Nature Methods
- 4, 245 - 250 (2007)
Published online: 4 February 2007; | doi:10.1038/nmeth1006
Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylationAstrid Jakobs1, 4, Jesko Koehnke1, 4, Fabian Himstedt1, Martin Funk2, Bernhard Korn3, Matthias Gaestel1 & Rainer Niedenthal11
Institut für Physiologische Chemie, Medizinische Hochschule Hannover, Carl Neuberg Str. 1, 30625 Hannover, Germany. 2
MediGene AG, Lochhamer Str. 11, 82152 Martinsried, Germany. 3
Deutsches Krebsforschungszentrum, Genomics and Proteomics Core Facilities, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany. 4
These authors contributed equally to this work.
Correspondence should be addressed to Rainer Niedenthal niedenthal.rainer@mh-hannover.de 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
