Proteomics of Isolated Mitotic Chromosomes Identifies the Kinetochore Protein Ska3/Rama1
- S. Ohta1,
- J.-C. Bukowski-Wills1,2,
- L. Wood1,
- F. de Lima Alves1,
- Z. Chen1,
- J. Rappsilber⇓1 and
- W.C. Earnshaw⇓1
- 1Wellcome Trust Centre for Cell Biology and
- 2Centre for Systems Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom
- Correspondence: bill.earnshaw{at}ed.ac.uk and juri.rappsilber{at}ed.ac.uk
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.
- Copyright © 2010, Cold Spring Harbor Laboratory Press