Identification of SUMO-interacting proteins by yeast two-hybrid analysis

Mary B. Kroetz, Mark Hochstrasser

Research output: Chapter in Book/Report/Conference proceedingChapter

25 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationSUMO Protocols
EditorsHelle D. Ulrich
Pages107-120
Number of pages14
DOIs
StatePublished - 2009

Publication series

NameMethods in Molecular Biology
Volume497
ISSN (Print)1064-3745

Keywords

  • Desumoylating enzymes
  • SIM (SUMO-interacting motif)
  • SUMO
  • SUMO proteases
  • Two-hybrid analysis

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