title: Identification of small ubiquitin-like modifier substrates with diverse functions using the Xenopus egg extract system.
abstract: Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA; §Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Aslanian
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Sun
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
authors.forename: Mingji
authors.initials: M
authors.lastname: Jin
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA;
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
authors.affiliation: §Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: From the ‡Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA; hunter@salk.edu.
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D059447:Cell Cycle Checkpoints / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D019742:Chemokines, CC / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010063:Ovum / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D058207:Sumoylation / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014981:Xenopus
chemical_list: D018797:Cell Cycle Proteins; D019742:Chemokines, CC; D002843:Chromatin; D018006:Myogenic Regulatory Factors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; C063552:transcription factor BTF3