Biochemical and cell biological characterisation of Sumo E1 activating enzyme Aos1/Uba2

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.