundance in musth urine, and (3) the role of bacteria in the increased abundance of these compounds exogenously in aged urine.
    Full-text · Chapter · Jan 2016 · Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
    • "binds methylated histone), Elp3p (the HAT subunit of elongator complex) and Spt5p (RNAPII core binding, transcription elongation by RNAPII). In particular, the identification of Spt16p and Pob3p, protein components of the yFACT nucleosome assembly complex, and Rfc1p, according to others studies [39], provides insights into the mechanism of NuA3-associated transcription and chromatin regulation. Previous studies have reported that Sas3p, core histones, Spt16p and Pdp3p, are co-purified with Yta7p tagged with Protein A [40]. "
    Full-text · Dataset · May 2015 · Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
    • "One such approach, developed in yeast, is to employ light sonication and to wash solubilized chromatin under very mild conditions in order to preserve protein interactions as extensively as possible (Lambert et al., 2009Lambert et al., , 2010). Another approach in yeast is to add light cross-linking prior to sonication and washing under mild conditions (Smart et al., 2009). Recently, an approach for systematic analysis of endogenous protein-protein interactions and protein-DNA binding events was developed (Mohammed et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components, as well as their mutual interactions. This unit presents a chromatin cross-linking approach (BioTAP-XL) that utilizes a special BioTAP-tagged transgenic protein bait along with mass spectrometry to identify protein complex components, and high-throughput sequencing to identify RNA components and DNA binding sites. Full protocols are provided for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of the approach in all cases is the generation of control data from input chromatin samples. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
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