The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A
Figure 4
kis1 encodes an essential protein.
(A, B) Identification of kis1. (A) A multicopy suppressor plasmid of kis1-1 contained an insert with the indicated region of chromosome II. (B) Ten-fold serial dilution assays on SD medium without leucine at 25 or 36°C. The kis1-1 mutants harbor plasmids which express indicated genes (mmm1, SPBC27B12.02 and erg32) or empty vector (empty). Plasmids in 1, 3, and 6 were derived from pREP1, whereas plasmids in 2, 4, 5, and 7 were derived from pREP41. The promoter in pREP1 is stronger than in pREP41. (C) Domain structure of Kis1. The asterisk indicates the mutation site of the kis1-1 mutant (R65C). Gray boxes indicate predicted coiled-coil regions. (D) Alignment of Kis1/Mis19 with predicted orthologs in yeast. Identical residues are boxed in black. Similar residues are shaded in gray. Spom, Schizosaccharomyces pombe; Scry, S. cryophilus; Soct, S. octosporus; Sjap, S. japonicus; Scer, Saccharomyces cerevisiae. The arrow indicates the mutation site of kis1-1. (E, F) Kis1/Mis19 is essential for growth. (E) Diploid cells heterozygous for kis1 (kis1+/kis1::kan) were sporulated and individual spores in each ascus were dissected on the nonselective medium (N/S). Two viable and two non-viable segregation patterns were obtained. Viable colonies were sensitive to kanamycin (G418) without exception, meaning that those are colonies of kis1+ haploid cells. (F) Growth of WT and kis1-1 cells at 36°C. (G) kis1-1 displays unequal chromosome segregation. Left: Centromeres of chromosome II were visualized with GFP (cen2-GFP: green), DAPI (red), and differential interference contrast (DIC). Cells were grown at 36°C for 6 h. Right: Population of cells with unequal segregation of cen2-GFP. n>200.
