FIG. 4.
Cells overexpressing CLA4 or STE20 have a vacuole inheritance defect. (A) Video microscopy and pedigree analysis of FM 4-64-stained GAL-CLA4 (YMG694) cells was performed on cells grown in YP plus 2% galactose. (B) FM 4-64-stained GAL-CLA4 (YMG694) cells were grown in YP plus 2% raffinose to mid-logarithmic stage. At time zero 2% galactose was added to half the culture and the presence of segregation structures in small and medium budded cells and of daughter vacuoles in large budded cells was examined. Representative pictures at the 6-h time point are shown. (C) Wild-type (W303-1A), GAL-CLA4 (YMG694), and GAL-cla4-K594R (YCB91) cells were treated as for panel B, and the percentage of large budded cells with a discrete daughter vacuole was quantified by fluorescence microscopy (>200 large budded cells were counted for each strain). (D) Mid-logarithmic-phase GAL-GFP-CLA4 (YCH5295) cells were grown in YP plus 2% raffinose. At the zero time point 2% galactose was added to induce GFP-CLA4 overexpression, samples were taken every hour, and Western blot analysis was performed. (E and F) FM 4-64-stained pRS316 (YCH4811), p316-GAL-GST-STE20 (YCH4981), and pRS316-GAL-GST-ste20-K649R (YCH4982) cells were grown in SC-URA plus 2% raffinose to the mid-logarithmic stage and treated as for panels B and C. Representative pictures at the 0- and 5-h time points are shown for GAL-STE20 cells. (G) Mid-logarithmic-phase GAL-STE20-GFP (YCB94) cells were treated as for panel D. Pedigree analysis: M, mother; d1 to -3, primary, secondary, and tertiary daughters.