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Vol. 17, Issue 1, 178-191, January 2006

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The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

Department of Biology, University of Rochester, Rochester, NY 14627

Submitted June 27, 2005; Revised October 5, 2005; Accepted October 12, 2005
Monitoring Editor: Tim Stearns

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1 mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5 mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

Abbreviations used: AD, activation domain; CFP, cyan fluorescent protein; cMT, cytoplasmic microtubule; DBD, DNA binding domain; HU, hydroxyurea; MAP, microtubule-associated protein; MT, microtubule; SPB, spindle pole body; TAP, tandem affinity purification.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Rita K. Miller (rmlr{at}mail.rochester.edu).

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