title: Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability.
abstract: Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1-3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Wood
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Booth
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Giulia
authors.initials: G
authors.lastname: Vargiu
authors.affiliation: Center for Innovative and Translational Medicine, Kochi University, Kochi, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Ohta
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Flavia
authors.initials: F
authors.lastname: deLima Alves
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Samejima
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Suita 565-0871, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Institute of Bioanalytics, Department of Biotechnology, Technische Universität Berlin, Berlin 13353, Germany.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, UK bill.earnshaw@ed.ac.uk.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D002645:Chickens; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D055786:Gene Knockout Techniques; D007210:Indoleacetic Acids / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008938:Mitosis; D059748:Proteolysis; D040901:Proteomics
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D007210:Indoleacetic Acids
keywords: AID system; CENP-T/W; SILAC; chromosomes; kinetochore; mass spectrometry