Abstract
A growing number of biological processes have been found to be regulated by the covalent attachment of the ubiquitin-like protein SUMO to key cellular targets. A critical step in the process of analyzing the role of SUMO in regulating the activity of these proteins is the identification of the lysine residues that are targeted by this modification. Unfortunately, current methods aimed at mapping these attachment-sites are laborious and often ineffective. We report here the development of a platform that combines the use of different C-terminal SUMO mutants with different protease digestion strategies to enable the rapid and efficient identification of SUMO attachment sites. We successfully apply this approach to several model SUMO substrates as well as to a mixture of SUMO conjugates purified from Saccharomyces cerevisiae. Although we specifically employ this strategy for the identification of SUMO attachment sites in yeast, this general approach can easily be adapted to map the sites of conjugation for other ubiquitin-like proteins from a wide range of organisms.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Binding Sites
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Escherichia coli / genetics
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Fungal Proteins / chemistry
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Fungal Proteins / genetics*
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Fungal Proteins / isolation & purification
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Fungal Proteins / metabolism*
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Glutathione / metabolism
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Lysine / analysis
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Lysine / chemistry
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Mass Spectrometry
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Models, Biological
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Molecular Sequence Data
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Mutation*
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Protein Binding
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / isolation & purification
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Repressor Proteins / metabolism
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism
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Sequence Homology, Amino Acid
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Small Ubiquitin-Related Modifier Proteins / chemistry
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Small Ubiquitin-Related Modifier Proteins / genetics*
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Small Ubiquitin-Related Modifier Proteins / isolation & purification
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Small Ubiquitin-Related Modifier Proteins / metabolism*
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Substrate Specificity
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Trypsin / pharmacology
Substances
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Fungal Proteins
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Recombinant Proteins
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Repressor Proteins
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SMT3 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Small Ubiquitin-Related Modifier Proteins
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Trypsin
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Glutathione
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Lysine