Chl4p and Iml3p interact with centromere DNA and outer kinetochore
proteins: insights into the molecular architecture of the yeast
kinetochore.
Isabelle Pot (1), Vivien Measday (1), Brian
Snydsman (2), Eric Muller (2), Trisha N. Davis (2), Philip Hieter
(1)
(1) Department of Biochemistry , University of British Columbia,
980 W. 28th Ave, Vancouver, BC V5Z 4H4, Canada; (2) Yeast Resource
Centre, Department of Biochemistry, University of Washington, Seattle,
WA 98195
The kinetochore, a multiprotein complex that assembles at
the centromere, is essential for maintaining chromosome transmission
fidelity during mitosis. The CBF3 complex binds centromere (CEN)
DNA. The outer kinetochore, which interacts with CEN DNA via CBF3
and mediates attachment to microtubules, consists of the Ctf19, Ctf3,
Ndc80 and Dam1 protein complexes. Phenotypic and genetic evidence
suggests that Chl4p and its two-hybrid interactor Iml3p are putative
outer kinetochore proteins. chl4 and iml3 mutants
missegregate chromosomes, maintain a dicentric plasmid, and test
positive in screens aimed at identifying candidate kinetochore genes. We
found that chl4 mutants are sensitive to a microtubule-destabilizing drug and synthetic lethal with several kinetochore genes.
We show that Chl4p and Iml3p specifically interact with CEN DNA
by chromatin immunoprecipitation, and co-immunoprecipitate with known
outer kinetochore proteins. By fluorescence deconvolution microscopy, we
also show that Chl4p and Iml3p have a kinetochore localization pattern.
Finally, we determine the requirements on specific components of the
kinetochore for CEN DNA interaction, proper localization, and
protein-protein interaction of Chl4p and Iml3p. Our experiments clearly
establish Chl4p and Iml3p as new members of the outer kinetochore and
provide insights into the molecular architecture of the kinetochore.
Return to YGM 2002 Home at SGD