Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps)

Nature Protocols
Kevin G MarkDavid P Toczyski

Abstract

Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

References

Dec 18, 2001·Journal of Mass Spectrometry : JMS·J Peng, S P Gygi
Jul 23, 2003·Nature Biotechnology·Junmin PengSteven P Gygi
Oct 15, 2003·Proceedings of the National Academy of Sciences of the United States of America·Amy L HitchcockPamela A Silver
Aug 2, 2005·Nature Cell Biology·Suzanne Elsasser, Daniel Finley
Jan 20, 2006·Molecular Biology of the Cell·Deanna M KoeppVeronica Rodriguez-Rivera
Apr 25, 2007·Biochemical and Biophysical Research Communications·Hong Bae JeonYung Joon Yoo
Sep 11, 2007·Nature Cell Biology·Jennifer A BenantiDavid P Toczyski
Sep 11, 2008·Journal of Proteome Research·David MeierhoferPeter Kaiser
Nov 8, 2008·Science·Hsueh-Chi Sherry YenStephen J Elledge
Nov 8, 2008·Science·Hsueh-Chi Sherry Yen, Stephen J Elledge
Jul 21, 2009·Cell·Mathew E SowaJ Wade Harper
Jul 22, 2009·Nature Structural & Molecular Biology·Joshua J SimsRobert E Cohen
Nov 26, 2009·Nature Structural & Molecular Biology·Yogesh KulathuDavid Komander
Dec 4, 2009·Nature·Nathan W PierceRaymond J Deshaies
Jun 22, 2010·Nature·Christian BehrendsJ Wade Harper
Jul 20, 2010·Nature Biotechnology·Guoqiang XuSamie R Jaffrey
Sep 24, 2010·Molecular & Cellular Proteomics : MCP·Maribel FrancoUgo Mayor
Oct 26, 2010·Molecular & Cellular Proteomics : MCP·Yi ShiJun Qin
Dec 9, 2010·Molecular & Cellular Proteomics : MCP·Jannie M R DanielsenMichael L Nielsen
Feb 1, 2011·Trends in Cell Biology·David G McEwan, Ivan Dikic
Sep 8, 2011·Genes & Development·Sjoerd J L van WijkIvan Dikic
Sep 13, 2011·Molecular Cell·Woong KimSteven P Gygi
Oct 4, 2011·Cell·Michael J EmanueleStephen J Elledge
Oct 21, 2011·Protein Science : a Publication of the Protein Society·Brenda A Schulman
Dec 20, 2011·Journal of Proteomics·Fernando Lopitz-OtsoaManuel S Rodriguez
Apr 25, 2012·Annual Review of Biochemistry·David Komander, Michael Rape
May 31, 2012·Molecular and Cellular Biology·Gustavo Martínez-NoëlPeter M Howley
Jul 31, 2012·PLoS Genetics·Benjamin D LandryJennifer A Benanti
Oct 3, 2012·Genetics·Daniel FinleyPeter Kaiser
Oct 12, 2012·PloS One·Marcus D WilsonJesper Q Svejstrup
Jul 12, 2014·BMC Cell Biology·Wananit WimuttisukJeffrey D Singer
Aug 17, 2014·Genes & Development·Vinod Pant, Guillermina Lozano

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Citations

Mar 9, 2018·Molecular Biology of the Cell·Edmond Y HuangJames A Olzmann
Dec 22, 2017·Nature Communications·Yiyang WangJun Yin
Jan 23, 2019·EMBO Reports·Sjoerd Jl van WijkMike Heilemann
Nov 12, 2016·The Biochemical Journal·Mary Iconomou, Darren N Saunders
Apr 30, 2017·Cellular and Molecular Life Sciences : CMLS·Hazel F O'Connor, Jon M Huibregtse
Feb 29, 2020·Pharmacological Reviews·Bo ZhaoJun Yin
Mar 17, 2020·Frontiers in Molecular Biosciences·Katarzyna Kliza, Koraljka Husnjak
Oct 22, 2020·Communications Biology·Masashi WatanabeShigetsugu Hatakeyama
Feb 21, 2021·Developmental Cell·Fernando Rodríguez-PérezMichael Rape
Mar 2, 2019·Trends in Biochemical Sciences·Michael MatternManuel S Rodriguez
Nov 14, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Yane-Shih WangRuey-Hwa Chen
Apr 27, 2021·Frontiers in Plant Science·Jamie N OrrIsabelle Colas

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