Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET).
We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.
Kim
Young-Pil
YP
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Daejeon 305-701, Republic of Korea.
Jin
Zongwen
Z
Kim
Eunkyung
E
Park
Sunyoung
S
Oh
Young-Hee
YH
Kim
Hak-Sung
HS
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D020690:GTPase-Activating Proteins; D008164:Luminescent Proteins; C092317:RANGAP1 protein, human; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C054562:yellow fluorescent protein, Bacteria; D049410:Luciferases, Renilla