KAR9P IS A NOVEL CORTICAL PROTEIN REQUIRED FOR CYTOPLASMIC MICROTUBULE ORIENTATION IN YEAST

kar9 was originally identified as a bilateral karyogamy mutant, in whi ch the two zygotic nuclei remained widely separated and the cytoplasmi c microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latteric h, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911-923.), We n ow report a general defect in nuclear migration and microtubule orient ation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations i n dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9 Delta dhc1 Delta, kar9 Delta jnm1 Delta, and kar9 Delta act5 Delta double mutant s were synthetically lethal, suggesting that these genes function in p artially redundant pathways to carry out nuclear migration. A function al GFP-Kar9p fusion protein localized to a single dot at the tip of th e shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortica l dot with both mother-daughter asymmetry and cell cycle dependence. I n small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localizatio n was observed, By indirect immunofluorescence, cytoplasmic microtubul es intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We pro pose that Kar9p is a component of a cortical adaptor complex that orie nts cytoplasmic microtubules.