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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)

Abstract

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 

Notes

Acknowledgments

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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