Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.
We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Hazel F
HF
O'Connor
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Nancy
N
Lyon
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Justin W
JW
Leung
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Poonam
P
Agarwal
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Caleb D
CD
Swaim
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.
Kyle M
KM
Miller
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA huibregtse@austin.utexas.edu.
Jon M
JM
Huibregtse
D000595:Amino Acid Sequence; D004260:DNA Repair; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000235:genetics; D006801:Humans; D009154:Mutation; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
D056827:Endosomal Sorting Complexes Required for Transport; D006657:Histones; D010445:Peptide Elongation Factors; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; C098415:BRD2 protein, human; D000092468:Bromodomain Containing Proteins; C493530:Htz1 protein, S cerevisiae; C554184:Psh1 protein, S cerevisiae; C578829:RNF126 protein, human; C469287:RSP5 protein, S cerevisiae
HECT E3s; RING E3s; Ubiquitin; Ubiquitin ligases