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.
Highlights
Mitotic division requires highly regulated morphological and biochemical changes to the cell
We further reasoned that highly synchronized cell populations would be required to identify substrates of the ubiquitin-proteasome system (UPS) specific to mitotic exit, so we generated a highly synchronizable human cell line expressing in vivo– biotinylated ubiquitin. bioUb is generated via expression of a synthetic linear construct of six ubiquitin moieties bearing a short biotinylatable tag in tandem with the Escherichia coli biotin ligase BirA
Our identification of a large number of UPS substrates targeted during mitotic exit, and even at specific times during mitotic exit, indicates the wealth of information that can be gathered from using synchronized cell populations
Summary
Plasmids and siRNA—bioUb6-BirA and BirA sequences [30] were inserted in pTRE-tight vector (Invitrogen) to generate pTRE-bioUb6BirA and pTRE-BirA plasmids. Human RacGAP1 cDNA (a kind gift from Masanori Mishima) was inserted into pVenus-C1. Human KIFC1 cDNA (a kind gift from Paola Coelho) was inserted into pVenus-N1. The lysate was diluted with 17.5 ml dilution buffer (1.43 M NaCl, 1ϫ EDTA-free protease inhibitor mixture, 50 mM N-ethylmaleimide in 1ϫ PBS) and applied to 1 ml High Capacity NeutrAvidin Agarose Resin (Thermo Scientific) in a binding step incubated on rollers for 1 h at room temperature and 2 h at 4 °C. The recorded spectra were analyzed using the MaxQuant software package (version 1.3.0.5) with a 1% false discovery rate for both peptides and proteins [35]. For quantification of Venus levels in single cell degradation assays, fluorescence was measured as pixel values within a region of interest (ROI) selected to include the entire cell when applied to all images in a series, and from which background pixel values were subtracted.
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