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Vol. 11, Issue 9, 2949-2959, September 2000
and
Department of Molecular Biology, Lewis Thomas Laboratory, Princeton
University, Princeton, New Jersey 08544
In Saccharomyces cerevisiae, positioning of the
mitotic spindle depends on the interaction of cytoplasmic microtubules
with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, and
KAR9 itself. STU2 encodes a component of
the spindle pole body. Bim1p is the yeast homologue of the human
microtubule-binding protein EB1, which is a binding partner to the
adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid
interactions were confirmed with the use of coimmunoprecipitation
experiments. Genetic analysis placed Bim1p in the Kar9p pathway for
nuclear migration. Bim1p was not required for Kar9p's cortical or
spindle pole body localization. However, deletion of
BIM1 eliminated Kar9p localization along cytoplasmic
microtubules. Furthermore, in the bim1 mutants, the
cytoplasmic microtubules no longer intersected the cortical dot of
Green Fluorescent Protein-Kar9p. These experiments demonstrate that
the interaction of cytoplasmic microtubules with the Kar9p cortical
attachment site requires the microtubule-binding protein Bim1p.
Permanent address: Institute of Molecular Biology,
Academia Sinica, Nankang, Taiwan.
Corresponding author. E-mail address:
mrose{at}molbio.princeton.edu.
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