Abstract
SUMOylation is a post-translational modification conserved in eukaryotic organisms that involves the covalent attachment of the small ubiquitin-like protein SUMO to internal lysine residues in target proteins. This tag usually alters the interaction surface of the modified protein and can be translated into changes in its biological activity, stability or subcellular localization, among other possible outputs. SUMO can be attached as a single moiety or as SUMO polymers in case there are internal acceptor sites in SUMO itself. These chains have been shown to be important for proteasomal degradation as well as for the formation of subnuclear structures such as the synaptonemal complex in Saccharomyces cerevisiae or promyelocytic leukemia nuclear bodies in mammals. In this work, we have examined SUMO chain formation in the protozoan parasite Trypanosoma brucei. Using a recently developed bacterial strain engineered to produce SUMOylated proteins we confirmed the ability of TbSUMO to form polymers and determined the type of linkage using site-directed mutational analysis. By generating transgenic procyclic parasites unable to form chains we demonstrated that although not essential for normal growth, SUMO polymerization determines the localization of the modified proteins in the nucleus. In addition, FISH analysis of telomeres showed a differential positioning depending on the polySUMOylation abilities of the cells. Thus, our observations suggest that TbSUMO chains might play a role in establishing interaction platforms contributing to chromatin organization.
Highlights
SUMOylation is a eukaryotic post-translational modification that involves the covalent attachment of a Small Ubiquitin-like Modifier (SUMO) to a diverse range of protein substrates
To facilitate the biochemical validation of T. brucei SUMO targets, we have previously developed an in bacteria SUMOylation system consisting of the heterologous co-expression of the SUMOylation machinery of the parasite (TbE1a/E1b, TbE2, TbSUMO), together with a potential substrate, in E. coli [28]
This observation led us to speculate that TbSUMO, similar to its human counterparts SUMO2/-3 or yeast Smt3, is capable of forming poly-SUMO chains
Summary
SUMOylation is a eukaryotic post-translational modification that involves the covalent attachment of a Small Ubiquitin-like Modifier (SUMO) to a diverse range of protein substrates. When testing this system in the absence of any substrate, we detected a ladder of wild-type SUMO (TbSUMO) that was no longer visible when all eight lysine residues present in the protein were mutated to arginine (TbSUMOallKR) (Fig 1A).
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