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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
Posttranslational regulation of Abcc2 expression by SUMOylation system
Satoko Minami1, Kousei Ito, Masashi Honma
1Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
American Journal of Physiology. Gastrointestinal and Liver Physiology
|December 17, 2008
Summary
Small ubiquitin-like modifier (SUMO)ylation regulates ATP-binding cassette transporter C 2 (Abcc2) expression. This study identifies Ubc9-mediated SUMOylation of Abcc2, impacting its protein levels but not localization.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The ATP-binding cassette transporter C 2 (Abcc2) facilitates the biliary excretion of organic anions from hepatocytes.
- Posttranslational modifications, including SUMOylation, are known to regulate Abcc2 function, but the precise mechanisms remain unclear.
Purpose of the Study:
- To identify novel proteins interacting with the Abcc2 linker region.
- To investigate the role of SUMOylation in Abcc2 regulation.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- In vitro SUMOylation assays.
- Small interfering RNA (siRNA) knockdown of Ubc9.
- Semiquantitative immunofluorescence analysis.
Main Results:
- Yeast two-hybrid screening identified SUMO-related enzymes and substrates interacting with the Abcc2 linker region.
- SUMOylation of the Abcc2 linker region (IKKE motif) by Ubc9 was confirmed in vitro and in rat hepatoma cells.
- Ubc9 suppression via siRNA reduced Abcc2 protein expression by 30% with minimal impact on subcellular localization.
Conclusions:
- This study demonstrates for the first time that Abcc2 expression is regulated by SUMOylation.
- Ubc9-mediated SUMOylation targets Abcc2, affecting its protein levels.
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