SIMs (SUMO-interaction motifs), which mediate the non-covalent binding of SUMO (small ubiquitin-related modifier) to other proteins, are usually involved in the recognition of SUMOylated substrates by downstream effectors that transmit the biological signal of the modification. In ubiquitin ligase Rad18 (radiation-sensitive 18) from Saccharomyces cerevisiae, a SIM, contributes to the recognition of SUMOylated PCNA (proliferating-cell nuclear antigen) as its physiological ubiquitylation target. In the present study we show that Rad18 is also capable of enhancing PCNA SUMOylation in a SIM-dependent manner in vitro, most probably by means of directing SUMO-loaded Ubc9 (ubiquitin-conjugating enzyme 9) towards the substrate. The process shares important features with Rad18-dependent ubiquitylation, such as an exquisite specificity for the modification site on PCNA and the requirement of DNA, and the reaction proceeds under conditions that are widely used in other in vitro assays for SUMO ligase activity. However, there is no evidence that Rad18 contributes to PCNA SUMOylation in vivo. The findings of the present study therefore illustrate the problematic nature of in vitro SUMOylation assays and highlight the danger of extrapolating from this type of experiment to the biological function of a SUMO-interacting protein.
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Research Article|
January 10 2014
SIM-dependent enhancement of substrate-specific SUMOylation by a ubiquitin ligase in vitro
Joanne L. Parker;
Joanne L. Parker
1
*Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD, U.K.
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Helle D. Ulrich
Helle D. Ulrich
2
*Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD, U.K.
†Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
2To whom correspondence should be addressed (email h.ulrich@imb-mainz.de).
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Publisher: Portland Press Ltd
Received:
October 18 2013
Revision Received:
November 12 2013
Accepted:
November 14 2013
Accepted Manuscript online:
November 14 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 457 (3): 435–440.
Article history
Received:
October 18 2013
Revision Received:
November 12 2013
Accepted:
November 14 2013
Accepted Manuscript online:
November 14 2013
Citation
Joanne L. Parker, Helle D. Ulrich; SIM-dependent enhancement of substrate-specific SUMOylation by a ubiquitin ligase in vitro. Biochem J 1 February 2014; 457 (3): 435–440. doi: https://doi.org/10.1042/BJ20131381
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