title: Ubiquitination-induced fluorescence complementation (UiFC) for detection of K48 ubiquitin chains in vitro and in live cells.
abstract: Proteins can be modified with eight homogenous ubiquitin chains linked by an isopeptide bond between the C-terminus of one ubiquitin and an amine from one of the seven lysines or the N-terminal methionine of the next ubiquitin. These topologically distinct ubiquitin chains signal for many essential cellular functions, such as protein degradation, cell cycle progression, DNA repair, and signal transduction. The lysine 48 (K48)-linked ubiquitin chain is one of the most abundant chains and a major proteasome-targeting signal in cells. Despite recent advancements in imaging linkage-specific polyubiquitin chains, no tool is available for imaging K48 chains in live cells. Here we report on a ubiquitination-induced fluorescence complementation (UiFC) assay for detecting K48 ubiquitin chains in vitro and in live cells. For this assay, two nonfluorescent fragments of a fluorescent protein were fused to the ubiquitin-interacting motifs (UIMs) of epsin1 protein. Upon simultaneous binding to a ubiquitin chain, the nonfluorescent fragments of the two fusion proteins are brought in close proximity to reconstitute fluorescence. When used in vitro, UiFC preferentially detected K48 ubiquitin chains with excellent signal-to-noise ratio. Time-lapse imaging revealed that UiFC is capable of monitoring increases in polyubiquitination induced by treatment with proteasome inhibitor, by agents that induce stress, and during mitophagy in live cells.
authors.affiliation: Center for Biomedical Engineering and Technology, Anhui Medical University, Hefei, China ; Department of Physiology, Anhui Medical University, Hefei, China.
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Chen
authors.forename: Yongwang
authors.initials: Y
authors.lastname: Zhong
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Shan
authors.initials: S
authors.lastname: Xu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: Ilia V
authors.initials: IV
authors.lastname: Baskakov
authors.forename: Mervyn J
authors.initials: MJ
authors.lastname: Monteiro
authors.forename: Mariusz
authors.initials: M
authors.lastname: Karbowski
authors.forename: Yuxian
authors.initials: Y
authors.lastname: Shen
authors.forename: Shengyun
authors.initials: S
authors.lastname: Fang
mesh_terms: D002470:Cell Survival; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D025801:Ubiquitin / Q000032:analysis* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin
references.citation: Curr Cancer Drug Targets. 2011 Mar;11(3):347-56
references.pmid: 21247385
references.citation: EMBO J. 2009 Dec 16;28(24):3903-9
references.pmid: 19927120
references.citation: Nat Cell Biol. 2001 Oct;3(10):939-43
references.pmid: 11584278
references.citation: J Biomol Screen. 2007 Dec;12(8):1050-8
references.pmid: 17989425
references.citation: Science. 2007 May 25;316(5828):1198-202
references.pmid: 17525341
references.citation: Nat Rev Mol Cell Biol. 2009 Aug;10(8):550-63
references.pmid: 19626045
references.citation: Nat Rev Mol Cell Biol. 2009 Nov;10(11):755-64
references.pmid: 19851334
references.citation: Nature. 2009 Mar 26;458(7237):438-44
references.pmid: 19325623
references.citation: Traffic. 2006 Mar;7(3):262-81
references.pmid: 16497222
references.citation: J Cell Biol. 2010 Dec 27;191(7):1367-80
references.pmid: 21173115
references.citation: Nat Cell Biol. 2005 Aug;7(8):742-9
references.pmid: 16056265
references.citation: J Biol Chem. 2011 Sep 30;286(39):33921-30
references.pmid: 21832065
references.citation: Am J Physiol Heart Circ Physiol. 2008 Dec;295(6):H2541-50
references.pmid: 18978187
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Mol Cell. 2004 Aug 27;15(4):535-48
references.pmid: 15327770
references.citation: J Biol Chem. 2003 Mar 14;278(11):8951-9
references.pmid: 12643283
references.citation: Chem Rev. 2009 Apr;109(4):1481-94
references.pmid: 19253967
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):745-50
references.pmid: 11805328
references.citation: J Biomol Screen. 2004 Dec;9(8):695-703
references.pmid: 15634796
references.citation: Nat Rev Mol Cell Biol. 2012 Jul 23;13(8):508-23
references.pmid: 22820888
references.citation: Anal Biochem. 2009 Jul 1;390(1):85-7
references.pmid: 19328766
references.citation: Cell. 2008 Aug 22;134(4):668-78
references.pmid: 18724939
references.citation: Int J Mol Med. 2012 Dec;30(6):1281-6
references.pmid: 22992760
references.citation: Science. 2010 Jun 4;328(5983):1244-5
references.pmid: 20522767
references.citation: FEBS J. 2012 Jun;279(12):2182-91
references.pmid: 22536838
references.citation: Mol Cell. 2012 Sep 14;47(5):797-809
references.pmid: 22819327
references.citation: Chem Soc Rev. 2009 Oct;38(10):2876-86
references.pmid: 19771334
references.citation: J Mol Biol. 2009 Dec 4;394(3):391-409
references.pmid: 19733184
references.citation: Biotechniques. 2006 Jan;40(1):61-6
references.pmid: 16454041
references.citation: J Biol Chem. 2007 Nov 23;282(47):33908-14
references.pmid: 17872946
references.citation: Biochem J. 2012 Jun 15;444(3):611-7
references.pmid: 22497224
references.citation: Mol Cell. 2011 Oct 21;44(2):325-40
references.pmid: 21906983
references.citation: Nat Methods. 2011 Jul 10;8(8):691-6
references.pmid: 21743460
references.citation: EMBO J. 2013 Feb 20;32(4):552-65
references.pmid: 23314748
references.citation: Nat Struct Mol Biol. 2010 Aug;17(8):939-47
references.pmid: 20622874
references.citation: J Biol Chem. 2010 Mar 19;285(12):8595-604
references.pmid: 20061386
references.citation: Mol Biol Cell. 2008 Nov;19(11):4588-601
references.pmid: 18716056
references.citation: Nat Rev Cancer. 2011 Aug 24;11(9):629-43
references.pmid: 21863050
references.citation: J Cell Biol. 2005 Nov 21;171(4):603-14
references.pmid: 16286508
references.citation: Oncogene. 2008 Jun 12;27(26):3739-45
references.pmid: 18212736
references.citation: Science. 2007 May 25;316(5828):1202-5
references.pmid: 17525342
references.citation: J Biomol Screen. 2008 Mar;13(3):229-37
references.pmid: 18270365
references.citation: J Biol Chem. 2004 Jan 30;279(5):3525-34
references.pmid: 14593114
references.citation: Mol Cell. 2010 Jun 11;38(5):627-35
references.pmid: 20541996
references.citation: Nature. 2007 Mar 15;446(7133):333-7
references.pmid: 17310145
references.citation: F1000 Biol Rep. 2011;3:26
references.pmid: 22162729
references.citation: Mol Cell. 2010 Aug 13;39(3):477-84
references.pmid: 20655260
references.citation: Nat Rev Mol Cell Biol. 2009 Aug;10(8):564-8
references.pmid: 19474794
references.citation: Cell. 2009 Apr 3;137(1):133-45
references.pmid: 19345192
references.citation: Annu Rev Pharmacol Toxicol. 2009;49:73-96
references.pmid: 18834306
references.citation: Nat Methods. 2012 Feb 05;9(3):303-9
references.pmid: 22306808
references.citation: Cancer Cell. 2009 Apr 7;15(4):270-82
references.pmid: 19345326
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20759-63
references.pmid: 18077395
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14422-7
references.pmid: 11724934
references.citation: J Cell Sci. 2012 Jan 15;125(Pt 2):277-86
references.pmid: 22357969
references.citation: Mol Cell Biol. 2004 Sep;24(18):8055-68
references.pmid: 15340068
references.citation: PLoS Biol. 2011 Mar;8(3):e1000605
references.pmid: 21468303
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20197-202
references.pmid: 19091944
references.citation: Development. 2007 Dec;134(23):4209-18
references.pmid: 17959720
references.citation: Annu Rev Biochem. 2012;81:291-322
references.pmid: 22482907