FIG. 3.

Effects of Ste20 mutations in Bem1- versus Cdc42-binding domains on viability, polarization, and localization. (A) Ste20^PP-GA shows a slight defect in performing the Cla4-redundant, essential function of Ste20. (Top) Strain KBY211 (ste20Δ cla4-75^ts) was transformed with the indicated GFP-STE20 plasmids, and then fivefold serial dilutions were spotted onto −Ura plates and incubated for 3 days at 25 or 37°C. (Bottom) KBY211 cultures harboring the indicated GFP-STE20 plasmids were grown in −Ura liquid medium to logarithmic phase at 23°C and then were diluted back and shifted to 37°C. Culture densities were measured over time by optical density at 660 nm (OD₆₆₀) as indicated. Results are means ± standard deviations from three trials; in each trial the same rank order of growth rate was observed. (B) Morphology of cells in panel A after 4 h at 37°C (similar results were observed after 3, 8, or 9 h). Note that the PP-GA mutant generally allows for normal bud neck constriction, compared to that of vector controls, but bud growth is less elongated than with wild-type Ste20, possibly implying a defect in apical polarization. Cells harboring CRIB domain mutants were heterogeneous but were on average more elongated than the PP-GA mutant and showed more examples of wide bud necks (especially the S338A mutant [data not shown]). (C) The Bem1-binding domain can contribute to localization of GFP-Ste20. Localization of the indicated GFP-Ste20 derivatives was examined in cells (PPY1249) overexpressing Bem1 from a high-copy-number plasmid (pPP1202). This method allowed occasional detection of the Ste20 Δ334-369 derivative at polarized growth sites (though rare in comparison to wild-type Ste20), whereas this phenomenon was never observed with the combined Ste20 Δ334-369 PP-GA derivative. The PP-GA mutation alone causes a mild decrease in the intensity of enrichment at bud tips rather than affecting the number of cells showing such enrichment.