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J. Cell Biol.,
Volume 144, Number 5, March 8, 1999 963-975
Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544
In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of
the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)-
Kar9p localizes to a single spot at the tip of the growing
bud and the mating projection. However, the cortical
localization of Kar9p does not require microtubules
(Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140:
377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing
drug, latrunculin-A. In both shmoos and mitotic cells,
Kar9p's cortical localization was completely dependent
on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2
, pea2, bud6, and bni1, required for normal polarization and actin
cytoskeleton functions and, of these, bni1 affected
Kar9p localization most severely. Like kar9, bni1
mutants exhibited nuclear positioning defects during
mitosis and in shmoos. Furthermore, like kar9, the
bni1 mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the
KAR9 pathway for nuclear migration. However, analysis of kar9 bni1 double mutants suggested that
Kar9p retained some function in bni1 mitotic cells. Unlike the polarization mutants, kar9 shmoos had a
normal morphology and diploids budded in the correct
bipolar pattern. Furthermore, Bni1p localized normally
in kar9. We conclude that Kar9p's function is specific
for cytoplasmic microtubule orientation and that
Kar9p's role in nuclear positioning is to coordinate the
interactions between the actin and microtubule networks.
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