Abstract
The bottleneck in studying protein sumoylation—the conjugation of the small ubiquitin-like modifier (SUMO)—is the detection of the low level of in vivo sumoylated proteins. The Ubc9 fusion-directed sumoylation (UFDS) system strongly enhances the in vivo sumoylation of a substrate protein at its specific sumoylation site. UFDS utilizes an expression plasmid for the protein of interest fused to the SUMO-conjugating enzyme Ubc9. When expressed in HEK293, COS-7, HeLa, or CHO cells, the fused target protein is conjugated with endogenous or coexpressed SUMO at its native sumoylation sites. This sumoylation requires neither SUMO ligase nor any extracellular stimulation and is easily detectable by fusion protein- or Ubc9-specific Western blotting with commercially available antibodies.
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References
Hay, R. T. (2005) SUMO: a history of modification. Mol. Cell 18, 1–12.
Melchior, F., Schergaut, M., and Pichler, A. (2003) SUMO: ligases, isopeptidases and nuclear pores. Trends Biochem. Sci. 28, 612–618.
Desterro, J. M., Rodriguez, M. S., Kemp, G. D., and Hay, R. T. (1999) Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. J. Biol. Chem. 274, 10618–10624.
Gong, L., Li, B., Millas, S., and Yeh, E. T. (1999) Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex. FEBS Lett. 448, 185–189.
Johnson, E. S., Schwienhorst, I., Dohmen, R. J., and Blobel, G. (1997a) The ubiquitinlike protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16, 5509–5519.
Okuma, T., Honda, R., Ichikawa, G., Tsumagari, N., and Yasuda, H. (1999) In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2. Biochem. Biophys. Res. Commun. 254, 693–698.
Desterro, J. M., Thomson, J., and Hay, R. T. (1997) Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett. 417, 297–300.
Johnson, E. S., and Blobel, G. (1997b) Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J. Biol. Chem. 272, 26799–26802.
Rodriguez, M. S., Dargemont, C., and Hay, R. T. (2001) SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659.
Hochstrasser, M. (2001) SP-RING for SUMO: new functions bloom for a ubiquitin-like protein. Cell 107, 5–8.
Kagey, M. H., Melhuish, T. A., and Wotton, D. (2003) The polycomb protein Pc2 is a SUMO E3. Cell 113, 127–137.
Kirsh, O., Seeler, J. S., Pichler, A., Gast, A., Muller, S., Miska, E., Mathieu, M.,Harel-Bellan, A., Kouzarides, T. et al. (2002) The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase. EMBO J. 21, 2682–2691.
Pichler, A., Gast, A., Seeler, J. S., Dejean, A., and Melchior, F. (2002) The nucleoporin RanBP2 has SUMO-1 E3 ligase activity. Cell 108, 109–120.
Uchimura, Y., Nakao, M.,and Saitoh, H. (2004) Generation of SUMO1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway. FEBS Lett. 564, 85–90.
Jakobs, A., Koehnke, J., Himstedt, F., Funk, M., Korn, B., Gaestel, M., and Niedenthal, R. (2007) Ubc9 fusion-directed sumoylation (UFDS): a method to analyze function of protein sumoylation. Nat. Methods 4, 245–250.
Jakobs, A., Himstedt, F., Funk, M., Korn, B., Gaestel, M., and Niedenthal, R. (2007) Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation. Nucleic Acids Res. 35(17):e109. Epub 2007 Aug 20.
Ehrhardt, C., Schmolke, M., Matzke, A., Knoblauch, A., Will, C., Wixler, V., and Ludwig, S. (2006) Polyethylenimine, a cost-effective transfection reagent. Signal Transduction 6, 179–184.
Acknowledgments
The author would like to thank Matthias Gaestel for critical reading of the manuscript, and Astrid Jakobs for preparing the figures shown for Ubc9 fusion-directed sumoylation. This work was supported by the Medical School Hannover, Institute of Physiological Chemistry.
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Niedenthal, R. (2009). Enhanced Detection of In Vivo SUMO Conjugation by Ubc9 Fusion-Dependent Sumoylation (UFDS). In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_5
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DOI: https://doi.org/10.1007/978-1-59745-566-4_5
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