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Mol Cell Proteomics. 2016 Aug;15(8):2802-18. doi: 10.1074/mcp.M116.057885. Epub 2016 May 26.

Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes.

Author information

1
From the ‡Center for Innovative and Translational Medicine, Medical School, Kochi University Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan; §Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3BF, UK; shinya.ohta@kochi-u.ac.jp.
2
§Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3BF, UK;
3
From the ‡Center for Innovative and Translational Medicine, Medical School, Kochi University Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan;
4
¶Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland;
5
‖Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan;
6
**Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia;
7
§Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3BF, UK; ‡‡Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany.

Abstract

Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression.

PMID:
27231315
PMCID:
PMC4974353
DOI:
10.1074/mcp.M116.057885
[Indexed for MEDLINE]
Free PMC Article

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