The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A
Figure 8
Genetic and physical interaction between Kis1/Mis19 and the Mis16-Mis18 complex.
(A) Genetic crossing of kis1-1 and the mis16-GFP-kan or mis18-GFP-kan mutant was performed, and spores were germinated on YE5S plates at 25°C. Colonies were then replica-plated onto YE5S plates with phloxine B (PB) or with kanamycin (G418) and incubated at 36°C or 25°C, respectively. The temperature-sensitive colonies (arrowheads) were sensitive to G418 without exception, indicating that the double mutant was inviable. mis16 and mis18 were found on the chromosome III, whereas kis1/mis19 was on chromosome II, excluding the possibility of genetic linkage between kis1/mis19 and mis16/mis18. (B) WT or kis1-1 cells expressing Mis18-GFP from plasmids were observed after growth at 25 or 36°C for 6 h. Percentages of cells without Mis18-GFP dots are shown (n≥100). (C) Localization of Kis1-GFP in WT, mis16-53, or mis18-262 cells grown at 25 or 36°C for 6 h. Frequencies are also given (n≥100). Scale bars, 5 µm. (D) Overexpression of Mis16-GFP suppressed the temperature sensitivity of kis1-1. Cells harboring plasmid pREP1-mis16-GFP (mis16 OP) were spotted on EMM plates and incubated at 25 or 32°C. Cells harboring pREP1-GFP were spotted as controls. (E) Coimmunoprecipitation (IP) of Kis1 and the Mis16–Mis18 complex. Cell extracts (input) were prepared from cells expressing (+) or not expressing (−) the indicated proteins. Kis1-myc (left) and Mis18-GFP (right) were immunoprecipitated with anti-Myc or anti-GFP, respectively. IP samples as well as 2.5% of the input were analyzed.
