title: Using in vivo biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells.
abstract: Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.
authors.affiliation: From the ‡Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, UK;
authors.forename: Mingwei
authors.initials: M
authors.lastname: Min
authors.affiliation: §CIC Biogune, Bizkaia Teknology Park, 48160 Derio, Basque Country, Spain; ¶Ikerbasque, Basque Foundation for Science, 48011, Bilbao, Spain;
authors.forename: Ugo
authors.initials: U
authors.lastname: Mayor
authors.affiliation: ‖Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.affiliation: From the ‡Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, UK; c.lindon@gen.cam.ac.uk.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lindon
mesh_terms: D019921:Biotinylation; D045744:Cell Line, Tumor; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D025941:Protein Interaction Mapping; D040901:Proteomics; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D020690:GTPase-Activating Proteins; C486566:KIFC1 protein, human; D025801:Ubiquitin; C115995:mgcRacGAP; D016547:Kinesins