A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation

Nathalie Eisenhardt; Viduth K Chaugule; Andrea Pichler

doi:10.1007/978-1-4939-6358-4_5

A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation

Methods Mol Biol. 2016:1475:67-78. doi: 10.1007/978-1-4939-6358-4_5.

Authors

Nathalie Eisenhardt¹, Viduth K Chaugule^{1

2}, Andrea Pichler³

Affiliations

¹ Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany.
² MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, DD1 5EH, Dundee, UK.
³ Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany. pichler@ie-freiburg.mpg.de.

PMID: 27631798
DOI: 10.1007/978-1-4939-6358-4_5

Abstract

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.

Keywords: E3 ligase; In vitro sumoylation; PIAS1; RanBP2; SUMO; SUMO chains; SUMO protease; Sp100; ZNF451.

MeSH terms

Biological Assay*
Cloning, Molecular
Escherichia coli / genetics
Escherichia coli / metabolism
Fluorescent Dyes / chemistry
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Humans
Molecular Chaperones / genetics
Molecular Chaperones / metabolism
Nuclear Pore Complex Proteins / genetics
Nuclear Pore Complex Proteins / metabolism
Protein Binding
Protein Inhibitors of Activated STAT / genetics
Protein Inhibitors of Activated STAT / metabolism
Protein Processing, Post-Translational*
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
SUMO-1 Protein / genetics
SUMO-1 Protein / metabolism*
Small Ubiquitin-Related Modifier Proteins / genetics
Small Ubiquitin-Related Modifier Proteins / metabolism*
Spectrometry, Fluorescence
Staining and Labeling / methods
Sumoylation
Ubiquitin-Conjugating Enzymes / genetics
Ubiquitin-Conjugating Enzymes / metabolism
Ubiquitins / genetics
Ubiquitins / metabolism*

Substances

Fluorescent Dyes
Molecular Chaperones
Nuclear Pore Complex Proteins
PIAS1 protein, human
Protein Inhibitors of Activated STAT
Recombinant Proteins
SUMO-1 Protein
SUMO1 protein, human
SUMO2 protein, human
SUMO3 protein, human
Small Ubiquitin-Related Modifier Proteins
Ubiquitins
ran-binding protein 2
Ubiquitin-Conjugating Enzymes
ubiquitin-conjugating enzyme UBC9