Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation
- PMID: 17277783
- DOI: 10.1038/nmeth1006
Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation
Abstract
Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion-directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity.
Similar articles
-
Is Transthyretin a Regulator of Ubc9 SUMOylation?PLoS One. 2016 Aug 8;11(8):e0160536. doi: 10.1371/journal.pone.0160536. eCollection 2016. PLoS One. 2016. PMID: 27501389 Free PMC article.
-
Enhanced detection of in vivo SUMO conjugation by Ubc9 fusion-dependent sumoylation (UFDS).Methods Mol Biol. 2009;497:63-79. doi: 10.1007/978-1-59745-566-4_5. Methods Mol Biol. 2009. PMID: 19107411 Review.
-
Ubc9 fusion-directed SUMOylation (UFDS).Biochem Soc Trans. 2007 Dec;35(Pt 6):1430-2. doi: 10.1042/BST0351430. Biochem Soc Trans. 2007. PMID: 18031239 Review.
-
Association of Ubc9, an E2 ligase for SUMO conjugation, with p53 is regulated by phosphorylation of p53.FEBS Lett. 2004 Aug 27;573(1-3):15-8. doi: 10.1016/j.febslet.2004.07.059. FEBS Lett. 2004. PMID: 15327968
-
Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation.Nucleic Acids Res. 2007;35(17):e109. doi: 10.1093/nar/gkm617. Epub 2007 Aug 20. Nucleic Acids Res. 2007. PMID: 17709345 Free PMC article.
Cited by
-
Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.PLoS Genet. 2013 Apr;9(4):e1003473. doi: 10.1371/journal.pgen.1003473. Epub 2013 Apr 18. PLoS Genet. 2013. PMID: 23637637 Free PMC article.
-
Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6.Genes Dev. 2016 Jun 1;30(11):1339-56. doi: 10.1101/gad.278275.116. Genes Dev. 2016. PMID: 27298337 Free PMC article.
-
Is Transthyretin a Regulator of Ubc9 SUMOylation?PLoS One. 2016 Aug 8;11(8):e0160536. doi: 10.1371/journal.pone.0160536. eCollection 2016. PLoS One. 2016. PMID: 27501389 Free PMC article.
-
SUMOylation regulates the intracellular fate of ZO-2.Cell Mol Life Sci. 2017 Jan;74(2):373-392. doi: 10.1007/s00018-016-2352-5. Epub 2016 Sep 7. Cell Mol Life Sci. 2017. PMID: 27604867 Free PMC article.
-
Identification of the non-structural influenza A viral protein NS1A as a bona fide target of the Small Ubiquitin-like MOdifier by the use of dicistronic expression constructs.J Virol Methods. 2010 Feb;163(2):498-504. doi: 10.1016/j.jviromet.2009.11.010. Epub 2009 Nov 14. J Virol Methods. 2010. PMID: 19917317 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials
Miscellaneous
