Identification of SUMO-interacting proteins by yeast two-hybrid analysis.
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 beta strand from a substrate SUMO-interacting motif (SIM) into a hydrophobic groove next to the SUMO beta2 strand. By mutating either the C-terminal diglycine motif or amino acids within the beta2 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.
Kroetz
Mary B
MB
Department of Cell Biology, Yale University, New Haven, CT, USA.
Hochstrasser
Mark
M
D001483:Base Sequence; D015698:Genomic Library; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins