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Chmielarska, Katarzyna (2005): Biochemical and cell biological characterisation of Sumo E1 activating enzyme Aos1/Uba2. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Small ubiquitin-related modifier (SUMO) is a protein that is attached to lysine residues in a variety of target proteins. Sumoylation of proteins can alter their intracellular localisation, stability, activity and interaction with other proteins. The pathway of sumoylation is analogous to that of ubiquitination. The reaction is ATP dependent and requires the E1-activating enzyme (Aos1/Uba2), the E2-conjugation enzyme (Ubc9) and for most target proteins SUMO E3 ligases. The aim of this study was to characterise the SUMO E1 enzyme, a heterodimer consisting of the subunits Aos1 and Uba2. On one hand I characterised an Uba2 splice variant, which lacks one exon encoding 50 amino acids. Using RT-PCR I could determine the tissue specific distribution of the Uba2 splice variant. I furthermore showed that Uba2 variant protein is still able to form an active E1 enzyme complex with Aos1. I could demonstrate that variant Aos1/Uba2 complex is fully active in RanGAP1 sumoylation with SUMO1 or SUMO2. This finding was surprising in light of the missing amino acids, and will have implications for the understanding of E1 function. A large part of my work was dedicated to the identification and characterization of a novel SUMO substrate called ELKS. According to literature, ELKS proteins have been linked to intracellular membrane traffic and NFB signaling pathways. I identified ELKS in membrane fractions as a binding partner for the Aos1 subunit of the SUMO E1 enzyme and confirmed in vivo interaction with ELKS antibodies that I generated. Because recombinant proteins did not interact directly, I searched for potential bridging factors. Neither SUMO nor Ubc9 or Rab6 (one ELKS partner) mediated interaction between ELKS and Aos1. Performing a large scale immunoprecipitation and analysis by mass spectrometry, I could find several candidates, including nucleoporin RanBP2, a SUMO E3 ligase. This suggested that ELKS may be a target for sumoylation. Indeed, I could show that ELKS was SUMO-modified in vivo and in vitro. Moreover, RanBP2 enhanced ELKS sumoylation. By mass spectrometry I identified two SUMO acceptor sites in ELKS. Mutation of these two residues had no effect on ELKS localisation, but strongly inhibited ELKS induced NFB activation. In conclusion, work described in this thesis implicates sumoylation as an important mechanism for ELKS function in NFkB signaling.