SUMO Protocols pp 107-120 | Cite as

Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis

  • Mary B. Kroetz
  • Mark Hochstrasser
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 497)


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 words

SUMO 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.


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Mary B. Kroetz
    • 1
  • Mark Hochstrasser
    • 2
  1. 1.Department of Cell BiologyYale UniversityNew HavenUSA
  2. 2.Department of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA

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