title: Proteomics of isolated mitotic chromosomes identifies the kinetochore protein Ska3/Rama1.
abstract: Despite many decades of study, mitotic chromosomes remain poorly characterized with respect to their structure and composition. Here, we have purified mitotic chromosomes from nocodazole-treated chicken DT40 cells. These chromosomes have a 0.7:1:1 ratio of nonhistone proteins to histones to DNA. They also contain a significant content of RNAs that have yet to be characterized. Overall, the isolated chromosomes contained >4000 polypeptides, >500 of which are either novel or uncharacterized. Elsewhere, we have developed an approach for comparing the results of multiple proteomics experiments. As a validation of this approach, one of 13 novel centromere proteins identified was found to occur in a complex with the previously described proteins Ska1 and Ska2. This novel protein, now known as Ska3/Rama1, occupies a unique domain in the outer kinetochore and was revealed by RNA interference (RNAi) experiments to be essential for cell cycle progression in human cells. The approach presented here offers a powerful way to define the functional proteome of complex organelles and structures whose composition is not simple or fixed.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom.
authors.forename: S
authors.initials: S
authors.lastname: Ohta
authors.forename: J-C
authors.initials: JC
authors.lastname: Bukowski-Wills
authors.forename: L
authors.initials: L
authors.lastname: Wood
authors.forename: F
authors.initials: F
authors.lastname: de Lima Alves
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Rappsilber
authors.forename: W C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D004247:DNA / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D006657:Histones; D008869:Microtubule-Associated Proteins; D020543:Proteome; D004247:DNA