The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

doi:10.1083/jcb.144.5.963

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© The Rockefeller University Press, 0021-9525/1999//963 $5.00
The Journal of Cell Biology, Volume 144, Number 5, , 1999 963-975

Regular Articles

The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

Rita K. Miller, Dina Matheos, and Mark D. Rose

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544

In the yeast Saccharomyces cerevisiae, positioning of the mitoticspindle requires both the cytoplasmic microtubules and actin.Kar9p is a novel cortical protein that is required for the correctposition of the mitotic spindle and the orientation of thecytoplasmic 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 localizationof Kar9p does not require microtubules (Miller, R.K., and M.D.Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interactswith other proteins at the cortex. To investigate Kar9p's corticalinteractions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p'scortical localization was completely dependent on polymerizedactin. Kar9p localization was also altered by mutations in fourgenes, spa2 ${Delta}$ , pea2 ${Delta}$ , bud6 ${Delta}$ , and bni1 ${Delta}$ , required for normal polarizationand actin cytoskeleton functions and, of these, bni1 ${Delta}$ affected Kar9p localization most severely. Like kar9 ${Delta}$ , bni1 ${Delta}$ mutantsexhibited nuclear positioning defects during mitosis and inshmoos. Furthermore, like kar9 ${Delta}$ , the bni1 ${Delta}$ mutant exhibited misorientedcytoplasmic microtubules in shmoos. Genetic analysis placedBNI1 in the KAR9 pathway for nuclear migration. However, analysisof kar9 ${Delta}$ bni1 ${Delta}$ double mutants suggested that Kar9p retained somefunction in bni1 ${Delta}$ mitotic cells. Unlike the polarization mutants,kar9 ${Delta}$ shmoos had a normal morphology and diploids budded inthe correct bipolar pattern. Furthermore, Bni1p localized normally in kar9 ${Delta}$ . We conclude that Kar9p's function is specific forcytoplasmic microtubule orientation and that Kar9p's role innuclear positioning is to coordinate the interactions betweenthe actin and microtubule networks.

Key Words: mitosis • nuclear migration • Spa2 • Bni1p • formin

Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; LAT-A, latrunculin-A; SC, synthetic complete; SPB, spindle pole body.

Address correspondence to Mark D. Rose, Department of MolecularBiology, Lewis Thomas Laboratory, Princeton University, Princeton,NJ 08544. Tel.: (609) 258-2804. Fax: (609) 258-6175. E-mail:mrose{at}molbio.princeton.edu

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