Proteomics of isolated mitotic chromosomes identifies the kinetochore protein Ska3/Rama1

Cold Spring Harb Symp Quant Biol. 2010;75:433-8. doi: 10.1101/sqb.2010.75.022. Epub 2011 Feb 2.


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.

MeSH terms

  • Animals
  • Cell Line
  • Chickens
  • DNA / metabolism
  • Histones / metabolism
  • Humans
  • Kinetochores / metabolism*
  • Microtubule-Associated Proteins / metabolism*
  • Mitosis*
  • Protein Binding
  • Proteome / metabolism
  • Proteomics / methods*


  • Histones
  • Microtubule-Associated Proteins
  • Proteome
  • DNA