SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. His-HA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a His-HA-TbSUMO(T106K) version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.

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.