Abstract
The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.
Highlights
Sumoylation, the covalent addition of small ubiquitin-related modifier (SUMO) to substrate proteins, is an essential process in metazoa
To execute SUMO conjugation, heterodimeric SUMO-activating enzyme (SAE) links the SUMO carboxyl terminus to a Cys residue in the active site, from where activated SUMO is transferred to active site Cys of SUMO-conjugating enzyme (SCE)
We show that SCE C94S forms an oxyester bond with SUMO, explaining its dominant negative effect on sumoylation
Summary
Sumoylation, the covalent addition of small ubiquitin-related modifier (SUMO) to substrate proteins, is an essential process in metazoa. SCE can transfer SUMO to substrates without the participation of additional components. Whereas most known substrates receive a single SUMO moiety, SUMO chains have been detected in vivo, and their formation can be observed in vitro, using purified components SAE, SCE and SUMO. Similar to substrate decoration with single SUMO moieties, SUMO chain formation can be enhanced by SUMO ligases [11,12]. We were interested in finding mechanistic differences between mono-sumoylation of a substrate and SUMO chain formation. We tested mutations in SCE for their effect on SUMO acceptance from SAE, on SUMO transfer to a substrate and on SUMO chain formation
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call