FIG. 5.
In vivo competition assay of nucleotide-binding mutant septins for neck localization. (A) Strains YEF473 (CDC11/CDC11), M-2630 (cdc11G32E-3HA/cdc11G32E), M-2634 (cdc11G32E-3HA/CDC11), and M-2633 (CDC11-3HA/CDC11) were grown to mid-log phase in YM-P medium at 23°C and processed for immunofluorescence with anti-HA and anti-Cdc11p antibodies before, or after, a 2-h shift to 37°C. More than 200 budded cells were counted for each sample. Asterisks indicate that means of the 37°C sample were significantly different from the mean of the corresponding 23°C sample (two-tailed Student's t test; P < 0.005). (B) YEF473A carrying low-copy-number plasmids expressing the indicated YFP-tagged septin was grown to mid-log phase in selective medium at 23° or 27°C, as indicated. Cells with medium buds were observed by fluorescence microscopy, and the YFP signal from the necks of over 200 cells was quantitated for each strain, as described in Materials and Methods. Bars indicate standard errors. Asterisks indicate means significantly different from the mean of the strain expressing the corresponding wild-type septin (two-tailed Student's t test; P < 0.005).