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Using in vivo-biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells

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Item Type:Article
Title:Using in vivo-biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells
Creators Name:Min, M. and Mayor, U. and Dittmar, G. and Lindon, C.
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 re-establish 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 in human cells that allows efficient purification of ubiquitin conjugates from synchronised cell populations. Coupling purification with mass spectrometry, we identify a series of mitotic regulators that are targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), 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.
Keywords:Affinity Proteomics, Cell Biology, Cell Cycle, Mass Spectrometry, Ubiquitin, APC/C, KIFC1/HSET, RacGAP1/Cyk4, Mitosis
Source:Molecular & Cellular Proteomics
ISSN:1535-9476
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:13
Number:9
Page Range:2411-2425
Date:September 2014
Official Publication:https://doi.org/10.1074/mcp.M113.033498
PubMed:View item in PubMed

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