Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis
- 3.1k Downloads
This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protein interactions that can be used to identify small ubiquitin-related modifier (SUMO) interacting proteins and to determine the nature of the SUMO—protein interactions that occur. SUMO binds to a protein in two different ways: covalently and noncovalently. In a covalent interaction an isopeptide bond forms between the glycine residue at the C terminus of the mature SUMO and a lysine side-chain on the substrate protein. Alternatively, SUMO can interact noncovalently with another protein, usually via insertion of a β strand from a substrate SUMO-interacting motif (SIM) into a hydrophobic groove next to the SUMO β2 strand. By mutating either the C-terminal diglycine motif or amino acids within the β2 strand of SUMO, these respective interactions can be abolished. The expression of the two-hybrid SUMO constructs with either of these mutations can help distinguish the type of interaction that occurs between a SUMO and a given protein. Sumoylation can be verified by independent methods, such as a SUMO mobility shift assay. Finally, the chapter will compare the two-hybrid approach with mass spectrometric analysis as a means of identifying SUMO-interacting proteins.
Key wordsSUMO two-hybrid analysis SIM (SUMO-interacting motif) desumoylating enzymes SUMO proteases
We would like to thank Rachael Felberbaum and Dan Su for critical reading of the manuscript. This work was supported by NIH grant GM053756.
- 12.Guthrie, C. and Fink G. R. (1991) Guide to Yeast Genetics and Molecular Biology Academic Press, San Diego, CA.Google Scholar
- 21.Takahashi, H., Hatakeyama, S., Saitoh, H., and Nakayama, K. I. (2005) Noncova-lent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein. J. Biol. Chem. 280, 5611–5621. 22. Kerscher, O. (2007) SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep. 8, 550–555.PubMedCrossRefGoogle Scholar