Positioning the
mitotic spindle in yeast has been shown to require both cytoplasmic
microtubules and actin. The spindle positioning gene, KAR9,
encodes a novel cortical protein and is required for the correct
orientation of cytoplasmic microtubules. GFP-Kar9p localizes as a single
dot at the tip of the growing bud and the tip of shmoo projections. The
cortical localization of Kar9p does not require microtubules, indicating
that Kar9p's ability to orient cytoplasmic microtubules is a property of
its cortical associations (Miller and Rose 1998 JCB 140:377). To better
understand Kar9p's interaction with the cortex and to determine the role
of the actin cytoskeleton in localizing Kar9p, we treated shmoos with
the actin depolymerizing drug, latrunculin-A. In latrunculin-A treated
shmoos, Kar9p was not localized to the cortex. These results suggest
that Kar9p mediates the interaction between actin and the cytoplasmic
microtubules. Mutations in four genes involved in actin cytoskeleton
functions, spa2, pea2, bni1 and bud6, significantly
affected Kar9p localization. In spa2 and pea2 mutant
shmoos, Kar9p was found as a cap of localization at the tip of the
shmoo. In bni1 and bud6 mutants, Kar9p's cortical
localization was greatly reduced. In many bni1 and bud6
shmoos a speckled pattern for Kar9p was observed. A variety of other
genes did not alter Kar9p localization. To understand Kar9p functions in
both mitosis and mating, we have examined Kar9p during both of these
life-cycle states. We have found that Kar9p exists in two forms.
Interestingly, the ratio of the two forms of Kar9p differ in mitotic
cells and shmoos.
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