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Published 24 December 2001. doi:10.1083/jcb.200109063
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© The Rockefeller University Press, 0021-9525/2001/12/1137 $5.00
The Journal of Cell Biology, Volume 155, Number 7, December 24, 2001 1137-1146

Article

Implication of a novel multiprotein Dam1p complex in outer kinetochore function



Iain M. Cheeseman1, Christine Brew2, Michael Wolyniak2, Arshad Desai4, Scott Anderson3, Nemone Muster3, John R. Yates3, Tim C. Huffaker2, David G. Drubin1 and Georjana Barnes1

1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853
3 Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
4 Max Planck Institute of Cell Biology and Genetics, 01307 Dresden, Germany

Address correspondence to Georjana Barnes, 401 Barker Hall #3202, University of California, Berkeley, CA 94720-3202. Tel.: (510) 642-5962. Fax: (510) 643-0062. E-mail: gbarnes{at}socrates.berkeley.edu

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ~245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ~0.5 µM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

Key Words: microtubule; spindle; mitosis; kinetochore; Saccharomyces cerevisiae


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