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Vol. 14, Issue 8, 3342-3355, August 2003
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* Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720-3202;
Howard Hughes Medical Institute, Chevy Chase, Maryland 20815-6789; and
Departments of Genome Sciences and Medicine, University of Washington,
Seattle, Washington 98195-7730
Submitted November 25, 2002;
Revised March 28, 2003;
Accepted March 28, 2003
Monitoring Editor: Tim Stearns
Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein–protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p–Ndc80p and Dam1p–Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.
Abbreviations used: GST, glutathione S-transferase; IVT, in vitro-coupled transcription/translation.
The online version of this article contains supplementary tabular material.
Online version is available at
www.molbiolcell.org.
Present address: Ludwig Institute for Cancer Research, La Jolla, CA
92093-0660.
|| Corresponding author. E-mail address: gbarnes{at}socrates.berkeley.edu.
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