FIG. 5.

Localization of GFP-tagged Cse4p proteins. Cells were fixed and prepared for microscopy as described in Materials and Methods. The top panels show GFP fluorescence, the middle panels show DAPI staining, and the bottom panels show the overlaid GFP and DAPI images. (Left) Yeast cells (KC100) carrying only wild-type Cse4GFP were grown at 30°C and shifted to 37°C for 5 h (three to four doublings) before fixation. (Middle) KC100 cells expressing only Cse4-107GFP grown at the permissive temperature (30°C). (Right) KC100 cells carrying cse4-107HA and cse4Δ55GFP on separate URA3 and TRP1 vectors, respectively, were streaked and grown at 38°C, the restrictive temperature for both mutations. A single colony was picked, inoculated into prewarmed medium, and grown for an additional 15 h (three to four doublings) at 38°C before fixation and microscopy.