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 language | English (US) |
|---|---|
| Title of host publication | SUMO Protocols |
| Editors | Helle D. Ulrich |
| Pages | 107-120 |
| Number of pages | 14 |
| DOIs | |
| State | Published - Feb 23 2009 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 497 |
| ISSN (Print) | 1064-3745 |
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Keywords
- Desumoylating enzymes
- SIM (SUMO-interacting motif)
- SUMO
- SUMO proteases
- Two-hybrid analysis
Cite this
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 proceeding › Chapter
}
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
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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 -