Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies.
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.
Wohlschlegel
James A
JA
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Johnson
Erica S
ES
Reed
Steven I
SI
Yates
John R
JR
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D008239:Lysine / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D005656:Fungal Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014357:Trypsin; D005978:Glutathione; D008239:Lysine