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© The Rockefeller University Press, 0021-9525/1998//377 $5.00
The Journal of Cell Biology, Volume 140, Number 2, , 1998 377-390

Article

Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast



Rita K. Miller and Mark D. Rose

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9{Delta} dhc1{Delta}, kar9{Delta} jnm1{Delta}, and kar9{Delta} act5{Delta} double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.

Abbreviations used in this paper: GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; pI, isoelectric point; SPB, spindle pole body; YPD, yeast peptone dextrose.

Address all correspondence to M.D. Rose, Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Tel.: (609) 258-2804. Fax: (609) 258-6175. E-mail: mrose{at}molecular.princeton.edu


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