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

Mary B. Kroetz, Mark Hochstrasser

Research output: Chapter in Book/Report/Conference proceedingChapter

23 Citations (Scopus)

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 - Feb 23 2009

Publication series

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

Fingerprint

Small Ubiquitin-Related Modifier Proteins
Ubiquitin
Yeasts
Proteins
Glycylglycine
Sumoylation
Amino Acid Motifs
Two-Hybrid System Techniques
Electrophoretic Mobility Shift Assay
Glycine
Lysine

Keywords

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

Cite this

Kroetz, M. B., & Hochstrasser, M. (2009). Identification of SUMO-interacting proteins by yeast two-hybrid analysis. In H. D. Ulrich (Ed.), SUMO Protocols (pp. 107-120). (Methods in Molecular Biology; Vol. 497). https://doi.org/10.1007/978-1-59745-566-4_7

Identification of SUMO-interacting proteins by yeast two-hybrid analysis. / Kroetz, Mary B.; Hochstrasser, Mark.

SUMO Protocols. ed. / Helle D. Ulrich. 2009. p. 107-120 (Methods in Molecular Biology; Vol. 497).

Research output: Chapter in Book/Report/Conference proceedingChapter

Kroetz, MB & Hochstrasser, M 2009, Identification of SUMO-interacting proteins by yeast two-hybrid analysis. in HD Ulrich (ed.), SUMO Protocols. Methods in Molecular Biology, vol. 497, pp. 107-120. https://doi.org/10.1007/978-1-59745-566-4_7
Kroetz MB, Hochstrasser M. Identification of SUMO-interacting proteins by yeast two-hybrid analysis. In Ulrich HD, editor, SUMO Protocols. 2009. p. 107-120. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-59745-566-4_7
Kroetz, Mary B. ; Hochstrasser, Mark. / Identification of SUMO-interacting proteins by yeast two-hybrid analysis. SUMO Protocols. editor / Helle D. Ulrich. 2009. pp. 107-120 (Methods in Molecular Biology).
@inbook{172fe77af92d41a080b9cebba92b4344,
title = "Identification of SUMO-interacting proteins by yeast two-hybrid analysis",
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.",
keywords = "Desumoylating enzymes, SIM (SUMO-interacting motif), SUMO, SUMO proteases, Two-hybrid analysis",
author = "Kroetz, {Mary B.} and Mark Hochstrasser",
year = "2009",
month = "2",
day = "23",
doi = "10.1007/978-1-59745-566-4_7",
language = "English (US)",
isbn = "9781934115800",
series = "Methods in Molecular Biology",
pages = "107--120",
editor = "Ulrich, {Helle D.}",
booktitle = "SUMO Protocols",

}

TY - CHAP

T1 - Identification of SUMO-interacting proteins by yeast two-hybrid analysis

AU - Kroetz, Mary B.

AU - Hochstrasser, Mark

PY - 2009/2/23

Y1 - 2009/2/23

N2 - 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.

AB - 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.

KW - Desumoylating enzymes

KW - SIM (SUMO-interacting motif)

KW - SUMO

KW - SUMO proteases

KW - Two-hybrid analysis

UR - http://www.scopus.com/inward/record.url?scp=59249094837&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=59249094837&partnerID=8YFLogxK

U2 - 10.1007/978-1-59745-566-4_7

DO - 10.1007/978-1-59745-566-4_7

M3 - Chapter

C2 - 19107413

AN - SCOPUS:59249094837

SN - 9781934115800

T3 - Methods in Molecular Biology

SP - 107

EP - 120

BT - SUMO Protocols

A2 - Ulrich, Helle D.

ER -